Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leukocyte Adhesion

Article abstract of DOI:10.1016/j.chembiol.2018.09.012

Small-molecule inhibitors of glycosylation can be applied in basic science studies, and clinical investigations as anti-inflammatory, anti-metastatic, and anti-viral therapies. This article demonstrates that thioglycosides represent a class of potent metabolic decoys that resist hydrolysis, and block E-selectin-dependent leukocyte adhesion in models of inflammation. Metabolic decoys are synthetic analogs of naturally occurring biosynthetic acceptors. These compounds divert cellular biosynthetic pathways by acting as artificial substrates that usurp the activity of natural enzymes. While O-linked glycosides are common, they are only partially effective even at millimolar concentrations. In contrast, we report that N-acetylglucosamine (GlcNAc) incorporated into various thioglycosides robustly truncate cell surface N- and O-linked glycan biosynthesis at 10–100 μM concentrations. The >10-fold greater inhibition is in part due to the resistance of thioglycosides to hydrolysis by intracellular hexosaminidases. The thioglycosides reduce β-galactose incorporation into lactosamine chains, cell surface sialyl Lewis-X expression, and leukocyte rolling on selectin substrates including inflamed endothelial cells under fluid shear. Treatment of granulocytes with thioglycosides prior to infusion into mouse inhibited neutrophil homing to sites of acute inflammation and bone marrow by ～80%–90%. Overall, thioglycosides represent an easy to synthesize class of efficient metabolic inhibitors or decoys. They reduce N-/O-linked glycan biosynthesis and inflammatory leukocyte accumulation.

Full text of DOI:10.1016/j.chembiol.2018.09.012

Article
Thioglycosides Are Efficient Metabolic Decoys of
Glycosylation that Reduce Selectin Dependent
Leukocyte Adhesion
Graphical Abstract
Authors
Shuen-Shiuan Wang, Xuefeng Gao,
Virginia del Solar, ..., Roger A. Laine,
Khushi L. Matta, Sriram Neelamegham
Correspondence
klmatta40@gmail.com (K.L.M.),
neel@buffalo.edu (S.N.)
In Brief
Small-molecule inhibitors of
glycosylation can be applied in basic
science studies, and clinical
investigations as anti-inflammatory, anti-
metastatic, and anti-viral therapies. This
article demonstrates that thioglycosides
represent a class of potent metabolic
decoys that resist hydrolysis, and block
E-selectin-dependent leukocyte
adhesion in models of inflammation.
Highlights
d
d
d
d
S-Glycosides act as superior metabolic decoys compared
with O-glycosides
Unlike O-glycosides, the S-glycosides resist hydrolysis by
cellular hexosaminidases
S-Glycosides reduce sialyl Lewis-X expression and E-
selectin binding to neutrophils
S-Glycosides block granulocyte migration to mouse bone
marrow and inflammation sites
Wang et al., 2018, Cell Chemical Biology 25, 1–14
December 20, 2018 ª 2018 Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2018.09.012

Please cite this article in press as: Wang et al., Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leuko-
cyte Adhesion, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.012
Cell Chemical Biology
Article
Thioglycosides Are Efficient Metabolic Decoys
of Glycosylation that Reduce Selectin
Dependent Leukocyte Adhesion
Shuen-Shiuan Wang,1 Xuefeng Gao,2 Virginia del Solar,1,3 Xinheng Yu,1 Aristotelis Antonopoulos,4 Alan E. Friedman,5
Eryn K. Matich,5 G. Ekin Atilla-Gokcumen,5 Mehrab Nasirikenari,6 Joseph T. Lau,6 Anne Dell,4 Stuart M. Haslam,4
1,3,7,
Roger A. Laine,2 Khushi L. Matta,1,2, and Sriram Neelamegham
*
*
1Department of Chemical and Biological Engineering, State University of New York, 906 Furnas Hall, Buffalo, NY 14260, USA
2TumorEnd LLC, Louisiana Emerging Technology Center, 340 East Parker Drive, Suite 246, Baton Rouge, LA 70803, USA
3Clinical & Translational Research Center and State University of New York, Buffalo, NY 14260, USA
4Department of Life Sciences, Imperial College London, London SW7 2AZ, UK
5Department of Chemistry, State University of New York, Buffalo, NY 14260, USA
6Department of Cellular and Molecular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
7Lead Contact
*Correspondence: klmatta40@gmail.com (K.L.M.), neel@buffalo.edu (S.N.)
https://doi.org/10.1016/j.chembiol.2018.09.012
SUMMARY
ferases (GTs) that transfer mono- or oligo-saccharides from
various donors to growing carbohydrate chains, and by glycosi-
Metabolic decoys are synthetic analogs of naturally dases that hydrolyze individual monosaccharides to trim gly-
cans. Small molecules developed to modify GT/glycosidase
activity can lead to novel strategies to regulate glycan structures,
occurring biosynthetic acceptors. These compounds
divert cellular biosynthetic pathways by acting as arti-
ficial substrates that usurp the activity of natural en-
zymes. While O-linked glycosides are common, they
are only partially effective even at millimolar concen-
trations. In contrast, we report that N-acetylglucos-
amine (GlcNAc) incorporated into various thioglyco-
sides robustly truncate cell surface N- and O-linked
cell function, and treat diseases.
While a number of small-molecule inhibitors of glycosidase ac-
tivity have been developed, fewer molecules disrupt GT activity
(Gloster and Vocadlo, 2012; Hudak and Bertozzi, 2014). In this
regard, most small-molecule GT inhibitors developed fall into
one of three categories: (1) acceptor analogs of nucleotide-sugar
donors or transition state mimetics of the acceptor-donor pair.
While such reagents haven been shown to be effective in
in vitro enzymology assays, their inability to permeate cell mem-
glycan biosynthesis at 10–100 mM concentrations.
The >10-fold greater inhibition is in part due to the
resistance of thioglycosides to hydrolysis by intracel- branes limits their biological utility (Schworer and Schmidt, 2002;
Zhu et al., 1995). (2) Modified monosaccharides that are cell
permeable. This large set of compounds, include monosaccha-
lular hexosaminidases. The thioglycosides reduce
b-galactose incorporation into lactosamine chains,
cell surface sialyl Lewis-X expression, and leuko-
cyte rolling on selectin substrates including inflamed
endothelial cells under fluid shear. Treatment of gran-
ulocytes with thioglycosides prior to infusion into
mouse inhibited neutrophil homing to sites of acute
ride analogs where selected hydroxyl or other groups are re-
placed by halogen, deoxy, thiol, or methyl substituents (Goon
and Bertozzi, 2002). Such molecules are often converted into
their corresponding nucleotide-sugar analogs within cells, at
high concentrations (Gloster et al., 2011). This results in deple-
tion of selected, natural nucleotide-sugars, and global depres-
inflammation and bone marrow by ꢀ80%–90%. Over-
sion in the activities of entire families of related GTs (Rillahan
et al., 2012; van Wijk et al., 2015). Thus, this approach may
lack specificity. (3) Surrogate acceptors that act as artificial sub-
all, thioglycosides represent an easy to synthesize
class of efficient metabolic inhibitors or decoys.
They reduce N-/O-linked glycan biosynthesis and in- strates for specific biochemical pathways (Brown et al., 2007;
Sarkar et al., 1997). These molecules compete with and reduce
carbohydrate biosynthesis on the natural glycoconjugate by
acting as alternate substrates for the GTs. Such decoys may
flammatory leukocyte accumulation.
INTRODUCTION
be designed to target specific pathways. However, to effectively
compete with natural substrates, they are typically applied at
Glycans are a complex post-translational modification that regu- relatively high concentrations. For example, peracetylated
late virtually all biological processes (Laine, 1994; Neelamegham benzyl-a-GalNAc (or ‘‘GalNAc-OBn’’) is added at 2–4 mM into
and Mahal, 2016; Varki, 2017). Commonly, cell surface carbohy- cell culture medium to inhibit O-linked glycosylation (Alfalah
drates appear either as O- or N-linked glycans on glycoproteins, et al., 1999; Huet et al., 1998; Kuan et al., 1989; Tsuiji et al.,
glycosphingolipids (GSLs), or glycosaminoglycans (GAGs). Such 2003), and xylosides are similarly used at 1–2 mM for inhibiting
structures are formed by the sequential action of glycosyltrans- GAG biosynthesis (Fritz et al., 1994; Okayama et al., 1973; Victor
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cyte Adhesion, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.012
Figure 1. Thio/S- and O-Glycosides as Metabolic Decoys
(A) Structures 1–6 correspond to S-glycosides; 7–12 are O-glycosides. Control molecules, GlcNAc with sulfhydryl group at anomeric position (13, ‘‘SH’’) and
GlcNAc (14), are also shown.
(B and C) Potential mechanism of S-glycoside (B) and O-glycoside (C) action as metabolic decoys. In both cases, peracetylated glycosides or decoys are taken
up by cells and deacetylated by cellular esterases. These compounds are processed through the Golgi where they form biosynthetic products, including Lewis-X
and sialyl Lewis-X-type structures. These glycosides divert natural biosynthetic pathways and truncate cell surface glycan biosynthesis. S-Glycosides are more
effective acceptor-decoys, compared with O-glycosides. Unlike the O-glycosides, they are not spontaneously hydrolyzed by cellular hexosaminidases.
See also Supplemental Information, compound characterization.
et al., 2009). At lower concentrations (ꢀ10–100 mM), these de- (compound 1) or O-linkage to yield ‘‘ONAP’’ (7). When added to
coys have little or no inhibitory activity, and thus are used as mo- human leukocytes, SNAP more effectively reduced expression
lecular probes that report on the cellular O-glycan (Kudelka et al., of the sialyl Lewis-X (sLeX, Siaa2-3Galb1-4(Fuca1-3)GlcNAc)
2016; Stolfa et al., 2016) or GAG biosynthesis pathways (Victor epitope on cells, compared with ONAP. SNAP also reduced cell
et al., 2009).
adhesion to E-selectin, the major human selectin which binds
In the current study, using a panel of N-acetylglucosamine sialofucosylated epitopes on leukocyte O-glycans, N-glycans,
(GlcNAc)-based metabolic decoys, we observed that the efficacy and GSLs (Mondal et al., 2016; Stolfa et al., 2016). In mouse,
of surrogate acceptors/decoys can be tuned by modifying the SNAP-treated leukocytes exhibited reduced migration to sites
linkage between the carbohydrate and aglycone. In particular, of inflammation and bone marrow. Overall, this report demon-
the study examined the effect of modifying the acetal group found strates the use of thioglycosides as a class of novel, potent inhib-
in traditional O-glycosides, to thioacetals in S-glycosides (Fig- itors that can disrupt lactosamine biosynthesis on cellular O- and
ure 1A). In this regard, previous enzymology investigations N-glycans.
suggest that selected S-aryl glycosides are susceptible to cleav-
age by hexosaminidases, albeit at lower rates compared with RESULTS
O-glycosides (Macauley et al., 2005). In contrast, we report,
here, a set of S-glycosides that are more stable with minimal Thioglycosides Reduce Cell Surface Sialyl Lewis-X
breakdown by cytoplasmic, lysosomal, and nuclear hexosa- Expression and E-Selectin Binding
minidases (Figures 1B and 1C). Due to this property, the S-glyco- A panel of GlcNAc-based thio/S- (1–6) and O-glycosides (7–12)
sides function in cell-based assays to disrupt cellular lactos- were synthesized (Figure 1A, Supplemental Information). The
amine biosynthesis on N- and O-linked glycans at ꢀ10-fold aglycone in these entities was varied. While some of the com-
lower concentrations compared with O-glycosides. In particular, pounds contained NAP (SNAP 1, 2, ONAP 7, 8), benzyl (6, 12),
many of the studies were performed with peracetylated com- or bromooctane (5) aglycones, others contained derivatives of
pounds where the 2-naphthalenemethanol (NAP) group was natural products, geranyl (3, 4, 11) or methoxyphenyl (9, 10).
linked to GlcNAc via an S-glycosidic linkage to yield ‘‘SNAP’’ Several of the compounds were peracetylated (1, 3, 5, 6, 7, 9)
2
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to enhance cell permeability (Sarkar et al., 1997). The acetyl bers of the S- and O-glycoside families. Neither compound, up to
groups are removed by esterases inside cells to yield free carbo- 200 mM, affected cell viability or proliferation based on trypan
hydrates (Figures 1B and 1C).
blue exclusion, LDS-751 live cell staining, hemocytometer
We determined if any of the compounds alter cell surface gly- counts, or the tetrazolium salt-based XTT assay (data not
cans important for inflammatory cell adhesion (Figure 2). Here, shown). Also, none of the treatments promoted apoptosis based
treatment of HL-60 promyelocytic leukemia cells with 100 mM on Annexin-V binding (data not shown).
of all S-glycosides for 40 hr reduced cell surface sialyl Lewis-X
Microfluidics-based leukocyte adhesion measurements
(sLeX) expression by 60%–90%, as measured using mAb determined if the reduced selectin binding observed in the static
HECA-452 (Figure 2A). The results with O-glycosides was assays, translate to physiologically relevant fluid shear condi-
more variable, with (11) and (7) reducing the sLeX epitope by tions (Figure 3). Here, glycoside or control treatments did not
20% and 40%, and (10) augmenting sLeX by 30%. Upon alter the density of rolling or adherent cells on substrates
comparing the GlcNAc-b-SNAP in its peracetylated (SNAP, 1) composed of E-selectin bearing interleukin-1b-simulated human
with free (2) form, it is apparent that peracetylation is not critical umbilical vein endothelial cells (HUVECs) (Figure 3A), immobi-
for inhibition function although it improves efficacy. Thus the lized E-selectin (Figure 3B), L-selectin (Figure 3C), or P-selectin
NAP group exhibits sufficient hydrophobicity for cell perme- (Figure 3D). However, SNAP increased the median cell-rolling
ability, and additional acetylation of glycan hydroxyl groups velocity on E-selectin bearing stimulated HUVEC monolayers
only has a marginal contribution. S-Glycosides were effective by 4.5-fold (from 1 to 4.5 mm/s, Figure 3E), on E-selectin sub-
at concentrations as low as 10 mM with maximum effectiveness strates by 2-fold (from 2.4 to 4.33 mm/s, Figure 3F), and on
above 50 mM (Figure 2B).
L-selectin by 3-fold (from 18 to 53 mm/s, Figure 3G). The glyco-
As sLeX is a critical determinant for selectin-dependent leuko- side did not affect rolling on P-selectin (Figure 3H). These obser-
cyte adhesion, the effect of the S- and O-glycosides on soluble vations were confirmed at multiple glycoside concentrations,
human selectin-immunoglobulin G (IgG) protein binding to HL- 25–100 mM (Figures S2F and S2G). Overall, SNAP reduced
60s was assayed. Here, consistent with the HECA-452 measure- leukocyte interactions on E-selectin and stimulated endothe-
ments, all S-glycosides reduced E-selectin IgG binding, with the lial cells.
blocking pattern being remarkably similar to the changes in sLeX
expression (Figures 2C and 2D). The O-glycosides did not exhibit SNAP Abrogated Neutrophil Homing to Sites of
this property. Similar to E-selectin, many of the S-glycosides Inflammation and the Bone Marrow in Mice
(1–3, 5, 6) also downregulated L-selectin binding although the ef- Previous studies show that increased rolling velocity on selectins
fect was smaller (Figure 2E). None of the glycosides affected ex vivo may be sufficient to reduce leukocyte accumulation
P-selectin binding (Figure 2F). The core-2 sLeX epitope at the in vivo (Marathe et al., 2010; Morikis et al., 2017). To determine
N terminus of PSGL-1 is the major P-selectin ligand on human the anti-inflammatory potential of the S-glycosides, a murine thi-
leukocytes (Lo et al., 2013; Wilkins et al., 1996), and this struc- oglycollate peritonitis model was used to contrast the effect of
ture is apparently not fully disrupted by the compounds. Func- SNAP versus ONAP (Figure 4). Here, murine bone marrow cells
tion-blocking anti-selectin mAbs and vehicle controls confirm (mBMCs) obtained from donor C57BL/6 were cultured ex vivo
the specificity of the measured interaction. Cytometry histo- with either ONAP, SNAP, or vehicle for 40 hr (Figure 4A). These
grams suggest that these observations are true for the entire cells were then differentially labeled with either a green fluores-
cell population, and not limited to a sub-population of HL-60s cent dye (CMFDA), red dye (CMTMR), or both, to result in three
(Figure S1).
differentially stained cell populations. The populations were
Similar to the undifferentiated HL-60s, SNAP also reduced mixed in equal proportion. Peritonitis was induced in recipient
sLeX expression and E-selectin binding to neutrophils obtained C57BL/6 using thioglycollate intraperitoneal injection for 1 hr,
by terminal-differentiation of HL-60s using DMSO (Figure S2). and then the labeled cell mixture was introduced intravenously.
These differentiated cells expressed five times greater cell sur- At 20 hr, 20%–30% fewer ONAP-treated cells were observed
face CD11b levels compared with undifferentiated HL-60s, in the bone marrow and inflamed peritoneum, compared with
with CD11b expression being unaffected by the glycoside treat- vehicle treatment (Figures 4B–4D). SNAP caused a more dra-
ment (Figure S2A). Thus, the glycosides do not alter the overall matic decrease, with cell accumulation being consistently
cell phenotype. Here, also, SNAP (S-glycoside) reduced sLeX reduced by ꢀ80%–90%. Deficiency in E- and L-selectin-depen-
expression by 70% (Figure S2B), E-selectin binding by 85% (Fig- dent cell adhesion may contribute to the observed reduced hom-
ure S2C), and L-selectin IgG recognition by 30% (Figure S2D), ing to sites of inflammation.
without altering P-selectin (Figure S2E). Neither SNAP nor
ONAP altered leukocyte surface expression of putative selectin Upregulation of PNA and ECL Lectin Binding upon
ligand scaffolds: CD11b, CD43, CD44, CD45, or CD162 (data Treatment with S-Glycosides
not shown). Overall, the S-glycosides are potent modifiers of Flow cytometry-based lectin binding studies were performed, to
glycan biosynthesis.
identify reagents that quantitatively report on the effect of glyco-
side treatment in heterologous cell types (Figure 5). Here, SNAP
augmented peanut agglutinin (PNA) binding to HL-60s by
ꢀ30-fold, suggesting the alteration of O-linked glycan biosyn-
Thio/S-Glycosides Reduce Leukocyte Adhesion under
Hydrodynamic Shear
Among the compounds tested, many of the studies contrasted thesis, and the increased expression of the Galb1,3GalNAc
the effectiveness of SNAP (1) with respect to ONAP (7) since epitope (Figure 5A). A ꢀ5-fold increase in Erythrina cristagalli
both molecules are peracetylated, and they are prototypic mem- lectin (ECL) binding (Figure 5B) and doubling of PHA-L binding
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Figure 2. Static Selectin IgG Binding and CLA Expression Altered by O- and S-Linked Decoys
HL60 cells were cultured with 0–100 mM GlcNAc decoys for 40 hr
Flow cytometry measured the binding of: (A and B) anti-cutaneous lymphocyte-associated antigen (CLA) mAb HECA-452, (C and D) E-selectin IgG, (E) L-selectin
IgG, and (F) P-selectin IgG to the cells. Dose-dependent data for HECA-452 expression and E-selectin IgG binding are presented in (B), (D) anti-E-selectin (clone
P2H3, B), L-selectin; Dreg-56 (C) and P-selectin (G1) (D), blocking mAbs confirm selectin IgG binding specificity. NC, negative-control (secondary antibody
alone); IC, isotype control; VC, vehicle control.
*p < 0.05 with respect to VC. S-Glycosides reduced E-selectin IgG binding and CLA expression more dramatically compared with the O-glycosides. All
S-glycosides, except 4, also partially reduced L-selectin IgG binding. Structures 1 (SNAP), 7 (ONAP), and 13 (SH) are indicated in the different panels as they are
the focus of subsequent studies.
See also Figures S1 and S2.
4
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Figure 3. Effect of SNAP on Cell Adhesion under Flow
HL60s were cultured with SNAP, ONAP, SH, or VC (0.2% DMSO) for 40 hr. Cells were then perfused at wall shear stress of 1 dyne/cm2 over substrates bearing:
(A and E) interleukin-1b stimulated HUVEC monolayers, (B and F) recombinant human E-selectin IgG, (C and G) L-selectin IgG, or (D and H) P-selectin IgG.
Interacting cells were classified into rolling or firmly adherent cells (top panels). Cell-rolling velocity was also recorded in the cumulative line plots (bottom panels).
Blocking antibodies used were against E-selectin (P2H3), L-selectin (Dreg-56), or PSGL-1 (KPL-1). SNAP increased cell-rolling velocity on stimulated HUVECs,
E-selectin IgG, and L-selectin IgG substrates, as indicated by red median velocity markings in (E–G).
*p < 0.05 for total interacting cells with respect to VC, SNAP, ONAP, and SH, except as indicated in (A).
(Figure 5C) was also measured indicating potential perturbations to undifferentiated HL-60s, HL-60s differentiated to neutrophils
in N-glycan structures, particularly the Galb1,4GlcNAc lactos- also exhibited >10-fold increase in PNA binding following culture
amine chains. In addition, anti-LeX mAb binding was marginally with SNAP (Figure S4B). Significantly, the concentration range
decreased (Figure 5D). More prominent changes in lectin binding where selectin binding function was altered in Figures 2, 3,
accompanied SNAP treatment, compared with ONAP (Figure 5). and 4 was similar to that necessary for PNA-lectin binding alter-
In such studies, PNA, ECL, and PHA-L lectin binding to O- and ation (20–100 mM, Figure S4C). Thus, all S-glycosides appear to
N-glycans is typically augmented upon de-sialylation (Stolfa alter cellular glycosylation via similar molecular mechanisms.
et al., 2016). However, the MAL-II binding data indicate that
the overall expression of a2,3 sialic acid terminated glycans Truncation of O-Glycan Biosynthesis by S-Glycosides
remains unchanged.
The increased PNA-lectin binding suggests that the S-glycosides
In general, the entire panel of S-glycosides exhibited a pattern may modify cell surface O-glycans. This was confirmed using a
of carbohydrate epitope alteration similar to SNAP, albeit to vary- panel of CRISPR-Cas9 knockout cell lines since PNA-lectin bind-
ing degrees depending on the aglycone (Figure S3). All O-glyco- ing to leukocytes was abolished in cells lacking O-glycansunder a
sides were similar to vehicle control except for ONAP (7), which variety of conditions, including upon SNAP treatment (Figures
displayed some carbohydrate modification potential, although S5A and S5B). In addition, the S-glycoside reduced the molecular
low relative to SNAP. The increased PNA-lectin binding upon massoftwoprominentleukocyteglycoproteins,PSGL-1, which is
SNAP treatment was observed across multiple cell lines, the major L-/P-selectin ligand (Figure 5E), and CD43/leukosialin
including human embryonic kidney HEK293T, breast cancer (Figure 5F). Both mucin glycoproteins displayed a ꢀ20% reduc-
T47D and ZR75-1, and prostate PC-3 cells (Figure S4A). Similar tion in molecular mass upon SNAP, but not ONAP, treatment.
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Figure 4. Murine Model of Acute Inflammation
(A) Bone marrow cells (mBMCs) were collected from 10- to 12-week-old wild-type C57BL/6 mice. These were cultured with 100 mM ONAP, SNAP, or 0.25%
DMSO (VC) for 40 hr ex vivo, differentially labeled with fluorescence reporters, mixed in approximately equal proportion, and then injected into recipient mice.
Peritonitis was induced 1 hr before injection. Twenty hours later, samples from the peritoneal lavage and bone marrow were collected and analyzed using
cytometry
(B) In ‘‘Mix 1,’’ ONAP-, SNAP-, and VC-treated cells were labeled with either green, red, or green and red dyes, respectively. The dyes were swapped in ‘‘Mix 2’’
and ‘‘Mix 3.’’ Panel presents raw cytometry data showing distinct labeled cell populations at the top-left, bottom-right, and top-right quadrants. Reduced cell
numbers upon SNAP treatment are indicated by blue arrows.
(C and D) In both the peritoneum (C) and bone marrow (D), the number of labeled 1A8/Gr-1+ cells was reduced dramatically (>90%) in the case of SNAP treatment
compared with either ONAP or VC. *p < 0.05 between indicated treatments.
To determine the biosynthetic steps affected by SNAP, substrates, N-terminal PSGL-1 peptide for ppGalNAcT and
radioactivity-based ppGalNAc-transferase (Figure S5C) and GalNAc-O-Bn for GalT. Enzymatic transfer of radioactivity
b1,3GalT (Figure 5G) enzymology assays were undertaken. from donor to substrate was then measured. Here, the transfer
Here, lysates of cells cultured with decoys or controls served of [14C]GalNAc to the peptide substrate was similar in all ly-
as the enzyme source. Cell lysates were mixed with radioactive sates, suggesting that the glycosides may not alter ppGalNAcT
donor (UDP-[14C]GalNAc or UDP-[14C]Gal) along with synthetic activity.
6
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Figure 5. Truncation of O-Glycan Biosynthesis by SNAP
(A–D) Wild-type HL-60s were cultured with 80 mM ONAP, SNAP, or vehicle controls for 40 hr. Flow cytometry measured cell surface carbohydrate structures using
either fluorescent antibodies or lectins: PNA (binds Galb1,3GalNAc) (A), ECL (binds Galb1,4GlcNAc) (B), PHA-L (binds complex N-glycans) (C), and CD15/Lewis-X
(mAb HI98) (D). Data in inset are mean fluorescence intensity SD for >3 independent experiments. *p < 0.05 with respect to VC. SNAP increased PNA, ECL, and
PHA-L bindings. ONAP has a smaller effect.
(E and F). Cell lysates run under standard reducing conditions, were probed with mAbs against mucinous proteins, anti-human PSGL-1 mAb TB5 (E) and anti-
human CD43 mAb L60 (F). SNAP reduced molecular mass of both glycoproteins indicating truncation on O-glycans.
(G) HL-60s were treated with 80 mM glycosides or vehicle control. Cell lysates were incubated with 10,000 dpm UDP-[14C]Gal (donor) in the presence or absence
of 0.5 mM GalNAc-OBn (acceptor). [14C]Gal is preferentially added to SNAP present in cell lysates rather than GalNAc-OBn (left half). Even in the absence of
GalNAc-OBn, radioactive products are formed on SNAP and ONAP (right half).
(H–J) HL-60s were cultured with 80 mM ONAP, SNAP, or GalNAc, with 100 mM GalNAc-OBn being added after 8 hr in some cases. Products secreted into culture
medium over 2 days were collected and quantitatively analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). SNAP, and to a lesser
extent ONAP, reduces glycan biosynthesis on GalNAc-OBn (H). Extended carbohydrate chains grew on SNAP (I) and ONAP (J). All assignments were verified by
MS/MS.
*p < 0.05, **p < 0.01 with respect to GalNAc; ynot detected. See also Figures S3–S5.
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In the GalT assay, however, two distinct products were of the HECA-452 epitope (Figure 2A). Notably, bi- and tri-antenn-
observed using thin-layer chromatography when the lysates con- ary glycans with the sLeX epitope (m/z = 3,140 and 3,950) are
tained ONAP or SNAP (left half, Figure 5G). One migrated identi- apparent in the vehicle, but not SNAP-treated cells.
cally to [14C]Galb1,3GalNAc-O-Bn, while the second migrated
The effect of SNAP on the GSLs was small compared with that
faster. This second product is likely [14C]Gal-GlcNAc-S-NAP on the N-glycans. Here, a majority of the GSL glycans were
(lane 2) and [14C]Gal-GlcNAc-O-NAP (lane 3) formed using unpro- similar in vehicle versus SNAP. However, a few truncated gly-
cessed SNAP and ONAP available in cell lysates, as these same cans were also apparent at m/z=1,188, 1,566, and 2,087 (red
entities were also prominently observed upon omitting GalNAc- peaks in Figure 6C, lower panel). Sialylated GSL glycans were
O-Bn in the reaction mixture (right half, Figure 5G). The extent of also reduced upon SNAP treatment compared with vehicle con-
[
14C]Gal transfer to SNAP was ꢀ2-fold greater than that to ONAP trol, e.g., the peak ratio at m/z 1,304/943 was reduced from 1.64
based on the more intense radioactive product. Even when in vehicle to 0.56 upon SNAP treatment; and at m/z 1,753/1,392
GalNAc-O-Bn was present in the reaction mixture (left half, Fig- from 1.28 in vehicle to 0.92 for SNAP (Figure 6C, upper and lower
ure 5G), [14C]Gal-GlcNAc-S-NAP was the dominant product in panels). Finally, consistent with the observations using GalNAc-
the SNAP runs, while equal amounts of [14C]Gal-GlcNAc-O-NAP O-Bn, O-glycans from SNAP-treated HL60s revealed a loss of
and [14C]Galb1,3GalNAc-O-Bn were formed in the ONAP run. In- core 1 and core 2 structures (Figure 6D).
dependent MS-based enzymology studies suggest that these ob-
servations are not simply because SNAP is a superior acceptor for SNAP Is a More Potent Surrogate Acceptor-Decoy
galactose compared with ONAP (Figure S5D). In these runs, Gal Compared to ONAP
transfer to ONAP was also diminished in the presence of SNAP, The more potent inhibitory effect of SNAP compared with ONAP
suggesting a potential inhibitory function for the S-glycoside could be due to the inherent intracellular stability of S-glycosides.
(Brockhausen et al., 2006). Overall, the S-glycosides may act In this regard, O-glycosides are commonly used to assay the ac-
both as acceptors of galactose and inhibitors of related enzymes. tivity of hexosaminidases, and it is known that mammalian cells
Mass spectrometry studies were undertaken to determine the have lysosomal, nuclear, and cytoplasmic hexosaminidases that
effect of SNAP and ONAP on O-glycosylation using the cellular maycleave such substrates(Stutz andWrodnigg, 2016). Todeter-
O-glycan reporter assay (Kudelka et al., 2016). Here, peracety- mine if such hexosaminidase activity is prominent in HL-60 cells,
lated GalNAc-O-Bn was fed to cells in the presence of glyco- we measured the high-performance liquid chromatography
sides or control, and products formed on these substrates was elution profile of 2-napthelenemethanol (HONAP [hydrolyzed
quantified using MS by assaying the culture medium (Figures ONAP]) and 2-naphthalenemethanethiol (HSNAP) standards
5H–5J, all structures validated using tandem MS [MS/MS]). spiked into the HL-60 cell culture supernatant (Figure 7A). This
Here, 38% of the GalNAc-O-Bn substrate was converted to profile was compared with that of cells cultured with vehicle (Fig-
other products when the medium contained GalNAc/control ure 7B), ONAP (Figure 7C), and SNAP (Figure 7D). Here, a promi-
(Figure 5H). Products formed included Galb1-3GalNAc-OBn/T- nent peak with retention time corresponding to HONAP was
antigen, mono- and di-sialylated T-antigen, and core-2 glycans. observed in Figure 7C upon culture with ONAP, but not one corre-
The fraction of GalNAc-O-Bn converted to product was reduced sponding to HSNAP in Figure 7D when SNAP was present. Based
to 14% upon culture with ONAP and 2.5% upon SNAP addition. on area under the curve, recoveries, and absorbance calibration
Thus, T-antigen biosynthesis was inhibited by the S-glycoside curves generated with chemical standards, we estimate that
decoy. Consistent with this notion, VVA lectin binding (recog- ꢀ30% of ONAP may be cleaved within cells. Neither liquid chro-
nizes GalNAca on O-glycans) to HL-60s treated with the entire matography-MS nor gas chromatography-MS were able to ionize
panel of S-glycosides was augmented by 3- to 100-fold (Fig- underivatized HONAP for MS detection, and thus dansylated-
ure S6). The effect of the O-glycosides was small, in comparison. HONAP standards were prepared (Figures 7E and 7F). In addition,
In addition to GalNAc-O-Bn, a variety of glycan products were NAP products secreted into cell culture medium were also deriv-
also elaborated on SNAP (Figure 5I) and ONAP (Figure 5J), atized with dansyl chloride (Figure 7G). Here, ESI-Q-TOF MS/MS
including sLeX structures, and extended LacNAc chains some- was able to verify the formation of HONAP by HL-60s, based on
times containing terminal fucose and sialic acid. Whereas elab- retention time compared with chemical standards (Figure 7F
orated glycans were observed on 23% of the SNAP substrate versus 7G, left panels), mass, and also fragmentation spectra
in the culture medium, this was lower at 17% for ONAP.
(right panels). Overall, ONAP and/or its derivatives are partially
hydrolyzed in cells, while SNAP is not.
SNAP Truncates N- and O-Glycan Biosynthesis, with a
Smaller Effect on Glycolipids
To determine the nature of competition between the glyco-
sides, equal amounts of peracetylated ONAP and SNAP were
MALDI-TOF MS glycome profiling was undertaken to determine mixed and fed to HL-60 cell culture medium. At 40 hr, glycoside
the precise N- and O-glycans, and GSLs that are altered by products were purified from culture supernatants (Figure 7H) and
SNAP (Figure 6). Here, we observed an increased abundance cell pellets (Figure 7I). They were permethylated and quantified
of GlcNAc-terminated (agalactosylated, truncated) carbohy- based on product ion counts using high-resolution liquid chro-
drate chains and LacNAc antennas with reduced sialic acid matography-MS/MS. As seen, the total prevalence of extra-
abundance (red peaks in Figure 6A, lower panel). These charac- and intracellular glycoside products was greater in the presence
teristic desialylated peaks that appear at m/z 2,530, 2,734, etc., of SNAP compared with ONAP, suggesting a role for hydroly-
may explain the increased ECL binding in Figure 5B. N-Glycans sis in regulating relative glycan biosynthesis. In particular, the
from SNAP-treated HL60s also displayed the absence of the concentration of SNAP substrate was 2.8-fold higher in the pellet
sLeX epitope (Figure 6B), consistent with the reduced expression compared with ONAP. In addition, whereas a variety of products
8
Cell Chemical Biology 25, 1–14, December 20, 2018

Please cite this article in press as: Wang et al., Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leuko-
cyte Adhesion, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.012
Figure 6. Whole-Cell Glycomics Profiling Following SNAP Treatment
(A and B) N-linked glycans, (C) glycosphingolipids, and (D) O-linked glycans isolated from HL60 cells that were cultured with 60 mM of vehicle control (upper
panels) or SNAP (lower panels). (B) MALDI-TOF MS spectra of N-glycans of the zoomed scan m/z 3,134–3,155 and of m/z 3,945–3,964 molecular ion clusters
derived either from vehicle control (upper panels) or SNAP (lower panels) of (A). Red peaks on SNAP-treated N-linked glycans and glycosphingolipids spectra
correspond to GlcNAc-terminated (agalactosylated) structures that appear upon SNAP treatment. Isolated glycans were permethylated and analyzed by MALDI-
TOF MS. All molecular ions are [M+Na]+. Putative structures are based on composition, tandem MS and biosynthetic knowledge. Structures that show sugars
outside a bracket have not been assigned.
were synthesized when ONAP was fed to cells alone (Figure 5J), with ONAP. Similar observations were made in independent vali-
glycan elaboration on this substrate was diminished in mixed dation runs performed using ESI-Q-TOF MS (Figure S7). Overall,
systems that contained SNAP (Figures 7H and 7I). Eight- to the hydrolysis of O-glycosides may partially explain the reduced
14-fold higher levels of glycans were formed on SNAP compared efficacy of such compounds, compared with the S-glycosides.
Cell Chemical Biology 25, 1–14, December 20, 2018
9

Please cite this article in press as: Wang et al., Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leuko-
cyte Adhesion, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.012
Figure 7. Hydrolysis of O-Glycosides in Cells
(A–D) HL60s were cultured with 80 mM SNAP, ONAP, or vehicle control. After 40 hr, the medium was extracted and analyzed using high-performance liquid
chromatography (224 nm detection). Standards were created by spiking culture media with 2-naphthalenemethanol (HONAP) and 2-naphthalenemethanethiol
(HSNAP) standards at 1:1 ratio (A). These compounds eluted at 17 and 29 min, respectively. Media with vehicle control had no peaks (B). Peak corresponding to
HONAP retention time was seen in supernatants from cells cultured with ONAP, black arrowhead in (C). Cells cultured with SNAP did not have product cor-
responding to HSNAP (D).
(E–G) HONAP was derivatized by dansyl chloride to serve as an MS standard (chemical reaction in schematic). No peak was observed in vehicle control (E).
Dansyl derivatization was performed for products extracted from cell culture medium in runs with ONAP, SNAP, and VC. Dansylated HONAP was only observed in
runs with ONAP (G) and this was verified by MS/MS, with respect to synthetic standard in (F). The equivalent product was not observed in cells cultured with SNAP
or vehicle. Thus, ONAP, but not SNAP, was hydrolyzed within cells.
(H and I) Cells were co-treated with a mix of 80 mM SNAP together with ONAP for 40 hr. Glycosylated products formed on ONAP and SNAP present in supernatant
(H) and cell pellet (I) were analyzed using LC-MS/MS. Larger glycan products were formed on SNAP compared with ONAP. ‘‘NAPX’’ is used to denote both O- and
S-glycosides. All structures were verified by MS/MS.
*p < 0.05, **p < 0.01, ***p < 0.01 with respect to ONAP; ynot detected.
See also Figure S7.
DISCUSSION
and prostate (PC3) cells. Such decoys were effective at low con-
centrations of ꢀ10–100 mM. In contrast, the more common O-gly-
This study demonstrates that thioglycosides or S-glycosides effi- cosides are only functional at >10-fold higher doses. The
ciently disrupt cellular biosynthetic pathways important for in- enhanced functional efficacy of S-glycosides was noted in all as-
flammatory processes by functioning as metabolic decoys. says, and it was most apparent in the competition/co-culture
This was observed in multiple cell types including leukocytes assay where SNAP and ONAP were simultaneously fed to cells.
(HL-60s and primary leukocytes), breast (T47D, ZR75-1) cells, Here, MS analysis of biosynthetic products present in culture
10 Cell Chemical Biology 25, 1–14, December 20, 2018

Please cite this article in press as: Wang et al., Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leuko-
cyte Adhesion, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.012
medium and cell pellet showed that extended glycosylation prod- to determine which of the hexosaminidases act on ONAP and
ucts formed on SNAP with 8- to 14-fold greater efficacy other O-glycosidase substrates as this may be a key mitigating
compared with ONAP. Indeed, while there is literature on the syn- factor that reduces the efficacy of such compounds.
thesis of S-glycosides, and alkyl/aryl 1-thioglycosides are used
The mammalian lactosamine motif is a common unit that is
as donors during the chemical synthesis of carbohydrates, rela- part of the molecular recognition epitope of various lectins,
tively fewer studies have applied such compounds in cell-based including selectins, siglecs, and galectins (Neelamegham and
assays (Macauley et al., 2005; Miura et al., 1999). The current re- Mahal, 2016; Varki, 2017). As GlcNAc-based S-glycosides
sults suggest that this may be a valuable avenue for investigation, truncate the growth of this common motif on different types
as it can yield entities that can modify cellular biosynthetic path- of glycoconjugates, these small molecules may be broadly
ways and related glycan structures. The scale up of such com- useful as inhibitors for a variety of studies, although this may
pounds is cost effective.
come at the cost of reduced specificity. To demonstrate
All S-glycosides used in this study truncate lactosamine chain such biological utility, we tested the ability of SNAP and
extensions on N- and O-glycans, although their relative efficacy ONAP to reduce selectin-dependent cell adhesion with focus
varied depending on the aglycone. In this regard, SNAP affected on E-selectin, an endothelial lectin that binds sialofucosylated
both type III Galb1,3GalNAc chains on O-glycans based on the glycans such as sLeX on all common glycoconjugate-types:
increased binding of VVA and PNA, and type II Galb1,4GlcNAc O-glycans, N-glycans, and GSLs (Stolfa et al., 2016). Here,
chains on N-glycans based on alterations in ECL binding (Stolfa all S-glycosides dramatically reduced cell surface leukocyte
et al., 2016). MS-based glycomics profiling also showed an in- sialyl Lewis-X expression on leukocytes, and E- and L-selectin
crease in GlcNAc-terminated N-glycans and reduction in sialyl- binding under static and flow conditions. These compounds
lactosamine structures on N-glycans. These glycomics profiles also increased leukocyte rolling velocity on stimulated endo-
are reminiscent of previous cases of congenital disorders of thelial cell monolayers, and treatment of leukocytes with these
glycosylation (Boztug et al., 2014; Hayee et al., 2011). The sLeX compounds abolished transplanted cell homing to the bone
structure was prominently absent in the SNAP samples. Lactos- marrow and sites of inflammation in the mouse. This effect of
amine extensions on O-glycans were also reduced upon small molecules to reduce inflammation by altering leukocyte
S-glycoside treatment as the carbohydrates synthesized on rolling rates is similar to results noted using two different sialyl
the GalNAc-O-Bn substrate were small. Immunoblotting showed Lewis-X analogs that completed phase II trials, GMI-1070 (Riv-
that the molecular mass of two mucinous proteins, PSGL-1 and ipansel) (Morikis et al., 2017) and TBC-1269 (Hicks et al., 2005),
CD43, was reduced upon S-glycoside addition. Thus, the overall and also the monosaccharide analog 4F-GalNAc (Marathe
glycan mass on extended O- and N-glycans was reduced. The et al., 2010). In the case of Rivipansel, a molecule currently in
effect of the compound on GSLs was relatively small.
phase III trials for reducing vaso-occlusive crisis following
Regarding the mechanism of action, the data suggest that the sickle cell disease, the partial reduction of E-selectin binding
thioglycosides may be superior decoys due to their enhanced interactions is considered to reduce cell activation via selectin
stability in cells, and reduced susceptibility to degradation by ligand interactions (Morikis et al., 2017). This alteration in cell
mammalian b-hexosaminidases (EC 3.2.1.52). In addition, these activation rather than the complete abrogation of selectin
glycosides may inhibit specific GalT activity (Brockhausen et al., ligand interaction is considered to be the mechanism of molec-
2006), although the mechanism for this process and specificity ular action. Consistent with this notion, SNAP also dramatically
toward different members of this enzyme class remains unsolved reduced cell migration to sites of inflammation and the bone
(Gao et al., 2010). Consistent with the dual role for S-glycosides marrow in the current study, by modifying leukocyte rolling in-
as decoys and inhibitors, while equal amounts of [C14]Gal were teractions in flow assays. Additional studies are needed in vivo
transferred to ONAP and GalNAc-O-Bn in the GalT assay, to complete the pharmacological characterization of the S-gly-
SNAP acted as an efficient decoy to take up almost ꢀ90% of cosides, compare their blocking efficacy to other small-mole-
the [C14]Gal and it dramatically reduced [C14]Galb1,3GalNAc- cule selectin antagonists (Dimitroff et al., 2003; Marathe
OBn formation. In this regard, hexosaminidases in cells are pre- et al., 2010; Rillahan et al., 2012; Sarkar et al., 1997; Zandberg
sent in the lysosomal, cytoplasmic, and nuclear compartments et al., 2012), and to test their inhibitory efficacy in relevant dis-
inhumans and may contribute to the degradation of O-glycosides eases models.
and their products, including those containing terminal GlcNAc
In conclusion, this study suggests that thioglycosides may
and GalNAc (Mahuran, 1999). Here, the lysosome contains be suitable for a broad range of basic science and trans-
various isoenzymes (HexA, HexB, and HexS) that are synthesized lational investigations, due to their enhanced efficacy within cells
by the homo- or hetero-dimerization of two subunits: a encoded compared with the O-glycosides. Using SNAP as a prototypic
by HEXA and b by HEXB. Mutations in HEXB often results in entity, it may be possible to vary the aglycone group to create
Sandhoff disease, while mutations in HEXA decreases GM2 molecular entities with varying specificity for diverse applica-
ganglioside degradation and causes Tay-Sachs disease (Sandh- tions. Advancing this concept, the glycan/GlcNAc entity of
off and Harzer, 2013). HEXC encodes for O-GlcNAcase (OGA) SNAP may also be changed to target other glycan biosynthetic
which acts as a nucleocytoplasmic b-hexosaminidase that re- pathways. Finally, it may be valuable to also vary the anomeric
moves O-GlcNAc modification on various proteins (Gao et al., linkage in order to further enhance functional effects. Such mod-
2001). Mammalian cells also contain a fourth hexosaminidase ifications to the glycan, linkage, and aglycone may result in novel
HexD in the nucleus and cytoplasm, encoded by HEXDC (Gutter- tools for biomedical research and drug candidates with favor-
nigg et al., 2009), which prefers to hydrolyze GalNAc compared able pharmacological activity to target leukocyte adhesion,
with GlcNAc (Alteen et al., 2016). Additional studies are needed inflammation, and cancer metastasis.
Cell Chemical Biology 25, 1–14, December 20, 2018 11

Please cite this article in press as: Wang et al., Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leuko-
cyte Adhesion, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.012
R.A.L., K.L.M., and S.N.; Co-corresponding authors, K.L.M. led the chemical
SIGNIFICANCE
synthesis effort and S.N. led functional studies.
There is active interest in rationally designing small-molecule
DECLARATION OF INTERESTS
inhibitors of cellular glycosylation. One such approach uses
metabolic decoys that function as mimetics of naturally
X.G., K.L.M., and R.A.L. are affiliated with TumorEnd LLC, a for-profit small
occurring glycoEnzyme (glycosylating enzyme) substrates.
business with interest in anti-inflammatory molecules. These investigators
When introduced into cells, such decoys attract the activity
of the glycoEnzymes and thus the natural glycoconjugates
are left under glycosylated. This manuscript demonstrates
that thesimplechemicalmodificationoftheanomericlinkage
of metabolic decoys, from acetal in traditional O-glycosides
to thioacetal group in thio/S-glycosides, dramatically en-
hances the stability of these compounds within cells and im-
proves inhibitor efficacy by >10-fold. This strategy to improve
metabolic decoy design may enhance their application in
basic science studies, and also clinical investigations as
anti-inflammatory, anti-metastasis and anti-viral therapies.
synthesized the tested compounds and requested but did not perform any
testing. K.L.M. authored PCT WO/2016/077567 related to the use of glyco-
sides for anti-inflammatory/viral activity. All other authors declare no conflicts
of interest.
Received: March 22, 2018
Revised: August 14, 2018
Accepted: September 25, 2018
Published: October 18, 2018
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14 Cell Chemical Biology 25, 1–14, December 20, 2018

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cyte Adhesion, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.012
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
Antibodies
SOURCE
IDENTIFIER
HECA-452 (anti-human CLA)
D12 (anti-human CD11b)
HI98 (anti-human CD15)
KPL-1 (anti-human CD162)
DREG-56 (anti-human CD62L)
L60 (anti-human CD43)
TB5 (anti-human CD162)
IA8 (anti-mouse Ly-6G)
P2H3 (anti-human E-selectin)
G1 (anti-human CD62P)
ECL lectin (Galb1,4GlcNAc)
BD Biosciences
BD Biosciences
BD Biosciences
BD Biosciences
BD Biosciences
BD Biosciences
GeneTex
RRID: AB_396243
RRID: AB_400112
RRID: AB_395801
RRID: AB_396324
Cat#: 555544
RRID: AB_394204
RRID: AB_377146
RRID: AB_1877163
RRID: AB_11219468
Cat#: 252-820
BioLegend
Thermo Fisher Scientific
Ancell
Vector Laboratories
Vector Laboratories
RRID: AB_2336437
RRID: AB_2336655
PHA-L lectin (binds complex glycan epitope:
Galb1,4GlcNAc b1,6(GlcNAc b1,2Mana1,3)Man a1,3)
MAL II lectin (binds a2,3sialic acid)
PNA lectin (binds Galb1,3GalNAc)
VVA lectin (binds GalNAca)
Chemicals, Peptides, and Recombinant Proteins
LC/MS solvents and common chemicals
L-selectin
Vector Laboratories
Vector Laboratories
Vector Laboratories
RRID: AB_2336569
RRID: AB_2336458
RRID: AB_2336854
Sigma-Aldrich
R&D Systems
R&D Systems
R&D Systems
R&D Systems
GenScript
728-LS-100
724-ES-100
137-PS-050
201-LB-025/CF
E-selectin
P-selectin
IL-1b
PSGL-1 peptide (Custom synthesis)
Glycosides
Syntheses described in
current manuscript
Experimental Models: Cell Lines
HL-60
ATCC
RRID: CVCL_0002
HL-60 Knock-outs
Stolfa, G. et al Sci. Rep.
2016, 6:30392
PC-3
ATCC
ATCC
ATCC
ATCC
RRID: CVCL_0035
RRID: CVCL_0553
RRID: CVCL_0588
RRID: CVCL_2959
T-47D
ZR-75-1
HUVEC
Experimental Models: Organisms/Strains
mice
Envigo RMS
Catalog 44 C57BL/6 wild-type
Software and Algorithms
DrawGlycan-NSFG
Cheng et al., 2017
Ceroni et al., 2008
https://github.com/kaichengub/DrawGlycan-SNFG
https://VirtualGlycome.org/drawglycan
GlycoWorkbench
https://github.com/alternativeTime/glycoworkbench
CONTACT FOR REAGENTS AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Sriram
Neelamegham (neel@buffalo.edu). Chemical compounds will be provided via TumorEnd LLC.
Cell Chemical Biology 25, 1–14.e1–e5, December 20, 2018 e1

Please cite this article in press as: Wang et al., Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leuko-
cyte Adhesion, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.012
EXPERIMENTAL MODEL AND SUBJECT DETAILS
10-12 week-old C57BL/6 wild-type mice of either sex were used. Animals were randomized prior to experimentation. All animal
studies were approved by the Roswell Park Cancer Institute Animal Care and Use Committee (RPCI-IACUC). HL-60 cells (female
promyeloblasts, RRID:CVCL_0002), T47D (female ductal carcinoma, RRID: CVCL_0553) and ZR-75-1 (female epithelial ductal car-
cinoma, RRID: CVCL_0588), and metastatic prostate PC-3 cells (male adenocarcinoma, RRID: CVCL_0035) were obtained from
ATCC. Human Umbilical Vein Endothelial Cells (HUVECs, cat #CC-2519A) were from Lonza.
METHODS DETAILS
Chemical Synthesis
The synthesis of GlcNAc derivatives is described below. These include a series of thio/S-glycosides: peracetylated GlcNAc-b-S-NAP
(1, abbreviated ‘SNAP’), GlcNAc-b-S-NAP (2), peracetylated GlcNAc-b-S-geranyl (3), GlcNAc-b-S-geranyl (4), peracetylated
GlcNAc-b-S-bromooctane (5) and peracetylated GlcNAc-b-S-benzyl (6). The O-glycosides include peracetylated GlcNAc-b-O-
NAP (7, abbreviated ‘ONAP’), GlcNAc-b-O-NAP (8), peracetylated GlcNAc-b-O-methoxyphenyl (9), GlcNAc-b-O-methoxyphenyl
(10), GlcNAc-b-O-geranyl (11) and GlcNAc-b-O-benzyl (12). Control molecules used are peracetylated GlcNAc-b-SH (13, abbrevi-
ated ‘SH’); and GlcNAc (14).
GlcNAcbZ (O-glycosides) Synthesis
2-Acetamido-2-deoxy-D-glucopyranose (1.77 g, 8.0 mmol) was suspended in acetyl chloride (AcCl, 8 mL) at 0ꢁC under nitrogen and
the mixture was stirred at room temperature (r.t.) overnight (Horton and Wolfrom, 1962; Orth et al., 2010). The solvent was removed
under vacuum, then the residue was diluted with dichloromethane (15 -25 mL) and extracted with ice water until the pH value of the
aqueous layer was neutral. The organic layer was dried and concentrated. The 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy glucopyra-
nosyl chloride obtained was a colorless solid. In the second step, the chloride (365 mg, 1.0 mmol) was stirred with 2-naphthalene-
˚
methanol (158 mg, 1.0 mmol), and freshly prepared molecular sieves 4A (500 mg) in CH2Cl2 (8 mL) for 6-8 h. To this mixture, InCl3
(110 mg, 0.5 mmol) was added and the reaction was stirred for additional 16-20 h at room temperature (Mukherjee et al., 2001).
The progress of the formation of the product was examined by TLC (ethylacetate/hexane 1:1 or dichloromethane/acetone 4:1).
The reaction mixture was then diluted with CH2Cl2 (8 mL), filtered over celite and washed with saturated sodium bicarbonate solution
(3 times), and dried over Na2SO4. It was filtered and concentrated in vacuo. The desired product was purified using silica gel flash
chromatography using solvent gradients ethylacetate/hexane 1:2 or acetone/dichloromethane 1:10. In most cases, compounds
were obtained as solid materials. 2-naphthalenemethanol was replaced by other aglycon alcohols to obtain other O-glycosides,
except for 9 & 10. Detailed reaction scheme is shown below (Matta, 2016).
Synthesis of Acetylated GlcNAcbSZ
For preparation of thiourea salt (Horton and Wolfrom, 1962; Ibatullin et al., 2001), thiourea (3.8 g, 5 mmol) was added to a mixture of
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy glucopyranosyl chloride (1.83 g, 5 mmol) in acetonitrile (10 mL). The reaction was refluxed
for 10 min. Then the solvent was evaporated. After crystallization from acetone, product was obtained as white solid. In the next step,
2-(bromomethyl)naphthalene (442 mg, 2 mmol) was added to a solution of thiourea salt (885 mg, 2 mmol) in acetonitrile (10 mL),
followed by TEA (0.7 mL, 5 mmol) (Ibatullin et al., 2001). The reaction was stirred at r.t. for 1 day, and then concentrated in vacuo.
The crude product was purified by flash chromatography using acetone /dichloromethane which afforded a white solid product.
In an alternate method reported earlier (Matta et al., 1973), the urea salt treated with potassium pyrosulfite in refluxing mixture of
water and chloroform gave 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-b-glycopyranose 13 (Matta et al., 1973). The latter on
treatment with alkyl bromide in acetone in the presence of anhydrous potassium carbonate in acetone (Claeyssens et al., 1970)
can give alkyl-thio glycosides. 2-(bromomethyl)naphthalene was replaced by other aglycone halides (Z-halide) in the above reaction
to furnish the remaining acetylated GlcNAcbSZ compounds from 13. The full reaction scheme is shown below.
e2 Cell Chemical Biology 25, 1–14.e1–e5, December 20, 2018

Please cite this article in press as: Wang et al., Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leuko-
cyte Adhesion, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.012
Synthesis Procedure for p-methoxyphenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-glucopyranoside 9
To a solution of b-D-glucosamine pentaacetate (10 g, 25 mmol) and p-methoxyphenol (9.5 g, 75 mmol) in CH2Cl2 (180 mL) chilled in
salt ice bath, was added BF3$OEt2 (4.7 mL, 37.5 mmol) dropwise (Fettke et al., 2009). The reaction was stirred at room temperature
for 24 h, and then was washed with water, NaHCO3 solution, and brine. The organic layer was dried (MgSO4) and concentrated. The
residue was purified with flash chromatography which afforded product as white solid. Compound 10 was obtained by deacetylation
of 9.
De-O-acetylation
Thio and O-glycosides were deacetylated using sodium methoxide in methanol. Typically, to a solution of 1 mmol starting material in
MeOH (10 mL), 5-10 drops NaOMe/MeOH 0.5 M solution was added. The reaction was allowed to stir for overnight. When reaction
was complete, the solution was filtered through a short pack of Dowex 50W resin, and concentrated. The resulted white solid was
washed with small amount of cold MeOH and filtered.
For large scale preparation of GlcNAc-bONAP has already been reported from Matta’s group (Xue et al., 2009) wherein one-step
b-selective glycosylation of N-acetylglucosamine (Vauzeilles et al., 2001) with NAP-bromide in presence of lithium bromide and so-
dium hydride was found to be effective in providing the target compound as solid material.
Cell Culture and Chemical Treatment
All cell lines were cultured according to ATCC (Manassas, VA), unless otherwise mentioned. HUVECs were cultured in EBM-2 media
(Lonza). Isogenic HL-60 clones lacking extended O-glycans ([O] cells), N-glycans ([N]) and glycolipids ([G]) were available from a pre-
vious study (Stolfa et al., 2016). These cells lack the genes COSMC (core-1 b3 galatoctosyltransferase molecular chaperone), MGAT1
(mannosyl a1,3-glycoprotein b1,2-N-acetylglucosaminyltransferase) and UGCG (UDP-Glucose Ceramide Glucosyltransferase),
respectively.
For cell treatment, all GlcNAc-based glycosides were dissolved in DMSO to make 40 mM stocks, and stored at -20ꢁC until use. In
typical runs, the GlcNAc O- and S-glycosides were added to 0.5-13106 cells/mL at 0-200 mM in normal culture medium for 2-days
(ꢀ40 h). All runs included control compounds (13, 14) and/or vehicle control (typically 0.2-0.25% DMSO). In some runs, HL-60s were
differentiated to terminal neutrophils by culturing cells with 1.3% DMSO over 5 days (Marathe et al., 2008). Here, on day-3, 80 mM
SNAP, ONAP, SH or 0.2% DMSO (vehicle) was added to the culture medium. Cells analysis was performed on day-5. In other cases,
100 mM peracetylated a-benzyl GalNAc (abbreviated ‘GalNAc-O-Bn’), available from a previous study (Stolfa et al., 2016), was added
along with ONAP or SNAP for subsequent MS analysis. In each case, at the treatment end-point, the cells were harvested and re-
suspended in HEPES buffers (30 mM HEPES, 110 mM NaCl, 10 mM KCl, 2 mM MgCl2, 10 mM glucose, pH 7.3) containing 0.1%
HSA (human serum albumin) and 1.5 mM Ca2+. Cell surface glycan analysis and functional assays were then performed using
flow cytometry, microfluidics based cell adhesion assays, Western blotting and enzymology as described previously ((Buffone
et al., 2013; Marathe et al., 2008; Mondal et al., 2015), see below).
Antibodies and Lectins
Antibodies employed for cytometry analysis include fluorescent rat anti-Cutaneous Lymphocyte Antigen/CLA mAb HECA-452 (IgM)
which recognized sialyl Lewis-X/sLeX and related antigens, mouse anti-human CD11b mAb D12 (IgG), mouse anti-CD15/Lewis-X
mAb HI98 (IgM), and isotype controls. Function blocking mAbs used include anti-human PSGL-1 mAb KPL-1, anti-P-selectin
mAb G1, anti-E-selectin mAb P2H3, and anti-L-selectin mAb DREG-56. Lectins used in this study include fluorescein-conjugated
Peanut agglutinin (PNA; binds the T-antigen or Galb1,3GalNAc), Erythrina cristagalli lectin (ECL; binds N-acetyl lactosamine
Galb1,4GlcNAc), Phaseolus vulgaris Leucoagglutinin (PHA-L; binds Galb4GlcNAcb6(GlcNAcb2Mana3)Mana3 on N-glycans), and
biotinylated Maackia amurensis lectin II (MAL-II; binds a(2,3) linked sialic acid). Recombinant human L-/CD62L, E-/CD62E and
P-/CD62P selectin IgG fusion proteins were also used.
Flow Cytometry
Typically, cells suspended in HEPES buffer with 0.1% HSA (human serum albumin) and 1.5 mM Ca2+ were incubated with 1-10 mg/mL
of fluorescent monoclonal antibodies or lectins for 20 min on ice prior to flow cytometry analysis using either a FACSCalibur or
LSRFortessa X-20 instrument (BD Biosciences). In the case of MAL-II binding measurements, FITC-conjugated anti-biotin Ab was
also added in a second incubation step as this lectin was biotinylated. For static selectin binding assays, 3 mg/mL human selec-
tin-IgG was first complexed with 10 mg/mL PerCP-conjugated anti-human Fc Ab (Jackson ImmunoResearch, West Grove, PA) in
HEPES buffer containing 1% goat serum and 1.5 mM Ca2+ for 10 min at r.t. In some cases, 5-10 mg/mL anti-selectin blocking
mAbs were also added in this step. The selectin-PerCP Ab complex was then incubated with 13106 HL-60 cells/mL for 10 min
at r.t., prior to 10-fold dilution of the mixture in HEPES buffer (with 0.1% HSA and 1.5 mM Ca2+) and cytometry analysis.
Microfluidics Based Leukocyte Adhesion Assay
Cell adhesion studies were performed as described previously (Buffone et al., 2013), using a 100 mm 3 400 mm cross-section custom
microfluidic flow cell placed on the stage of a Zeiss AxioObserver Z1 microscope. Here, the flow cell substrate was composed of
either 4 h IL-1b stimulated HUVEC monolayers, or recombinant L-, E- or P-selectin IgG at physiological levels (Mondal et al.,
2015). Control or glycoside treated HL-60s at 23106 cells/mL resuspended in HEPES buffer contained 1.5 mM Ca2+ and 0.1%
HSA were perfused over these substrates at a wall shear stress of 1 dyn/cm2. Cell rolling density, firm adhesion density and cell rolling
Cell Chemical Biology 25, 1–14.e1–e5, December 20, 2018 e3

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cyte Adhesion, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.012
velocity was measured as described previously. Ten mg/mL function blocking mAbs were applied in some cases to block the function
of either the immobilized selectins on the flow cell substrate or the selectin-ligand PSGL-1 on the leukocytes.
Western Blot Analysis
HL-60 cells were cultured with 80 mM SNAP, ONAP, SH or 0.2% DMSO (vehicle). These cells were washed and resuspended in 90 mL
Laemmli sample buffer (Bio-rad) containing b-mercaptoethanol. After denature by boiling at 95ꢁC for 5 min, debris was pelleted by
centrifugation. 20 mL supernatant was resolved using 4-20% gradient SDS-PAGE. Next, the proteins were transfer onto 0.2 mm nitro-
cellulose membrane, and probed with either anti-human PSGL-1 mAb TB5 (GeneTex, Irvine, CA) or anti-human CD43 mAb L60 (BD
Biosciences). A secondary horse-radish peroxidase (HRP) coupled anti-mouse Ab was incubated with the membrane prior to chem-
iluminescence development using ECL substrate (Thermo-Pierce).
Galactosyltransferase Assay
107 HL-60 cells with different treatments (80mM SNAP, ONAP, SH, or 0.2% DMSO) were lysed using either RIPA buffer or sonication
(3 cycles of 10s on and 10s off, 40% amplitude), both in the presence of HaltTM protease inhibitor (Thermo). The cell debris was pel-
leted by centrifugation at 18,000 g for 15 min, and the supernatant was collected. In some runs, a 25 mL reaction was prepared with
60 mg lysate-supernatant and 10,000 dpm UDP-[C14]Gal (258.00 mCi/mmol uridine di-phosphate-galactose, PerkinElmer, Boston,
MA) in reaction buffer (100 mM HEPES, 7 mM ATP, 20 mM manganese acetate), either with or without the substrate 0.5 mM Gal-
NAc-O-Bn (de-acetylated). Following overnight reaction, 1 mL reaction mixture spots were placed on thin layer chromatography/
TLC plates (Selecto Scientific, Suwanee, GA). Radioactive product was resolved from unreacted C-14 nucleotide-sugar using
CHCl3:CH3OH:H2O (5:4:1) solvent, and TLC image was recorded using a Storm 860 phosphorimager (GE Healthcare). In other
runs, 60 mg of vehicle-treated lysate was mixed with 500 mM ONAP/SNAP substrate and 1 mM UDP-Gal in reaction buffer in the
same reaction buffer (25 mL volume). Following overnight reaction, proteins were precipitated by addition of 1 mL 70% acetonitrile
and vortexing. Supernatant collected following centrifugation (14,000 g x 5 min) was evaporated under high vacuum, resuspended in
50 mL 50% MeOH and injected into LC-MS (Thermo Scientificꢀ Q Exactiveꢀ Hybrid Quadrupole-Orbitrap Mass Spectrometer) using
C18 separation.
ppGalNAcT Assay
The enzymatic reaction was performed in 20 mL reaction buffer (100 mM HEPES, 7 mM ATP, 20 mM manganese acetate, HaltTM pro-
tease inhibitor) containing 60 mg lysate-supernatant, 50,000 dpm UDP-[C14]GalNAc (55 mCi/mmol, ARC Inc., St. Louis, MO) and
50 mg PSGL-1 N-terminal peptide substrate (AQTTPRAATEAQTTRLRATESHHHHHH, GenScript, Piscataway, NJ). Following over-
night reaction at r.t., reaction volume was increased 10-fold using 500 mM NaCl in phosphate-buffered saline (PBS) and mixed
with 10 mL MagneHisTM Ni-Particles (Promega, Madison, WI) for 45 min at r.t. Followed 5-wash cycles in washing buffer (100 mM
NaCl, 10 mM imidazole in PBS) using magnetic separation at each step, the entire mixture was brought up to 200 ml in the washing
buffer and mixed with 4mL scintillation cocktail. The UDP-[C14]GalNAc radioactivity associated with the peptide immobilized with the
beads was quantified using a standard scintillation counter.
Animal Studies
Mouse bone marrow cells (mBMCs) were isolated from the tibia and femur of 10-12 week old C57BL/6 donor mice. These mBMCs
were cultured ex vivo, with 100 mM ONAP, SNAP or vehicle (0.25% DMSO) in IMDM media containing 10 ng/mL G-CSF, 1 ng/mL IL-3
and 10% FBS. Following culture for 40 h, the cells were labeled using the green fluorescent dye CMFDA (1 mM) (Setareh Biotech,
Eugene, OR), orange dye CMTMR (5 mM) or both dyes together by incubating the dye with the cells for 15 min at 37ꢁC. The exact
dye used for individual cell types was varied to ensure that dye labeling did not influence the study findings. Following this, the three
labeled cell types were mixed in approximately equal amounts and infused i.v. into recipient mouse injected with 4% thioglycollate
i.p. to induce peritonitis (Marathe et al., 2010). Samples from the peritoneal lavage and bone marrow were collected 20h thereafter.
Granulocytes in the collected samples were identified based on their characteristic forward-side scatter profile, and labeling with
APC conjugated anti-mouse Ly-6G clone 1A8 (BioLegend, San Diego, CA). Granulocytes tagged with green and/or red fluorescent
dyes were enumerated using a BD LSR-II flow cytometer. Final data are normalized with respect to the cell ratio in the injected
mixture.
MALDI TOF MS/MS Glycomics Profiling
N-linked, O-linked and GSL derived glycans were extracted from vehicle control and SNAP treated HL60s as described previously
(Mondal et al., 2015). All glycans were permethylated prior to MALDI-TOF MS and MALDI-TOF-TOF MS/MS analysis. Released gly-
cans from GSLs were deuteroreduced prior to permethylation. Data were annotated using the glycobioinformatics tool, GlycoWork-
Bench (Ceroni et al., 2008). The proposed assignments for the selected peaks were based on 12C isotopic composition together with
knowledge of the biosynthetic pathways. The proposed structures were confirmed using MS/MS.
LC-MS/MS Glycan Analysis
HL-60s (0.33106/mL) were cultured for 48 h in advance-DMEM without phenol red (ADMEM). In some cases, 60-100mM SNAP,
ONAP, or peracetylated benzyl-a-GalNAc was added alone to the culture medium, while in other cases two compounds were mixed
e4 Cell Chemical Biology 25, 1–14.e1–e5, December 20, 2018

Please cite this article in press as: Wang et al., Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leuko-
cyte Adhesion, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.012
and added together. 0.2% DMSO served as vehicle. Cell culture medium was purified using Sep-Pak C18 columns (Waters, Milford,
MA), with glycosides being released by elution with 50-75% MeOH. The released product was permethylated prior to MS analysis
(Stolfa et al., 2016). In some cases, to examine glycans formed within cells, cell pellets were collected in the above runs, and lysed
using 75% MeOH followed by sonication. Following removal of cell debris by centrifugation at 18,000 g for 15 min, the supernatant
was dried using a CentriVap centrifugal evaporator. Three mL ADMEM was added to resuspend the glycans. Separation using C18
Sep-Pak and permethylation was performed as above.
For LC-MS analysis, the above samples were filtered through 0.2 mm PES (Polyethersulfone) syringe filters, centrifuged at 16,000 g
at 4ꢁC for 15 min to remove any debris, and the supernatant was analyzed. Two instruments were used: i. An Orbitrap-XL MS
(Thermo) equipped with a nano-LC column (PepMap C18 2 mm; 75 mm3150 mm, Thermo) and ESI (electrospray ionization) source;
ii. A 6530 Accurate Mass Dual Agilent Jet Stream ESI-Q-ToF (quadrupole-time-of-flight) with a Phenomenex Gemini C18 column
(5 mm, 4.6 x 50 mm). In the Orbitrap-XL runs, the mobile phases were, A: water and B: acetonitrile (CH3CN), both containing 0.1%
(v/v) formic acid. Data were acquired over 80 min at a flow rate of 300 nL/min using the following linear gradient: (i) increase from
0% to 20% B (0-5 min); (ii) 20% to 40% B (5-45 min); (iii) 40% to 70% B (45-65 min); (iv) 70% to 100% B (65-75 min); and (v) isocratic
elution at 100% B (75-80 min). MS1 data were acquired using the Orbitrap detector (60,000 resolution), and MS/MS in CID mode (ion
trap with 30% collision energy). For ESI-Q-ToF separation, the mobile phases were, A: 10% CH3CN and B: 90% CH3CN, both with
0.1% formic acid and 0.1% ammonium formate. Flow rate was set to 0.5 mL/min. MS analysis started at isocratic 100% A (0-5 min),
linear ramp to 100% B (5-40 min) and finally isocratic 100% B (40-50 min). MS data were collected over 50-1700 m/z in positive ESI
mode at high resolution. Targeted LC-MS/MS analyses were carried out by varying collision energies from 0-75 eV. Data were an-
notated using the glycoinformatics tools DrawGlycan-SNFG (Cheng et al., 2017).
2-Napthalenemethanol Detection and Analysis
In a variation of the Folch method (Folch et al., 1957), 1.8 mL of HL-60 culture media was transferred to a glass vial and mixed with
methanol and chloroform in a ratio 3:4:8 (media:methanol:chloroform). The mixture was vortexed for 1 min and allowed to settle for
1 min, with this vortex-settling cycle being repeated thrice. In the final step, the sample was centrifuged at 500 g for 10 min, and
4.4 mL of the chloroform layer was collected and vacuum-dried.
In one experiment, this product was resuspended in 500 mL MeOH and resolved using an Agilent 1100 HPLC equipped with a
reversed-phase ZORBAX Eclipse XDB-C18 column (5 mm, 4.6 mm 3 150 mm). Mobile phase A: water and B: methanol, both con-
taining 0.1% (v/v) formic acid. Flow rate was 0.5 mL/min and detector wavelength was 224 nm. Data were acquired over 40 min using
the following gradient: (i) isocratic at 40% B (0-3 min), (ii) increase from 40% to 70% B (3-13 min); (iii) isocratic 70% B (13-23 min); (iv)
increases from 70% to 85% B (23-25 min); (v) isocratic 85% B (25-35 min), (vi) decrease from 85% to 40% B (35-37 min), and finally
(vii) isocratic 40% B (37-40 min). Pure 2-naphthalenemethanol and 2-napthalenemethanethiol (Sigma) added to culture media served
as standards for this run. Calibration curves were linear over the 5–150 mM range for HONAP (r2 = 0.9997) and HSNAP (r2 = 0.9985).
The limits of detection (LoD) and quantification (LoQ) were 3.8 mM and 11.4 mM for HONAP and 9.1 mM and 27.6 mM for HONAP.
Quantifications were carried out in triplicates.
In another experiment, dansyl derivatization was performed by resuspending the above vacuum-dried sample with 100 mL of an
amine mixture (0.4% v/v triethylamine or TEA and 6 mg/mL 4-dimethylaminopyridine or DMAP). After vortexing for 1 min, 100 mL of
dansyl chloride (6 mg/mL) were added and the reaction was stirring at 600 rpm for 1 h at r.t. in dark. The solvent and triethylamine
were then eliminated under high vacuum and the residue was resuspended in 750 mL MeOH. This sample was injected into an
LC-ESI-Q-ToF system (Agilent) using LC and MS parameters listed in the previous section. 2- Napthalenemethanol was dansyl
derivatized to serve as MS standard.
QUANTIFICATION AND STATISTICAL ANALYSIS
All data are presented as Mean S.D (nR3). Individual data points are plotted in each panel to give a measure of n in each group/
condition. Two-tailed Student’s T-test was used for dual-comparisons. Analysis of variance (ANOVA) followed by the Student-
Newman-Keuls post-test was used for multiple comparisons. P<0.05 was considered to be statistically significant.
DATA AND SOFTWARE AVAILABILITY
Two software are used to annotate MS spectra: GlycoWorkBench and DrawGlycan-SNFG. Both resources are provided open-
source at github (see Key Resources Table).
Cell Chemical Biology 25, 1–14.e1–e5, December 20, 2018 e5