Trisaccharide Sulfate and Its Sulfonamide as an Effective Substrate and Inhibitor of Human Endo- O-sulfatase-1

Article abstract of DOI:10.1021/jacs.0c00005

Human endo-O-sulfatases (Sulf-1 and Sulf-2) are extracellular heparan sulfate proteoglycan (HSPG)-specific 6-O-endosulfatases, which regulate a multitude of cell-signaling events through heparan sulfate (HS)-protein interactions and are associated with th

Full text of DOI:10.1021/jacs.0c00005

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Article
A Trisaccharide Sulfate and its Sulfonamide as Effective
Substrate and Inhibitor of Human Endo-O-sulfatase-1
Li-Ting Chiu, Narayana Murthy Sabbavarapu, Wei-Chen Lin, Chiao-Yuan Fan, Chih-
Chung Wu, Ting-Jen Rachel Cheng, Chi-Huey Wong, and Shang-Cheng Hung
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c00005 • Publication Date (Web): 21 Feb 2020
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A Trisaccharide Sulfate and its Sulfonamide as Effective Substrate and Inhibitor of
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Human Endo-O-sulfatase-1
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Li-Ting Chiu,†,‡ Narayana Murthy Sabbavarapu,† Wei-Chen Lin,† Chiao-Yuan Fan,† Chih-
Chung Wu,† Ting-Jen Rachel Cheng,† Chi-Huey Wong,*,†,§ and Shang-Cheng Hung*,†,‖
†Genomics Research Center, Academia Sinica, 128, Section 2, Academia Road, Taipei 115,
Taiwan
‡Institute of Biochemistry and Molecular Biology, National Yang Ming University, 155,
Section 2, Linong Street, Taipei 115, Taiwan
§Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road
BCC 338, La Jolla, CA 92037, USA
‖Department of Applied Science, National Taitung University, 369, Section 2, University Road,
Taitung 95092, Taiwan
Keywords: heparan sulfate, heparin, human endo-O-sulfatase-1 (Sulf-1), substrate specificity,
inhibitor
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ABSTRACT
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Human endo-O-sulfatases (Sulf-1 and Sulf-2) are extracellular heparan sulfate
proteoglycan (HSPG)-specific 6-O-endosulfatases, which regulate a multitude of cell-signaling
events through heparan sulfate (HS)-protein interactions and are associated with the onset of
osteoarthritis. These endo-O-sulfatases are transported onto the cell surface to liberate the 6-
sulfate groups from the internal D-glucosamine residues in the highly sulfated subdomains of
HSPGs. In this study, a variety of HS oligosaccharides with different chain lengths and N- and
O-sulfation patterns via chemical synthesis was systematically studied about the substrate
specificity of human Sulf-1 employing the fluorogenic substrate 4-methylumbelliferyl sulfate
(4-MUS) in a competition assay. The trisaccharide sulfate IdoA2S-GlcNS6S-IdoA2S was
found to be the minimal-size substrate for Sulf-1; and substitution of the sulfate group at the 6-
O position of the D-glucosamine unit with the sulfonamide motif effectively inhibited the Sulf-
1 activity with IC50 = 0.53 μM, Ki = 0.36 μM, and KD = 12 nM.
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INTRODUCTION
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Human sulfatases are a highly conserved family of enzymes that catalyze the hydrolysis
of sulfate esters in a wide range of substrates including glycosaminoglycans (GAGs),
glycolipids, and steroids.1 Based on their subcellular localization and their structural and
functional features, numerous sulfatases have been identified and classified as exo- and endo-
O-sulfatases in both eukaryotic and prokaryotic species. Sulfatases target individual substrates
with exquisite specificity in vivo to effect hydrolytic desulfation. For example, exo-O-
sulfatases located in lysosomes hydrolyze sulfate esters at nonreducing-end residues in the
degradative pathways of GAGs (Figure 1). Mutagenesis data provided key mechanistic aspect
of sulfatases in the catabolism of sulfate-bearing metabolites, featuring the special amino acid
residue C-formylglycine in the active site of the enzyme.2,3 C-formylglycine is the product
of a unique post-translational modification of a conserved cysteine residue. Its impairment
results in the global inactivation of human lysosomal sulfatases, contributing to multiple
sulfatase deficiency and lysosomal storage diseases such as mucopolysaccharidoses and
metachromatic leukodystrophy.4 In contrast to lysosomal sulfatases, the extracellular endo-6-
O-sulfatases (Sulf-1 and Sulf-2) are responsible for the endolytic cleavage of the 6-sulfate
groups in the trisulfated (N-, 2-O-, and 6-O-) disaccharides during post-biosynthetic
modification of heparan sulfate proteoglycans (HSPGs), thereby regulating a cascade of events
in embryonic development and signal transduction pathways.5,6
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Figure 1. Preferred site of 6-sulfate cleavage by exo-O-sulfatase and endo-O-sulfatase in the
heparan sulfate chain.
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Heparan sulfate (HS) is an important type of information rich GAG polymer
interspersed on the cell surface and in the extracellular matrix as an integral part of
proteoglycans.7 The HS polysaccharide chains are composed of alternating 14-linked -D-
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glucosamine (GlcN) and -D-glucuronic acid (GlcA) or -L-iduronic acid (IdoA) residues. The
biosynthesis of HS, as well as its structural analogue heparin, is a non-template driven process
facilitated by several enzyme isoforms of varying specificities that enable the assembly and
modification of the polysaccharide chains while attached to a protein core. The action of the
biosynthetic enzymes responsible for chain modifications are normally incomplete, leading to
structural heterogeneity.8 These irregular enzymatic reactions, which include N-deacetylation,
5-C-epimerization of GlcA toward IdoA, and sulfations at 2-N, 3-O, and 6-O of GlcN and at
2-O of the uronic acid, could create 48 theoretical possibilities for the repeating disaccharide
(Figure 1). The significant diversity of these saccharides in nature encumbers the interrogation
of their interactions with HS-binding proteins. Deciphering the structure-activity relationship
of HS oligosaccharides bearing different lengths and sulfation patterns with binding proteins
has been very challenging. Chemical9-19 and chemoenzymatic20-23 syntheses are currently the
common routes by which well-defined HS oligosaccharides with exquisite stereochemistry and
functionalization as required by these assays could be accessed.
HSPGs regulate a diverse array of promiscuous biological processes such as cellular
adhesion and biological signaling.24 The structure and function of HSPGs can be altered by
enzymes and thus modulate the bioavailability of various signaling ligands that require HS
chains to affect interaction with their cell-surface receptors. Recent studies revealed that both
Sulf-1 and Sulf-2 are functionally redundant with different roles in the biological perspective
by interfering with a few HS-binding growth factors.25 The expressions of these endosulfatases
are appreciably dysregulated in many cancers. For instance, up-regulation of endogenous
human Sulf-1 is implicated in the attenuation of FGF-2 signaling, leading to growth and
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invasion of pancreatic cancer cells.26,27 Numerous studies demonstrated that increased
expression of Sulf-1 is correlated with gastric carcinoma,28 invasive breast carcinoma,29
urothelial carcinoma,30 neoplasia31 as well as osteoarthritis (OA).32,33 In vivo assays in
transgenic mice revealed that overexpression of Sulf-1 activates the TGF-induced pathway
and enhances tumor progression and metastasis in hepatocellular carcinoma (HCC).
Translational validation of this data strongly supported an oncogenic role of Sulf-1 in human
HCC.34 GlcN-6-sulfonamide derivatives have been reported as weak inhibitors of Sulf-1 and
Sulf-2 in initial studies.35,36 Thus, continual efforts are needed to design more potent inhibitors
of these enzymes.
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Although the crystal structures of three human sulfatases, arylsulfatases A, B and C,
have been determined,37 that of Sulfs are still elusive. Indeed, a complete understanding of
Sulf’s recognition and specificity of its natural substrates is largely lacking. Early studies on
the substrate specificity of Sulfs involved the characterization of depolymerized HS
disaccharide constructs after enzymatic remodeling, which was completely based on analytical
tools such as mass spectrometry38,39 and HPLC.40-42 In this direction, 35S-radiolabeled HS
polysaccharides were prepared using recombinant HS biosynthetic enzymes.41 To further
elaborate the current understanding of the substrate preference of human Sulf-2, high
throughput liquid chromatography/mass spectrometry based glycomics technology43 was
employed and validated. However, a thorough survey of the literature did not yield any report
about the identification of specific substrate for Sulf-1. To understand how Sulfs recognize
their HSPG substrates and also to develop oligosaccharide mimetics as inhibitors to facilitate
the drug discovery process, we present herein the chemical synthesis of well-defined HS
oligosaccharides of various lengths with defined sulfation patterns. These oligosaccharides are
used to elucidate the substrate specificity of Sulf-1, employing a competition-based assay
against the fluorogenic pseudosubstrate 4-methylumbelliferyl sulfate (4-MUS). Based on the
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newly identified substrates with minimal size, sulfonamide variants of specific sulfate groups
were also prepared and further evaluated as inhibitors of Sulf-1.
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RESULTS
Synthesis of the di- and tetrasaccharide donors 4 and 7. Our work commenced with
the synthesis of the glycosyl donors 4 and 7 from the common disaccharide 3 (Scheme 1),
which was generated by -stereoselective glycosylation from the individual monosaccharides
1 and 2 according to our previous report.9 Cu(OTf)2-catalyzed acetolysis of compound 3
Scheme 1. Preparation of the di- and tetrasaccharide donors 4 and 7a
a
Reagents and conditions: (a) (1) 1.5 mol% Cu(OTf)2, Ac2O, 0 C, 2 h; (2) TMSSTol,
ZnI2,CH2Cl2, 2 h; 4: 77% (2 steps), 7: 78% (2 steps); (b) DDQ, CH2Cl2/H2O (18/1, v/v), rt, 4
h; 75%; (c) 4, NIS, TfOH, 3 Å molecular sieves, CH2Cl2, 60 C to–40 C, 4 h; 80%. Ac,
acetyl; Bn, benzyl; Bz, benzoyl; 2-NAP, 2-naphthylmethyl; PBB, p-bromobenzyl; TBDPS,
tert-butyldiphenylsilyl; Tf, trifluoromethylsulfonyl; TMS, trimethylsilyl; Tol, 4-methylphenyl.
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followed by addition of trimethylsilyl p-toluenyl thioether (TMSSTol) in the presence of ZnI2
yielded the thioglycoside donor 4 (77% in two steps). Cleavage of the 2-naphthylmethyl (2-
NAP) group at the 4’-O position of 3 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)
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furnished the 4’-alcohol
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(75%). Coupling with compound
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upon N-
iodosuccinimide/trifluoromethanesulfonic acid (NIS/TfOH) activation provided the expected
tetrasaccharide 6 (80%). The -glycosidic bond was formed by neighboring group (2-O-Bz)
participation. Compound 6 was then subjected to acetolysis and thioglycoside formation,
affording the desired donor 7 (78%) in two steps.
Synthesis of HS oligosaccharides 18, 19, and 20. With the orthogonally protected
thioglycoside donors 4 and 7 in hand, we turned our attention to the preparation of HS
oligosaccharides with various chain lengths (Scheme 2). The synthesis of the linker-attached
disaccharide 8 began by coupling compound 4 with 5-(N-benzyl-N-benzyloxycarbonyl)amino-
1-pentanol [HO(CH2)5N(Bn)Cbz] under a combination of NIS and TfOH. The corresponding
-form product 8 was obtained in 77% yield. Treatment of compound 8 with DDQ afforded
the disaccharide acceptor 9 (75%), which was subsequently coupled with the donors 4 and 7 in
exclusive -stereoselectivity to form the tetrasaccharide 10 (81%) and hexasaccharide 11
(82%), respectively. Removal of all Ac and Bz groups in compounds 8, 10, and 11 using
sodium methoxide followed by oxidation with (2,2,6,6-tetramethylpiperidin-1-yl)oxyl
(TEMPO) gave the lactones 12 (67%), 13 (70%), and 14 (80%), respectively. The cyclic ester
was confirmed through the correlation between the 2-H and 6-C of the internal L-iduronyl
residue in the HMBC spectra and by the relatively high IR C=O stretching band observed
around 1790 cm–1. Further 6-O-desilylation by tetra-n-butylammonium fluoride (TBAF),
lactone ring opening under basic conditions, and O-sulfonation of all free hydroxy groups with
SO3•Et3N furnished the sulfate derivatives 15, 16, and 17 in three steps in 66%, 78%, and 30%
overall yields, respectively. Finally, the individual target compounds 18 (90%), 19 (85%), and
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Scheme 2. Synthesis of HS oligosaccharides 18, 19 and 20a
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a Reagents and conditions: (a) HO(CH2)5N(Bn)Cbz, NIS, TfOH, 3Å MS, CH2Cl2/CH3CN (1/2,
v/v), 78 C to –40 C, 3 h; 77%; (b) DDQ, CH2Cl2/H2O (18/1, v/v), 4 h; 75%; (c) NIS, TfOH,
3Å molecular sieves, CH2Cl2, 78 C to –40 C, 3 h; 10 (n=2, from 4+9): 81%, 11 (n=3, from
7+9): 82%; (d) (1) NaOMe, CH2Cl2/MeOH (1/1, v/v), rt, 18 h; (2) TEMPO, BAIB,
H2O/CH2Cl2 (1/2, v/v), rt, 16 h; 12: 67% (2 steps), 13: 70% (2 steps), 14: 80% (2 steps); (e) (1)
1M TBAF, THF, 50 oC, 1 d; (2) LiOH, rt, 3 h; (3) SO3•Et3N, DMF, 16 h; 15: 66 % (3 steps),
16: 78% (3 steps), 17: 30% (3 steps); (f) (1) 1 M PMe3/THF, THF, NaOH(aq), rt, 5 h; (2)
SO3•pyridine, Et3N, NaOH(aq), MeOH, rt, 1 d; (3) Pd(OH)2/C, H2 (balloon), MeOH, phosphate
buffer (pH=7), rt, 2 d; 18: 90 % (3 steps), 19: 85% (3 steps), 20: 70% (3 steps). BAIB,
[bis(acetoxy)iodo]benzene; Cbz, benzylooxycarbonyl; THF, tetrahydrofuran.
20 (70%) were generated from 15–17 through a 3-step transformation including the Staudinger
reduction of the azido groups to the amino groups, the N-sulfation of all primary amines, and
the Pd(OH)2/C-mediated hydrogenolysis to cleave all benzyl, PBB, 2-NAP, and Cbz groups.
Due to frequent peak overlap, the direct assignments of protons in the NMR spectra for the
tetrasaccharide 19, and hexasaccharide 20 were difficult. However, this could be made possible
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by the analysis of 2D COSY NMR spectrum of compound 18 (Figure S1) and the side-by-side
comparison with the NMR data of HS analogues 19 and 20 (Figure S2). The molecular weights
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of compounds 18 (M+H+, calculated for C17H29N2O20S3Na4 769.0067, found 769.0076), 19
+
(M+H+, calculated for C29H44N3O39S6Na8 1433.9058, found 1433.9055) and 20 (M-4H4-,
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4-
calculated for C41H66N4O58S9 457.4951, found 457.4981) were further confirmed by high
resolution electrospray ionization mass spectra.
Screening of substrates for Sulf-1 based on competition assay with 4-MUS. To
identify its substrate, assays were performed with human Sulf-1 based on insights and
integrated workflow devised by Dierks et al.44 where HS oligosaccharides of different chain
lengths were used as competing substrates in the presence of 4-MUS. The experiments were
separately conducted with compounds 18-20 as well as the commercially available heparin
3000 to assess their competing effect on 4-MUS cleavage by Sulf-1 to yield the fluorescent 4-
methylumbelliferone (4-MU) (Figure 2A). The fluorescence intensities at the excitation and
emission wavelengths of 355 nm and 460 nm, respectively, were measured and the generic 4-
MUS hydrolysis in pure water was considered as 100%. When purified Sulf-1 was incubated
with the disaccharide 18 (320 M) as a competitor of 4-MUS (4.35 mM), the resulting
fluorescence (94.5%) was quite like the control, illustrating that 18 is unable to block the entry
of 4-MUS into the Sulf-1 active site (Figure 2B). On the other hand, experimental
measurements with tetrasaccharide 19, hexasaccharide 20, and heparin 3000 demonstrated that
four or more sugar units strongly compete with 4-MUS.
To better understand the structural requirements by Sulf-1 in its substrates, an array of
–
–
the HS tetrasaccharides (i.e., compounds 21-35) having N-Ac/SO3 and 6-O-SO3 at the GlcN
–
units and 2-O-H/SO3 at the GlcA/IdoA units45 was assayed as competing binders against 4-
MUS (Figure 2C). This systematic study revealed that compound 31 is also a good binder in
addition to compound 19, indicating that the 2-O-sulfated IdoA unit near the nonreducing
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(C)
-
OSO3
O
-
OSO3
O
-O2C
-
-
-O2C
OSO3
O
OSO3
O
OH
-O2C
-O2C
OH
O
OH
O
O(CH2)5NH2
O
O(CH2)5NH2
O
O
O
HN
R
O
O
HO
HO
HO
HN
R
O
HO
O
O
O
HO
HN
R
HN
R
HO
O
HO
O
R1
R1
R1
R1
-
24: R = Ac, R1 = H (fluorescence: 98.6%)
19: R = SO3 , R1 = SO3- (fluorescence: 0%)
21: R = Ac, R1 = H (fluorescence: 100%)
22: R = Ac, R1 = SO3- (fluorescence: 71.7%)
25: R = Ac, R1 = SO3- (fluorescence: 100%)
26: R = SO3 , R1 = H (fluorescence: 71.9%)
-
27: R = SO3 , R1 = SO3- (fluorescence: 96.0%)
-
23: R = SO3 , R1 = H (fluorescence: 98.2%)
-
-
OSO3
-
-O2C
-
O
-
OSO3
O
OSO3
O
OSO3
O
O
-O2C
-O2C
O
-O2C
O
OH
O(CH2)5NH2
O
O
O
HN
R
HO
O(CH2)5NH2
O
O
HO
HO
HO
HO
O
O
HO
HO
HN
R
R1
HN
R
O
O
HO
O
HN
R
HO
O
R1
R1
R1
32: R = Ac, R1 = H (fluorescence: 96.4%)
28: R = Ac, R1 = H (fluorescence: 100%)
33: R = Ac, R1 = SO3- (fluorescence: 35.5%)
: R = Ac, R = SO3- (fluorescence: 29.0%)
1
29
-
34: R = SO3 , R1 = H (fluorescence: 81.9%)
30: R = SO3 , R1 = H (fluorescence: 29.4%)
-
35: R = SO3 , R1 = SO3- (fluorescence: 100%)
-
: R = SO3 , R = SO3- (fluorescence: 0%)
-
1
31
Figure 2. (A) Fluorogenic assay of 4-MUS and competitors as Sulf-1 substrates. (B)
Fluorescence intensities of the disaccharide 18, tetrasaccharide 19, hexasaccharide 20 and
heparin 3000 at 320 M as competitors in the hydrolysis of 4-MUS (4.35 mM) with Sulf-1.
The intensity of 4-MUS (4.35 mM) with Sulf-1 in pure water was set as 100%. (C) Fluorogenic
screening of various sulfonated tetrasaccharides 21-35 at 0.32 mM as competing substrates for
Sulf-1.
end is essential and the N-sulfate groups at GlcN and the 2-O-sulfate groups of GlcA/IdoA at
the reducing end play important roles in the interaction with Sulf-1. Further assays of both 19
and 31 exhibited the EC50 values of 87.8 M and 151 M, respectively.
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Figure 3. The proposed substrate 36 with minimum sugar units for Sulf-1.
Synthesis of the HS trisaccharide 36 and its competition assay. From the
aforementioned results, the HS trisaccharide 36 (Figure 3), lacking the GlcN unit at the
nonreducing end of 19, was chosen for further investigation. Compound 36 was prepared from
our previously synthesized L-ido donor 3746 and the disaccharide acceptor 9 (Scheme 3).
Coupling of 37 and 9 in the presence of NIS and TfOH furnished the desired -linked
trisaccharide 38 (82%), which then underwent sequential deacylation under Zemplén’s
conditions and TEMPO oxidation to afford the lactone 39 in 70% yield (2 steps). Ring opening
of the lactone 39 in methanol and triethylamine delivered the methyl ester 40 (89%), which
was subjected to O-sulfation with SO3•Et3N yielding the 2,2”-di-O-sulfated derivative 41
(79%). Desilylation of 41 with HF•Pyr at the 6’-O position gave the desired 6’-alcohol 42
(87%), which was further O-sulfated to provide the trisulfate 43 in 84% yield. A 3-step
conversion of compound 43 via consecutive demethylation (H2O2/LiOH) of two carboxylic
ester groups, Staudinger reduction (PMe3) of the azido group, and sulfation (SO3•Pyr) of the
primary amino group afforded the corresponding N- and O-sulfated product 44 in 52% overall
yield. Global deprotection of compound 44 under hydrogenolysis conditions was successfully
carried out and the target molecule 36 was obtained in 74% yield after purification through
Sephadex G25 and ion-exchange chromatography with Dowex 50WX8-Na+ resin. The
chemical structures of the final compound 36 and intermediates were confirmed by extensive
two-dimensional NMR experiments (see the Supplementary Information). The evidence was
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further corroborated with high resolution ESI-MS ([M-2H]2–, calculated for C23H38N2O29S4
467.0216, found 467.0247).
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Scheme 3. Preparation of heparan sulfate trisaccharide 36a
a Reagents and conditions: (a) NIS, TfOH, 3 Å molecular sieves, CH2Cl2, 78 C to –40 C, 4
h, 82%; (b) (1) NaOMe, CH2Cl2/MeOH (1/1, v/v), rt, 18 h; (2) TEMPO, BAIB, H2O/CH2Cl2
(1/2, v/v), rt, 16 h, 70% (2 steps); (c) MeOH, Et3N, CH2Cl2, 40 C, 18 h, 89%; (d) SO3•Et3N,
DMF, 60 C, 18 h, 79%; (e) HF•Pyr, Pyr, THF, 72 h, 87%; (f) SO3•Et3N, DMF, 60 C, 18 h,
o
84%; (g) (1) LiOH(aq), H2O2, THF, 37 C, 18 h; (2) PMe3/THF, THF, NaOH(aq), rt, 14 h; (3)
SO3•Pyr, Et3N, NaOH(aq), MeOH, rt, 18 h, 52% (3 steps); (h) Pd(OH)2/C, H2 (balloon), MeOH,
phosphate buffer (pH=7), rt, 3 d, 74%.
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Figure 4. (A) Fluorescence intensities of the trisaccharide 36 at 320 M as a competing
substrate in the hydrolysis of 4-MUS at 4.35 mM with Sulf-1. The intensity of 4-MUS (4.35
mM) with Sulf-1 in pure water was set to 100%. The EC50 values of 36 is 3.8 M. (B)
Determination of the substrate specificity towards Sulf-1 via HPLC measurement of sulfate
amount indicating 36 is a better substrate than the disaccharide 18 and the tetrasaccharide 19.
Three molecules have been assayed at the same concentration (480 µM). (C) Concentration-
2-
dependent and (D) time-dependent release of SO4 of 36 at 480 M with human Sulf-1. (E)
Determination of kinetic parameters of 36 towards human Sulf-1.
Fluorogenic assay was next carried out to evaluate the trisaccharide 36 (0.32 mM) as a
competing substrate for Sulf-1. The data has revealed that 36 has significant potential to reduce
the hydrolysis of 4-MUS (4.35 mM) in the substrate mixture (Figure 4A). The EC50 value of
36 measured by ELISA is 3.8 M, indicating that 36 is a better binder than 19.
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An HPLC-based assay for the measurement of sulfate ions after digestion of HS with
Sulf-1 in vitro was also developed. To directly calculate the contribution of sulfate release,
compound 36 (480 M) was treated with human Sulf-1 and the amount of the sulfate analyte
was compared with that of untreated 36 as a control (Figure 4B). The sulfate concentration
(381.1 M) was determined by HPLC using the strong AS9-HC anion exchange column and a
conductivity detector. Based on this assay, the sulfate concentrations upon the hydrolysis of
disaccharide 18 and tetrasaccharide 19 were determined as 5.9 M and 46.2 M, respectively.
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Moreover, 480 M of 36 was treated with varying amounts of Sulf-1 (1, 2, and 5 L) for 16 h,
and 49.9, 93.5, and 155.8 μM SO42– were obtained, respectively. As observed, 2 L of Sulf-1
could hydrolyze the 6-O-sulfate group of 36 (Figure 4C) to a notable extent. The kinetic
character of the specific 6-desulfation phenomenon was further evaluated by treating 480 M
of 36 with 2 L of Sulf-1 at different time points. Here, the Sulf-1 activity reached a maximum
at 2 h (Figure 4D). The amounts of released sulfate ions at different substrate concentrations
were analyzed, and the kinetic parameters Vmax and Km (Figure 4E) were determined as 14.1
μM/h and 25.8 μM, respectively. These data indicate that compound 36 has markedly higher
activity compared with the artificial substrate 4-MUS (Km value was measured as 10 mM).
These findings also suggest that binding of the HS substrate blocks the entry of 4-MUS to the
enzyme active site and emphasize that the trisaccharide 36 is an excellent substrate for human
Sulf-1.
Design and synthesis of Sulf-1’s inhibitors and their inhibitory activity. We
envisioned inhibitors of Sulf-1 for having selected the 6-O positions in the D-glucosamine
unit(s) for installing the sulfonamide group(s). The di-, tri-, and tetrasaccharide analogues 45-
47 (Figure 5) were rationally designed for this purpose. Scheme 4 illustrates the preparative
route of compound 46. Treatment of the 6’-alcohol 42 with ClSO2NHBn in pyridine afforded
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Figure 5. Rational design of the di-, tri-, and tetrasaccharide analogs 45-47 as Sulf-1’s
inhibitors.
compound 48 (95%), which underwent a 3-step conversion (ester demethylation, Staudinger
reduction, and N-sulfation) to give the desired N-sulfate 49 in 32% overall yield.
Hydrogenolysis of compound 49 led to the target compound 46, which was isolated in 43%
yield after purification through Sephadex G25 and ion-exchange with Dowex 50WX8-Na+
resin.
Scheme 4. Preparation of the trisaccharide sulfonamide 46a
a Reagents and conditions: (a) ClSO2NHBn, Pyr, rt, 10 min, 95%; (b) (1) LiOH(aq), H2O2, THF,
37 oC, 18 h; (2) PMe3/THF, THF, NaOH(aq), rt, 14 h; (3) SO3•Pyr, Et3N, NaOH(aq), MeOH, rt,
18 h, 32% (3 steps); (c) Pd/C, H2 (balloon), MeOH, rt, 3 d, 43%.
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The syntheses of the di- and tetrasaccharide analogues 45 and 47 are outlined in Scheme
5. Ring opening of the lactones 12 and 13 under basic conditions in MeOH and Et3N yielded
the methyl esters 50 (83%) and 51 (97%), respectively. O-Sulfation of compounds 50 and 51
individually provided the corresponding O-sulfates 52 (75%) and 53 (68%), which were
subjected to desilylation, affording the alcohols 54 and 55 in 60% and 76% yields, respectively.
Sulfamoylation of compounds 54 and 55 at the primary hydroxy groups resulted
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Scheme 5. Synthesis of the di- and tetrasaccharide analogs 45 and 47a
a
Reagents and conditions: (a) MeOH, Et3N, CH2Cl2, 40 C, 18 h, 50: 83%, 51: 97%; (b)
SO3•Et3N, DMF, 60 C, 18 h, 52: 75%, 53: 68%; (c) HF•Pyr, Pyr, THF, 72 h, 54: 60%, 55:
76%; (d) ClSO2NHBn, Pyr, rt, 10 min, 56: 61%, 57: 87%; (e) (1) LiOH(aq), H2O2, THF, 37
oC, 18 h; (2) PMe3/THF, THF, NaOH(aq), rt, 14 h; (3) SO3•Pyr, Et3N, NaOH(aq), MeOH, rt,
18 h, 58: 56% (3 steps), 59: 39% (3 steps); (f) Pd/C, H2 (balloon), MeOH, rt, 3 d, 45: 29%, 47:
22%.
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in the successful formation of the benzyl sulfamates 56 (61%) and 57 (87%), which underwent
a similar 3-step transformation as 48→49 to furnish the N-sulfated derivatives 58 and 59 in
56% and 39% overall yields, respectively. Finally, hydrogenolysis to remove all Bn, Cbz, 2-
NAP, and PBB groups concomitantly gave the expected target products 45 (29%) and 47 (22%).
The inhibitory activity of compounds 45-47 toward Sulf-1 were further examined with
the fluorescent assay by monitoring the hydrolysis of 4-MUS to 4-MU. Only 20% of Sulf-1
activity was inhibited with the disaccharide analog 45 at 0.7 mM. Conversely, the tri- and
tetrasaccharide analogues 46 and 47 showed more potent inhibition with IC50 values of 0.53
M and 29.6 M, respectively. In addition, using the 4-MUS activity assay, kinetic
measurements were performed to determine the inhibitory activity of 46, confirming the
selectivity and potency of this inhibitor. The kinetic studies of the inhibitory activity showed
that the Ki value (0.36 μM, Figure 6) is in good agreement with the IC50 value (0.53 μM).
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Figure 6. Kinetic measurements to determine the Ki value (0.36 μM) of compound 46 using
the 4-MUS fluorogenic assay.
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Figure 7. Characterization of the binding kinetics of compound 46 with hSulf1d417-726 by
surface plasmon resonance (SPR).
With highest inhibitory activity, the binding affinity of the inhibitor 46 with human
Sulf-1 was further measured by surface plasmon resonance (SPR). The hydrophilic domain-
deleted mutant of Sulf-1, namely human Sulf-1d417–726, lacking two furin-type proteinase
cleavage sites responsible for forming the disulfide-bonded heterodimers of 75- and 50-kDa
subunits with retained enzyme activity was constructed according to the literature report.47
Compound 46 was immobilized on the CM5 sensor chip, and aliquots of human Sulf-1d417-726
at gradient concentrations were applied onto the chip. From the concentration-dependent
titration curves (Figure 7) resulting from stable complexes upon fast association and very slow
dissociation, the dissociation constant (KD) was determined as 12 nM, indicating that 46 could
possibly be applied as a probe to identify new Sulfs and inhibitors through its attachment to the
magnetic nanoparticles.
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CONCLUSIONS
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Identification of high affinity ligands for HS-binding proteins to decipher structure-
activity relationships remains a very challenging task. In this direction, a practical enzymatic
assay in vitro has been developed to screen possible substrate preference for Sulf-1. Using the
HS oligosaccharides with various chain lengths and sulfation patterns as competitors of the
fluorogenic pseudosubstrate 4-MUS, the HS trisaccharide 36 (IdoA2S-GlcNS6S-IdoA2S) was
identified as the minimal-size substrate for Sulf-1. HPLC data analysis also showed that Sulf-
1 digested the substrate 36 effectively and blocked the entry of 4-MUS to the active site. Based
on the 4-MUS activity assay, it was further demonstrated that replacement of the 6-O-sulfate
group in 36 with the 6-O-sulfonamide group (46) effectively inhibited the human Sulf-1
activity. This preliminary study allowed us to identify the most potent inhibitor of human Sulf-
1 (i.e., compound 46) with IC50 = 0.53 μM, Ki = 0.36 μM, and KD = 12 nM. Overall, the
methods developed here and the molecular details of Sulf-1 recognition elucidated in this study
should offer important insights into the development of Sulf-1 inhibitors.
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EXPERIMENTAL SECTION
Chemical synthesis. The complete experimental details and compound
characterization data can be found in the Supporting Information.
Competition assay with 4-MUS substrate. A solution containing 320 μM substrate
was incubated with Sulf-1 and 4-MUS in 50 mM Tris, 15 mM HEPES, 225 mM NaCl, 5 mM
CaCl2, and 5 mM MgCl2 (pH 7.4) for 1 h at 37 °C, the fluorescent intensity was measured at
355/460 nm. A control mixture without the competing substrate was defined as 100%
fluorescent intensity, and the sample with substrate was determined as percentage of
fluorescent intensity: (Fluorescent intensity of substrate sample) / (Fluorescent intensity of
control sample).
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Sulf-1 activity assay. Human Sulf-1 was overexpressed and purified according to the
procedure reported previously.48 The activity of Sulf-1 was determined by incubating 4-MUS
(4.35 mM) with Sulf-1 in 50 mM Tris, 15 mM HEPES, 225 mM NaCl, 5 mM CaCl2, and 5
mM MgCl2 (pH 7.4) for 1 h at 37 °C, and the fluorescent intensity was measured at 460 nm
following excitation at 355 nm in ELISA reader (CLARIOstar plate reader).
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HPLC assay. A solution of the substrate (480 μM) was incubated with or without Sulf1
in 50 mM Tris, 15 mM HEPES, 225 mM NaCl, 5 mM CaCl2, and 5 mM MgCl2 (pH 7.4) for
16 h at 37 °C. Release of the sulfate ion from the substrate was quantified via HPLC. Each
sample (5 μL) was injected to AS9HC anion-exchange column (Dionex), and the sulfate ion
was eluted by 9 mM Na2CO3 then analyzed by a conductivity detector (PROD, AERS500, 4
mm, Thermal) with reagent-free controller (PROD, RFC-10) and compared with a standard
curve with Na2SO4 (Merck) from 0 to 500 μM.
Sulf-1 inhibition assay. The inhibition activity was measured by the released 4-MU
from 4-MUS in the presence of human Sulf-1 and the inhibitor. Sulf-1 was incubated with the
inhibitor (0‒10 μM) and 4-MUS (0‒20 mM) in 50 mM Tris, 15 mM HEPES, 225 mM NaCl, 5
mM CaCl2, and 5 mM MgCl2 (pH 7.4) for 1 h at 37 °C. The initial rates of 4-MUS hydrolysis
were determined by measuring the fluorescent intensity at 460 nm following excitation at 355
nm (CLARIOstar, BMG LABTECH). The data obtained were fitted to the Michaelis–Menten
equation using the GraphPad to determine the enzyme inhibition parameters.
Determination of dissociation constant (KD) using surface plasmon resonance.
Compound 46 was immobilized on the surface of a Biacore sensor chip (GE Healthcare)
according to the manufacturer’s guidelines using Biacore T200 system. The kinetic analysis
study was performed at different concentrations of hSulf1d417-726 protein upon serial dilution to
250, 125, 63, 31 and 15 nM in running buffer [20 mM NaOAc (pH 5.0), 150 mM NaCl, and
0.05% Tween 20] and injected on to the chip surface with a flow rate of 25 μL/min at 25°C. In
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each run, the chip sensor surface was regenerated by injecting 4 M NaCl. The titration curves
fit well to a 1:1 Langmuir binding model for calculations of equilibrium constants.
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ASSOCIATED CONTENT
Supporting Information. Full experimental procedures and characterization of
prepared compounds, NMR spectra of all new compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*chwong@gate.sinica.edu.tw; *schung@gate.sinica.edu.tw
ORCID
Chi-Huey Wong: 0000-0002-9961-7865
Shang-Cheng Hung: 0000-0002-8797-729X
Notes. The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by the Ministry of Science and Technology of Taiwan (MOST
106-2745-M-001-001-ASP, MOST 106-2113-M259-009, MOST 106-0210-01-15-02, MOST-
106-2113-M-001-009-MY2, MOST 107-0210-01-19-01 and MOST 108-3114-Y-001-002),
and the Academia Sinica (AS-IA-104-L04 and AS-SUMMIT-108).
REFERENCES
(1)
Diez-Roux, G.; Ballabio, A. Sulfatases and Human Disease. Annu. Rev.
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5
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Genomics Hum. Genet. 2005, 6, 355–379.
(2) Dierks, T.; Schmidt, B.; Borissenko, L. V.; Peng, J.; Preusser, A.; Mariappan,
7
8
9
M.; von Figura, K. Multiple Sulfatase Deficiency is Caused by Mutations in the Gene Encoding
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
the Human Cα-formylglycine Generating Enzyme. Cell 2003, 113, 435–444.
(3)
Cosma, M. P.; Pepe, S.; Annunziata, I.; Newbold, R. F.; Grompe, M.; Parenti,
G.; Ballabio, A. The Multiple Sulfatase Deficiency Gene Encodes an Essential and Limiting
Factor for the Activity of Sulfatases. Cell 2003, 113, 445–456.
(4)
von Figura, K.; Schmidt, B.; Selmer, T.; Dierks, T. A Novel Protein
Modification Generating an Aldehyde Group in Sulfatases: Its Role in Catalysis and Disease.
Bioessays 1998, 20, 505–510.
(5)
Parenti, G.; Meroni, G.; Ballabio, A. The Sulfatase Gene Family. Curr. Opin.
Genet. Dev. 1997, 7, 386–391.
(6)
Hanson, S. R.; Best, M. D.; Wong, C.-H. Sulfatases: Structure, Mechanism,
Biological Activity, Inhibition, and Synthetic Utility. Angew. Chem. Int. Ed. 2004, 43, 5736–
5763.
(7)
Sweet Side of Development. Nat. Rev. Mol. Cell. Biol. 2005, 6, 530–541.
(8) Peterson, S.; Frick, A.; Liu, J. Design of Biologically Active Heparan Sulfate
and Heparin Using an Enzyme-Based Approach. Nat. Prod. Rep. 2009, 26, 610–627.
(9) Zulueta, M. M. L.; Lin, S.-Y.; Lin, Y.-T.; Huang, C.-J.; Wang, C.-C.; Ku, C.-C.;
Hacker, U.; Nybakken, K.; Perrimon, N. Heparan Sulphate Proteoglycans: The
Shi, Z.; Chyan, C.-L.; Irene, D.; Lim, L.-H.; Tsai, T.-I; Hu, Y.-P.; Liu, J.-Y.; Chang, W.; Arco,
S. D.; Wong, C.-H.; Hung, S.-C. -Glycosylation by D-Glucosamine-Derived Donors:
Synthesis of Heparosan and Heparin Analogs that Interact with Mycobacterial Heparin-
Binding Hemagglutinin. J. Am. Chem. Soc. 2012, 134, 8988–8995.
(10) Chang, C.-H.; Lico, L. S.; Huang, T.-Y.; Lin, S.-Y.; Chang, C.-L.; Arco, S. D.;
22
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Journal of the American Chemical Society
1
2
3
4
5
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Hung, S.-C. Synthesis of the Heparin-Based Anticoagulant Drug Fondaparinux. Angew. Chem.
Int. Ed. 2014, 53, 9876–9879.
7
8
9
(11) Hansen, S. U.; Miller, G. J.; Cliff, M. J.; Jayson, G. C.; Gardiner, J. M. Making
the Longest Sugars: A Chemical Synthesis of Heparin-related [4]n Oligosaccharides From 16-
mer to 40-mer. Chem. Sci. 2015, 6, 6158–6164.
10
11
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21
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26
27
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30
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46
47
48
49
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53
54
55
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57
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59
60
(12) Mende, M.; Bednarek, C.; Wawryszyn, M.; Sauter, P.; Biskup, M. B.; Schepers,
U.; Bräse, S. Chemical Synthesis of Glycosaminoglycans. Chem. Rev. 2016, 116, 8193−8255.
(13) Zong, C.; Huang, R.; Condac, E.; Chiu, Y.; Xiao, W.; Li, X.;Lu, W.; Ishihara,
M.; Wang, S.; Ramiah, A.; Stickney, M.; Azadi, P.; Amster, I. J.; Moremen, K. W.; Wang, L.;
Sharp, J. S.; Boons, G.-J. Integrated Approach to Identify Heparan Sulfate Ligand
Requirements of Robo1. J. Am. Chem. Soc. 2016, 138, 13059−13067.
(14) Zong, C.; Venot, A.; Li, X.; Lu, W.; Xiao, W.; Wilkes, J. L.; Salanga, C. L.;
Handel, T. M.; Wang, L.; Wolfert, M. A.; Boons, G.-J. Heparan Sulfate Microarray Reveals
That Heparan Sulfate−Protein Binding Exhibits Different Ligand Requirements. J. Am. Chem.
Soc. 2017, 139, 9534−9543.
(15) Huang, T.-Y.; Irene, D.; Ku, C.-C.; Zulueta, M. M. L.; Tai, T.-J.; Lain, S.-H.;
Cheng, C.-P.; Tsai, P.-X.; Lin, S.-Y.; Chen, Z.-G.; Hsiao, C.-D.; Chyan, C.-L.; Hung, S.-C.
Structure of the Complex between a Heparan Sulfate Octasaccharide and Mycobacterial
Heparin-Binding Hemagglutinin. Angew. Chem. Int. Ed. 2017, 56, 4192–4196.
(16) Dey, S.; Wong, C.-H. Programmable One-Pot Synthesis of Heparin
Pentasaccharides Enabling Access to Regiodefined Sulfate Derivatives. Chem. Sci. 2018, 9,
6685–6691.
(17) Dey, S.; Lo, H.-J.; Wong, C.-H. An Efficient Modular One-Pot Synthesis of
Heparin-Based Anticoagulant Idraparinux. J. Am. Chem. Soc. 2019, 141, 10309–10314.
(18) Sakamoto, K.; Ozaki, T.; Ko, Y.-C.; Tsai, C.-F.; Gong, Y.; Morozumi, M.;
23
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Page 24 of 28
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2
3
4
5
6
7
8
9
Ishikawa, Y.; Uchimura, K.; Nadanaka, S.; Kitagawa, H.; Zulueta, M. M. L.; Tamura, J.-I.;
Hung, S.-C.; Kadomatsu, K. Glycan Sulfation Patterns Define Autophagy Flux at Axon Tip via
PTPR-cortactin Axis. Nat. Chem. Biol. 2019, 15, 699–709.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(19) Pawar, N. J.; Wang, L.; Higo, T.; Bhattacharya, C.; Kancharla, P. K.; Zhang, F.;
Baryal, K.; Huo, C.-X.; Liu, J.; Linhardt, R. J.; Huang, X.; Hsieh-Wilson, L. C. Expedient
Synthesis of Core Disaccharide Building Blocks from Natural Polysaccharides for Heparan
Sulfate Oligosaccharide Assembly. Angew. Chem. Int. Ed. 2019, 58, 18577–18583.
(20) Xu, Y.; Masuko, S.; Takieddin, M.; Xu, H.; Liu, R.; Jing, J.; Mousa, S. A.;
Linhardt, R. J.; Liu, J. Chemoenzymatic Synthesis of Homogeneous Ultralow Molecular
Weight Heparins. Science 2011, 334, 498–501.
(21) Wang, Z.; Hsieh, P.-H.; Xu, Y.; Thieker, D.; Chai, E. J. E.; Xie, S.; Cooley, B.;
Woods, R. J.; Chi, L.; Liu, J. Synthesis of 3-O-Sulfated Oligosaccharides to Understand the
Relationship between Structures and Functions of Heparan Sulfate. J. Am. Chem. Soc. 2017,
139, 14, 5249–5256.
(22) Zhang, X.; Pagadala, V.; Jester, H. M.; Lim, A. M.; Pham, T. Q.; Goulas, A. M. P.;
Liu, J.; Linhardt, R. J. Chemoenzymatic Synthesis of Heparan sulfate and Heparin Oligosaccharides
and NMR Analysis: Paving the Way to a Diverse Library for Glycobiologists. Chem. Sci. 2017, 15,
5656–5668.
(23) Zhang, X.; Lin, L.; Huang, H.; Linhardt, R. J. Chemoenzymatic Synthesis of
Glycosaminoglycans. Acc. Chem. Res. 2019, 52, DOI: 10.1021/acs.accounts.9b00420.
(24) Whitelock, J. M.; Iozzo, R. V. Heparan Sulfate: A Complex Polymer Charged
with Biological Activity. Chem. Rev. 2005, 105, 2745–2764.
(25) Rosen, S. D.; Lemjabbar-Alaoui, H. Sulf-2: An Extracellular Modulator of Cell
Signaling and a Cancer Target Candidate. Expert. Opin. Ther. Targets 2010, 14, 935–949.
(26) Li, J.; Kleeff, J.; Abiatari, I.; Kayed, H.; Giese, N. A.; Felix, K.; Giese, T.;
24
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5
6
7
8
9
Büchler, M. W.; Friess, H. Enhanced Levels of Hsulf-1 Interfere with Heparin-Binding Growth
Factor Signaling in Pancreatic Cancer. Molecular Cancer 2005, 4, 14.
(27) Abiatari, I.; Kleeff, J.; Li, J.; Felix, K.; Buchler, M. W.; Friess, H. Hsulf-1
Regulates Growth and Invasion of Pancreatic Cancer Cells. J. Clin. Pathol. 2006, 59, 1052-
1058.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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40
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(28) Hur, K.; Han, T.-S.; Jung, E.-J.; Yu, J.; Lee, H.-J.; Kim, W. H.; Goel, A.; Yang,
H.-K. Up-Regulated Expression of Sulfatases (SULF1 and SULF2) as Prognostic and
Metastasis Predictive Markers in Human Gastric Cancer. J. Pathol. 2012, 228, 88-98.
(29) Zhu, C.; He, L.; Zhou, X.; Nie, X.; Gu, Y. Sulfatase 2 Promotes Breast Cancer
Progression Through Regulating Some Tumor-Related Factors. Oncol. Rep. 2016, 35, 1318–
1328.
(30) Lee, H.-Y.; Yeh, B.-W.; Chan, T.-C.; Yang, K.-F.; Li, W.-M.; Huang, C.-N.; Ke,
H.-L.; Li, C.-C.; Yeh, H.-C.; Liang, P.-I.; Shiue, Y.-L.; Wu, W.-J.; Li, C.-F. Sulfatase-1
Overexpression Indicates Poor Prognosis in Urothelial Carcinoma of the Urinary Bladder and
Upper Tract. Oncotarget 2017, 8, 47216-47229.
(31) Bret, C.; Moreaux, J.; Schved, J.-F.; Hose, D.; Klein, B. SULFs in Human
Neoplasia: Implication as Progression and Prognosis Factors. J. Transl. Med. 2011, 9, 72.
(32) Otsuki, S.; Taniguchi, N.; Grogan, S.; D'Lima, D.; Lotz, M. Expression of Novel
Extracellular Sulfatases Sulf-1 and Sulf-2 in Normal and Osteoarthritic Articular Cartilage.
Arthritis Rheum. 2008, 58, S874–S875.
(33) Otsuki, S.; Hanson, S. R.; Miyaki, S.; Grogan, S. P.; Kinoshita, M.; Asahara, H.;
Wong, C.-H.; Lotza, M. K. Extracellular Sulfatases Support Cartilage Homeostasis by
Regulating BMP and FGF Signaling Pathways. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10202-
10207.
(34) Dhanasekaran, R.; Nakamura, I.; Hu, C.; Chen, G.; Oseini, A. M.; Seven, E. S.;
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7
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9
Miamen, A. G.; Catherine D. Moser, C. D.; Zhou, W.; van Kuppevelt, T. H.; van Deursen, J.
M.; Mounajjed, T.; Fernandez-Zapico, M. E.; Roberts, L. R. Activation of the TGFβ/SMAD
Transcriptional Pathway Underlies a Novel Tumor Promoting Role of Sulfatase1 in
Hepatocellular Carcinoma. Hepatology 2015, 61, 1269-1283.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(35) Schelwies, M.; Brinson, D.; Otsuki, S.; Hong, Y.-H.; Lotz, M. K.; Wong, C.-H.;
Hanson, S. R. Glucosamine-6-sulfamate Analogues of Heparan Sulfate as Inhibitors of
Endosulfatases. ChemBioChem 2010, 11, 2393–2397.
(36) Miller, D. C.; Carbain, B.; Beale, G. S.; Alhasan, S. F.; Reeves, H. L.; Baisch,
U.; Newell, D. R.; Golding, B. T.; Griffin, R. J. Regioselective Sulfamoylation at Low
Temperature Enables Concise Syntheses of Putative Small Molecule Inhibitors of Sulfatases.
Org. Biomol. Chem. 2015, 13, 5279–5284.
(37) Ghosh, D. Human Sulfatases: A Structural Perspective to Catalysis. Cell. Mol.
Life Sci. 2007, 64, 2013–2022.
(38) Saad, O. M.; Ebel, H.; Uchimura, K.; Rosen, S. D.; Bertozzi, C. R.; Leary, J. A.
Compositional Profiling of Heparin/Heparan Sulfate Using Mass Spectrometry: Assay for
Specificity of a Novel Extracellular Human Endosulfatase. Glycobiology 2005, 15, 818–826.
(39) Staples, G. O.; Shi, X. F.; Zaia, J. Glycomics Analysis of Mammalian Heparan
Sulfates Modified by the Human Extracellular Sulfatase HSulf-2. Plos One 2011, 6, e16689.
(40) Ai, X.; Do, A.-T.; Kusche-Gullberg, M.; Lindahl, U.; Lu, K.; Emerson, Jr., C. P.
Substrate Specificity and Domain Functions of Extracellular Heparan Sulfate 6-O-
endosulfatases, QSulf-1 and QSulf-2. J. Biol. Chem. 2006, 281, 4969–4976.
(41) Pempe, E. H.; Burch, T. C.; Law, C. J.; Liu, J. Substrate Specificity of 6-O-
endosulfatase (Sulf-2) and Its Implications in Synthesizing Anticoagulant Heparan Sulfate.
Glycobiology 2012, 22, 1353–1362.
(42) Nagamine, S.; Tamba, M.; Ishimine, H.; Araki, K.; Shiomi, K.; Okada, T.; Ohto,
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T.; Kunita, S.; Takahashi, S.; Wismans, R. G. P.; van Kuppevelt, T. H.; Masu, M; Keino-Masu,
K. Organ-Specific Sulfation Patterns of Heparan Sulfate Generated by Extracellular Sulfatases
Sulf-1 and Sulf-2 in Mice. J. Biol. Chem. 2012, 287, 9579–9590.
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54
55
56
57
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59
60
(43) Huang, Y.; Mao, Y.; Buczek-Thomas, J. A.; Nugent, M. A.; Zaia, J.
Oligosaccharide Substrate Preferences of Human Extracellular Sulfatase Sulf-2 Using Liquid
Chromatography-Mass Spectrometry Based Glycomics Approaches. Plos One 2014, 9,
e105143.
(44) Frese, M.-A.; Milz, F.; Dick, M.; Lamanna, W. C.; Dierks, T. Characterization
of the Human Sulfatase Sulf-1 and Its High Affinity Heparin/Heparan Sulfate Interaction
Domain. J. Biol. Chem. 2009, 284, 28033–28044.
(45) Hu, Y.-P.; Zhong, Y.-Q.; Chen, Z.-G.; Chen, C.-Y.; Shi, Z.; Zulueta, M. M. L.;
Ku, C.-C.; Lee, P.-Y.; Wang, C.-C.; Hung S.-C. Divergent Synthesis of 48 Heparan Sulfate-
Based Disaccharides and Probing the Specific Sugar–Fibroblast Growth Factor-1 Interaction.
J. Am. Chem. Soc. 2012, 134, 20722–20727.
(46) Hu, Y.-P., Lin, S.-Y., Huang, C.-Y., Zulueta, M. M. L., Liu, J.-Y., Chang, W.;
Hung, S.-C. Synthesis of 3-O-Sulfonated Cell-Surface Heparan Sulfate Octasaccharides that
Inhibit Herpes Simplex Virus Type 1 Host-Cell Interaction. Nat. Chem. 2011, 3, 476–477.
(47) Tang, R.; Rosen, S. D. Functional Consequences of the Subdomain
Organization of the Sulfs. J. Biol. Chem. 2009, 284, 21505–21514.
(48) Lamanna, W. C.; Frese, M. A.; Balleininger, M.; Dierks, T. Sulf Loss Influences
N-, 2-O-, and 6-O-Sulfation of Multiple Heparan Sulfate Proteoglycans and Modulates
Fibroblast Growth Factor Signaling. J. Biol. Chem. 2008, 283, 27724–27735.
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