Discovery of rare sulfated: N -unsubstituted glucosamine based heparan sulfate analogs selectively activating chemokines

Article abstract of DOI:10.1039/d0sc05862a

Achieving selective inhibition of chemokines with structurally well-defined heparan sulfate (HS) oligosaccharides can provide important insights into cancer cell migration and metastasis. However, HS is highly heterogeneous in chemical composition, which limits its therapeutic use. Here, we report the rational design and synthesis of N-unsubstituted (NU) and N-acetylated (NA) heparan sulfate tetrasaccharides that selectively inhibit structurally homologous chemokines. HS analogs were produced by divergent synthesis, where fully protected HS tetrasaccharide precursor was subjected to selective deprotection and regioselectively O-sulfated, and O-phosphorylated to obtain 13 novel HS tetrasaccharides. HS microarray and SPR analysis with a wide range of chemokines revealed the structural significance of sulfation patterns and NU domain in chemokine activities for the first time. Particularly, HT-3,6S-NH revealed selective recognition by CCL2 chemokine. Further systematic interrogation of the role of HT-3,6S-NH in cancer demonstrated an effective blockade of CCL2 and its receptor CCR2 interactions, thereby impairing cancer cell proliferation, migration and invasion, a step towards designing novel drug molecules.

Full text of DOI:10.1039/d0sc05862a

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Discovery of rare sulfated N-unsubstituted
glucosamine based heparan sulfate analogs
Cite this: Chem. Sci., 2021, 12, 3674
selectively activating chemokines†
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Prashant Jain,‡b Chethan D. Shanthamurthy,‡b Shani Leviatan Ben-Arye,a
c
b
a
*
*
Robert J. Woods, Raghavendra Kikkeri and Vered Padler-Karavani
Achieving selective inhibition of chemokines with structurally well-defined heparan sulfate (HS)
oligosaccharides can provide important insights into cancer cell migration and metastasis. However, HS
is highly heterogeneous in chemical composition, which limits its therapeutic use. Here, we report the
rational design and synthesis of N-unsubstituted (NU) and N-acetylated (NA) heparan sulfate
tetrasaccharides that selectively inhibit structurally homologous chemokines. HS analogs were produced
by divergent synthesis, where fully protected HS tetrasaccharide precursor was subjected to selective
deprotection and regioselectively O-sulfated, and O-phosphorylated to obtain 13 novel HS
tetrasaccharides. HS microarray and SPR analysis with a wide range of chemokines revealed the
structural significance of sulfation patterns and NU domain in chemokine activities for the first time.
Particularly, HT-3,6S-NH revealed selective recognition by CCL2 chemokine. Further systematic
interrogation of the role of HT-3,6S-NH in cancer demonstrated an effective blockade of CCL2 and its
receptor CCR2 interactions, thereby impairing cancer cell proliferation, migration and invasion, a step
towards designing novel drug molecules.
Received 24th October 2020
Accepted 15th January 2021
DOI: 10.1039/d0sc05862a
rsc.li/chemical-science
uronic acid composition, sulfation patterns and oligosaccha-
ride length.4
Introduction
This has prompted the synthesis of well-described, homo-
geneous GAG structures, and more specically HS oligosac-
charides, to regulate chemokine activity. For example,
Gallagher et al. reported that the CXCL4 chemokine requires HS
2-O-sulfated iduronic acid (IdoA) for tetramerization and
binding to its cell surface receptors.5 Elsewhere, Lindahl et al.
had shown that interleukin-8 (CXCL8 or IL-8) prefers the IdoA(2-
OSO3ꢀ)-GlcNSO3ꢀ(6OSO3ꢀ) repeating unit to activate neutro-
phil trafficking,6 while Gardiner et al. reported the elegant role
of 6-O-sulfation in switching the binding between CXCL12 and
IL-8.7 Hesieh-Wilson et al. demonstrated that the trisulfated
Chemokines are endogenous signaling peptides essential for
immuno-surveillance, homeostasis, inammation, infection
and tissue repair.1 Chemokines and their receptor activities
depend on how they bind and oligomerize in the presence of
glycosaminoglycans (GAGs).2 Humans express 47 chemokines
and 20 receptors, and most of the chemokines are highly basic
proteins. It has been therefore hypothesized elsewhere that
chemokine–GAG binding is non-specic. However, aer the
discovery of acidic CCL3 and CCL4 chemokines binding to
GAGs, it had become clearer that their interaction proceeds in
a sequence-dependent manner.3 Moreover, chemokines have
shown different interaction strengths with various GAGs,
including heparan sulfate (HS), chondroitin sulfate and der-
matan sulfate, illustrating that microheterogeneity in GAG
structures can modulate binding patterns, for example through
IdoA(2-OSO3ꢀ)-GlcNSO3ꢀ(6-OSO3ꢀ)-conjugated
polymer
strongly inhibited RANTES (CCL5)-CCR3-receptor-mediated cell
migration.8 In addition, Seeberger et al. showed that CCL21
strongly binds to a hexasaccharide containing the GlcNSO3ꢀ(6-
OSO3ꢀ)-IdoA(2-OSO3ꢀ) repeating unit as compared to CXCL12,
while CCL19 does not bind to it at all.9 Boons et al. discovered
that CCL2 binds to highly sulfated HS compounds and exhibits
no preference for the uronic acid component, while both CCL2
and CCL13 displayed promiscuous binding with most of the HS
glycans.10 These data suggest that well-dened HS oligosac-
charides can provide structural details to target chemokine–
GAG interaction to modulate its activities. However, HS is highly
heterogeneous in its structure and the majority of the HS
libraries that have been used for chemokine studies are
aDepartment of Cell Research and Immunology, The Shmunis School of Biomedicine
and Cancer Research, The George S. Wise Faculty of Life Sciences, Tel Aviv
University, Tel Aviv, 69978, Israel. E-mail: vkaravani@tauex.tau.ac.il
bDepartment of Chemistry, Indian Institute of Science Education and Research, Pune-
411008, India. E-mail: rkikkeri@iiserpune.ac.in
cComplex Carbohydrate Research Center, University of Georgia, Athens 30606, GA,
USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0sc05862a
‡ Equal contribution.
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Fig. 1 Structures of heparan sulfate tetrasaccharide analogs (1–13).
composed of N-sulfated and N-acetylated (NA) glucosamine building blocks 21 and 18. Notably, the use of 4-O-chloroacetate
domains.8–10 Native HS also expresses an N-unsubstituted (NU) (ClA) at non reducing GlcN residue as a temporary protecting
domains, but this region has not been fully investigated in group in 17 was found to be vital for chain elongation than
existing studies.11 To address this gap, and to decipher the other previously reported 4-O protecting groups such as Lev13f
/
sulfation code of chemokine heparin binding, here we report Fmoc13m/TCA.13r Disaccharides (21 and 18) and monosaccharide
the divergent synthesis of a limited number of NU- and NA precursors were synthesized from glucosamine and iduronic
domains HS tetrasaccharides (Fig. 1).
acid building blocks 14, 16, 19 & 20, as previously described.13h,14
High-throughput screening of these synthetic HS ligands Next, we adopted [2 + 2] glycosylation approach with 21 (glycosyl
with a wide range of chemokines revealed selective chemokine donor) and 18 (glycosyl acceptor) to obtain 1,6 anhydrous tet-
binder, which can be used to block chemokine activity to target rasaccharide 22 in excellent yield. Acetolysis of the reducing end
cancer biology. However thus far, only few heparin binding IdoA residue of 22 with the aid of acetic anhydride and copper
proteins have been reported to bind NU-domain-containing HS triuoromethanesulfonate as catalyst followed by phenyl tri-
ligands,12 and here we provide the rst such example where methylsulde and ZnI2 treatment afforded 24 as a thiophenol
chemokines activity was interrogated systematically with NU glycosyl donor. Linker glycosylation of 24, followed by sequen-
domain ligands.
tial deacetylation and TEMPO mediated oxidation of 25, yielded
27 (72% for three steps). Finally, C-2 azide of glucosamine
moieties in 27 was converted into acetate in the presence of Zn/
AcOH/Ac2O to develop tetrasaccharide 28, which was further
used to synthesize HS analogs with N-acetate backbone (Scheme
1).
A divergent synthetic approach was followed to develop
a combinatorial library of HS oligosaccharides. For instance,
silyl protecting group TBDPS was deprotected selectively using
70% HF$py for 6-O-sulfate derivatives precursors (29 & 36).
Similarly, NAP deprotection with the help of DDQ yielded 3-O-
sulfated precursors (31 & 34). The lactone ring was rst opened
for the derivatives carrying 2-O-sulfated IdoA, followed by the
benzyl esterication to yield 39 & 42 in moderate yield (Scheme
Results and discussion
N-unsubstituted and N-acetylated HS analog library with
regioselective sulfate modications at 2-O (IdoA), 3-O (GlcN),
and 6-O (GlcN) were obtained by single tetrasaccharide 27 and
its N-acetate counterpart 28 (Scheme 1) using the divergent
synthetic approach. Synthesis of 27 required a carefully
designed technique that allowed us to do selective site modi-
cations along the tetrasaccharide backbone in a controlled
manner. Efforts led by various research groups revolutionized
heparin sulfate synthesis in the past decade.13 Using a similar
strategy as Hung13h et al. with slight modications in the pro-
tecting group, synthesis of 27 was carried out by disaccharide
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Scheme 1 (a) (i) PTSA, CH2Cl2/MeOH (1 : 2), rt, 6 h; (ii) TBDPSCl, imidazole, DMAP, CH2Cl2, 0 ꢁC, 12 h; (iii) (ClAc)2O, CH2Cl2/py (4 : 1), 0 ꢁC,
20 min. (b) (i) BH3$THF, TMSOTf, CH2Cl2, 0 ꢁC, 6 h; (ii) TBDPSCl, imidazole, DMAP, CH2Cl2, 0 ꢁC, 12 h. (c) NIS, TMSOTf, 4 A˚ MS, ꢀ78 ꢁC to ꢀ20 ꢁC,
CH2Cl2, 30 min. (d) Thiourea, MeOH/py (1 : 1), 80 ꢁC, 1 h. (e) NIS, TMSOTf, 4 A˚ MS -10 ꢁC, CH2Cl2, 30 min. (f) Ac2O, Cu(OTf)2, rt, 12 h. (g) TMSSPh,
ZnI2, CH2Cl2, rt, 2 h. (h) Benzyl (3-hydroxypropyl)carbamate, NIS, TfOH, 4 A˚ MS, rt, CH2Cl2, 30 min. (i) NaOMe, CH2Cl2/MeOH (1 : 1), rt, 12 h. (j)
TEMPO, CH2Cl2/MeOH (1 : 1), rt, 12 h. (k) Zn, THF/AcOH/Ac2O (3 : 2:2), rt, 12 h.
2). Subsequent, selective deprotection was carried out in
In this analysis, CCL13 (an inammatory chemokine),
a similar manner for oligosaccharides having multiple O-2,6 or CXCL12 and CCL21 (homeostatic chemokine) shared several of
O-2,3 sulfate modications (45, 47 & 50). SO3$NEt3 was used for the conserved binding patterns, which suggest that these che-
introducing sulfate group in the backbone (30, 32, 35, 37, 40, 43, mokines share several homologous binding pockets. For
46, 48 & 51), whereas for the phosphate derivative (38 & 52) instance, group A chemokines bind to non-sulfated HT-0S-NH
diphenylphsphoryl chloride (DPPC) was utilized. Finally, global and phosphorylated ligands (HT-6,2P and HT-6P) (Fig. 2a),
deprotection of all the sulfated derivatives, including non- suggesting that the HS-based structure–activity relationship of
sulfated analogs (29 & 36) yielded desired HS tetrasaccharide these chemokines do not solely depend on the sulfate group.
1–13 with the amine linker at the reducing for the generation of Moreover, group A chemokines showed strong binding with di-
HS microarray. Final HS oligosaccharides and intermediates sulfated analogs such as HT-2S-NH and HT-6S-NAc. However,
were characterized by 1H, 13C, DEPT and 31P NMR. Additionally, its respective NU and NA counterpart (HT-6S-NH and HT-2S-
molecular weights for all the compounds were conrmed by NAc) displayed weak binding (Fig. 2a), illustrating that NU and
high-resolution mass spectroscopy.
NA domains display switchable binding patterns via sulfation
To unravel HS–chemokines binding patterns, heparin codes. It is noted that highly sulfated HS ligands displayed
microarray was fabricated and examined with various bio- moderate to strong binding regardless of sulfation pattern or
tinylated chemokines, at three different concentrations, fol- NU/NA domains. These trends clearly demonstrate that group A
lowed by detection with Cy3-tagged streptavidin. We chemokines are sensitive to di-sulfation codes, while the highly
interrogated the binding of HS analogs on three homeostatic sulfated HS ligands may improve the binding strength, but with
chemokines (CCL28, CXCL12 and CCL21) and six inammatory poor selectivity.
chemokines [CXCL8 (IL-8), CXCL10 (IP-10), CCL2 (MCP-1) CCL7
In group B, CCL28 chemokine displayed weak binding with
(MCP-3), CCL13 (MCP-4) and CCL5 (RANTES)]. To rationalize non-sulfated analogs (ranked 36% for HT-0S-NH and 22% HT-
the binding patterns of oligosaccharides, each HS–chemokines 0S-NAc) and moderate to strong binding with sulfated ligands.
interaction was ranked according to percentage of maximal Unlike, group A chemokines, CCL28 displayed poor binding
binding. Based on ranking and chemokine binding patterns, with 2-O-sulfated NU ligand (ranked 43% for HT-2S-NH).
they were segregated into four groups (A, B, C and D) (Fig. 2a). Whereas, HT-6S-NAc (ranked 79%) and HT-3S-NH (ranked 62%)
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Scheme 2 (a) (i) LiOH$H2O, THF/H2O (1 : 1), rt, 2 h; (ii) BnBr, TBAI, NaHCO3, DMF, 60 ꢁC, 2 h. (b) 70% HF$py, py, 0 ꢁC, 12 h. (c) DDQ, CH2Cl2/H2O
(18 : 1), rt, 1 h. (d) SO3$NEt3, DMF, 60 ꢁC, 72 h. (e) DPPC, DMAP, NEt3, CH2Cl2/py (1 : 1), 0 ꢁC, 12 h.
di-sulfated ligands, H-2,6S-NH (ranked 79%), HT-2,6-NAc ligands and weak binding with di-sulfated ligands, nonsulfated
(ranked 66%), HT-3,6S-NH (ranked 54%) tetra-sulfated ligands or phosphorylated ligands. Among high-sulfate ligands, group D
and HT-2,6P (ranked 78%) phosphate ligand displayed chemokines displayed high selectivity to NU domain over NA
moderate to strong binding (Fig. 2a). These results suggest that domain. Notably, CXCL8 and CCL2 displayed strong binding
group A and group B chemokine binding patterns may require preference to HT-3,6S-NH (ranked 94% for CXCL8 and 95% for
a more complex HS library to establish selectivity.
CCL2) ligand, whereas, CCL5 showed strong binding to HT-2,6S-
In group C, CCL7 and CXCL10 displayed a sulfation pattern- NH (ranked 95%) ligand (Fig. 2a), suggesting that NU domain is
based binding. Unlike group A and group B chemokines, CCL7 highly signicant in modulating these chemokines activities,
and CXCL10 displayed weak binding with non-sulfated and particularly of group D chemokines. It had previously been
phosphate HS ligands (ranked between 2–56%). Among six di- shown that the interaction of heparin tetrasaccharides with
sulfated ligands, only HT-3S-NH (ranked 78% for CCL7 and CCL5 is modulated by sulfation pattern and pH, however these
65% for CXCL10) and HT-6S-NAc (ranked 73% for CCL7) dis- studies also emphasized the dynamic and oen non-specic
played strong binding as compared to the other analogs. Simi- nature of the ionic GAG-protein contacts.15 Nevertheless, to
larly, among four tetra-sulfated HS ligand, HT-2,3S-NH (ranked provide further insights into the interactions between CCL5 and
35% for CCL7 and 27% for CXCL10) displayed weak binding, HT compounds of varying sulfation patterns, we used a co-crystal
whereas HT-2,6S-NH (ranked 91% for CCL7 and 89% for structure of CCL5 co-complexed with a heparin trisaccharide
CXCL10), HT-2,6S-NAc (ranked 86% for CCL7 and 64% for (PDB ID: 5DNF, Chain I), in which sulfate groups were added or
CXCL10) and HT-3,6S-NH (ranked 89% for CCL7 and 96% for deleted from the ligand (Fig. 2b). This analysis revealed that
CXCL10) ligands displayed strong binding (Fig. 2a). These sulfation at the 6 position in GlcNH is preferred over sulfation at
results illustrate that group C chemokines have some selectivity the 3 position, because the 6S group can interact with both R59
to di-sulfated ligands. Larger HS-disulfated library could further and K55, whereas the 3S only interacts with R59. For this reason,
allow to ne-tune their binding patterns characteristics.
both GlcNH[6S]-IdoA[2S]-GlcNH[6S] and GlcNH[3S,6S]-IdoA-
Finally, all members of D group chemokines (CXCL8, CCL5 GlcNH[3S,6S] (related to HT-2,6S-NH and HT-3,6S-NH, ranked
and CCL2) displayed exclusive strong binding to high-sulfated 95% and 82% by glycan microarrays, respectively) are stronger
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Fig. 2 Chemokine glycan microarray binding assay. (a) Arrays were fabricated on epoxide-activated slides as described.16 Binding was tested at 3
serial dilutions, then detected with the relevant biotinylated secondary antibody (1 mg mlꢀ1) followed by Cy3-streptavidin (1.5 mg mlꢀ1) (Table S1†).
Arrays were scanned, relative fluorescent units (RFU) obtained, and maximum RFU determined and set as 100% binding. Then rank binding (per
printed glycan per concentration, per each chemokine dilution, per printed block) was determined. Since each glycans was printed at 2
concentration, 100% binding was set separately for each concentration. Then, binding to all the other glycans at the same concentration was
ranked in comparison to the maximal binding, and the average rank binding and SEM for each glycan across the two glycan concentrations and
three examined dilutions of each chemokine was calculated (n ¼ 6; 2 glycan concentrations, across 3 chemokine dilutions). This analysis allowed
to compare the glycan binding profiles of the different chemokines and dissect their binding preferences. The mean rank is shown as a heat map
of all the examined binding assays together (red highest, blue lowest and white 50th percentile of ranking). (b) Sulfate groups were added or
deleted from the ligand in the crystal structure of the co-complex of a heparin trisaccharide with CCL5 (PDB ID: 5DNF, Chain I) using Chimera
(UCSF Chimera – a visualization system for exploratory research and analysis).17 No other changes to the orientation of the ligand or protein were
made. Based on glycan microarray screening, binding to CCL5 was high for GAG fragments HT-2,6-NH and HT-3,6S-NH (red), but low to HT-
2,3S-NH (blue), consistent with expectations based on the co-complex crystal structure models.
binders than GlcNH[3S]-IdoA[2S]-GlcNH[3S] (related to HT-2,3S- NH and HT-2,6S-NH ligands, respectively. Thus, for the rst
NH, ranked 4%) (Fig. 2a).
time, we were able to identify key NU domain sulfation pattern
To quantitatively evaluate the binding patterns between HS that can modulate chemokines activity.
ligands and chemokines, SPR experiment was performed with
Given the high affinity binding of HT-3,6S-NH to CCL2 che-
H-3,6S-NH and chemokines (CXCL10, CXCL8, CCL5 and CCL2), mokines, and the link between CCL2 and cancer metastasis,
which showed strong selective and sensitive binding in micro- investigating inhibitor effect on CCL2 cancer cell signaling is
array experiments. The equilibrium binding constants (KD) considered a novel approach to demonstrate the therapeutic
measured from steady state ts are listed in Table S2.† HT-3,6S- potential of HS mimetics.18 To this end, we rst studied cancer
NH displayed strong binding with inammatory chemokine cells proliferation in the presence of HT-3,6S-NH (H-3) ligand and
CCL2 (1.89 mM) (Fig. 3a–d). This strong binding is attributed to CCL2. Native heparin was used as a positive control. Cell prolif-
the fast association (Kon) as compared to chemokine–CCL2 eration was analyzed by WST assay using MCF-7 cell line, as they
interaction. In contrast, HT-2,6SNH displayed strong binding to express high level of the CCL2 specic chemokine receptor
CCL5 (2.34 mM) (Fig. 3a–d). Interestingly, CXCL8, CCL7 and (CCR2).19 It was observed that high concentration of HT-3,6S-NH
CXCL10 showed weak binding constants (10–15 mM) for both ligand moderately inhibited cell proliferation (Fig. 3e). To
NU domain ligands (Fig. S2†). Furthermore, SPR analysis of understand the mechanism of inhibition, we performed cell-
CCL5 and CCL2 displayed 3-fold stronger binding with H-3,6S- cycle analysis in the presence and absence of HS ligand and
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Fig. 3 SPR analysis of chemokines binding profile on sensor chip having HT ligands: (a & b) SPR binding analysis of the interaction between HT-
2,6S-NH with CCL5 and CCL2 respectively; (c & d) SPR binding analysis of the interaction between HT-3,6S-NH with CCL5 and CCL2,
respectively. Concentrations of chemokines were 0.05–2 mM. A global fit according to a 1 : 1 binding model was applied (black curves); (e) MCF-7
cell proliferation was quantified by WST assay after 72 h treatment with HT-3,6S-NH (H-3) at different concentration with CCL2 chemokine. The
bar graphs indicated percentage of cell growth. L corresponds to 10 mg mlꢀ1 concentration; H corresponds to 50 mg mlꢀ1 concentration of
ligands ((H-3) and Heparin (Hep)). CCL2 chemokines (50 ng); (f) cell migration assay: area repopulated in 8 h with CCL2 chemokine is considered
as 100% wound heal and data expressed as mean ꢂ SD (n ¼ 3; *P < 0.05, *P < 0.01); (g) Boyden chamber assay was performed in presence of HT-
3,6S-NH and Hep (50 mg mlꢀ1) with or without CCL2 (50 ng); (h) bright field images of Boyden chamber assay; (i) MAPK pathway analysis: MCF-7
cells were treated with CCL2 (50 ng) with or without HT-3,6S-NH (H-3) ligands (50 mg mlꢀ1) and cell lysate was prepared at 30 min time points
and P-p44/42 and total p44/42 was imaged.
CCL2 chemokine, then quantied the DNA content of each cell
state by ow-cytometry. The cell cycle analysis clearly revealed
Conclusions
that addition of CCL2 induced S and G2/M phase cell-cycles,
while high concentration of HT-3,6S-NH and heparin reduced
G2/M state from 15% to 10%, indicating that HT-3,6S-NH
moderately to poorly activate the cell cycle. Further studies with
HT-3,6S-NH multivalent probes are ideal for modulating cancer
cell proliferation.
We next examined cell migration by wound healing assay
(Fig. 3f) and by Boyden-chamber assay (Fig. 3g and h). Addition of
HT-3,6S-NH ligand reduced chemokine activity, where a 24%
reduction in cell migration rate and 41% reduction in the wound
healing was observed. In addition, a substantial reduction in cell
migration was observed in the Boyden chamber assay. Finally, the
mechanism of invasiveness was examined further by analyzing the
level of phosphorylation of MAP kinase pathway. Western blot
analysis of p42/44 showed that MCF-7 cells treated with HT-3,6S-
NH/CCL2 expressed low level of MAPK compared to CCL2 treated
cells (Fig. 3i). Overall, these results suggest that HT-3,6S-NH is
a potential ligand that can modulate CCL2 chemokine activities.
Here, we describe the divergent synthesis of 13 new HS ligands,
displaying different sulfate/phosphate patterns with NU/NA
glucosamine residues. The binding interactions between HS
ligands and chemokines on nano-printed microarray platform
displayed several cryptic binding pockets for sulfation patterns
with NU domain, which was not identied with previous HS
synthetic ligands. Among them, HT-3,6S-NH ligand displayed
a marked selectivity and sensitivity to CCL2 chemokine. The
biological relevance of such structural binding studies was
illustrated by incubating HT-3,6S-NH ligand with cancer cells
showing the HS ligand inhibited cancer cells proliferation,
migration and invasion. Thus, NU domain is important to
regulate specic chemokine biological activities, thereby
demonstrating potential novel therapeutic applications of HS
ligands. To the best of our knowledge, we have identied CCL2
and CCL5 chemokines as only the fourth and h proteins
currently known to recognize NU-domain HS ligands with
different sulfation patterns.
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Conflicts of interest
There are no conicts of interest to declare.
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