4-Deoxy-4-fluoro-xyloside derivatives as inhibitors of glycosaminoglycan biosynthesis

Article abstract of DOI:10.1016/j.bmcl.2010.10.085

Various 4-deoxy-4-fluoro-xylosides were prepared using click chemistry for evaluating their potential utility as inhibitors of glycosaminoglycan biosynthesis. 2,3-Di-O-benzoyl-4-deoxy-4-fluoro-β-d-xylopyranosylazide, obtained from l-arabinopyranose by six

Full text of DOI:10.1016/j.bmcl.2010.10.085

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7269–7273
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-Deoxy-4-fluoro-xyloside derivatives as inhibitors
of glycosaminoglycan biosynthesis
Yasuhiro Tsuzuki a, , Thao Kim Nu Nguyen b, , Dinesh R. Garud c, Balagurunathan Kuberan b,d,e
,
Mamoru Koketsu f,
⇑
a Department of Chemistry, Faculty of Engineering, Gifu University, Gifu 501-1193, Japan
b Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA
c Department of Chemistry, Sir Parashurambhau College, Tilak road, Pune 411030, India
d Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA
e Interdepartmental Program in Neuroscience, University of Utah, Salt Lake City, UT 84112, USA
f Department of Materials Science and Technology, Faculty of Engineering, Gifu University, Gifu 501-1193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Various 4-deoxy-4-fluoro-xylosides were prepared using click chemistry for evaluating their potential
utility as inhibitors of glycosaminoglycan biosynthesis. 2,3-Di-O-benzoyl-4-deoxy-4-fluoro-b- -xylopyr-
Received 16 August 2010
Revised 6 October 2010
Accepted 18 October 2010
Available online 23 October 2010
D
anosylazide, obtained from L-arabinopyranose by six steps, was treated with a wide variety of azide-
reactive triple bond-containing hydrophobic agents in the presence of Cu2+ salt/ascorbic acid, a step known
as click chemistry. After click chemistry, benzoylated derivatives were deprotected under Zemplén condi-
tions to obtain 4-deoxy-4-fluoro-xyloside derivatives. A mixture of a:b-isomers of twelve derivatives were
Keywords:
then separated on a reverse phase C18 column using HPLC and the resulting twenty four 4-deoxy-4-fluoro-
xylosides were evaluated for their ability to inhibit glycosaminoglycan biosynthesis in endothelial cells. We
identified two xyloside derivatives that selectively inhibit heparan sulfate and chondroitin sulfate/derman
sulfate biosynthesis without affecting cell viability. These novel derivatives can potentially be used to
define the biological actions of proteoglycans in model organisms and also as therapeutic agents to combat
various human diseases in which glycosaminoglycans participate.
Glycosaminoglycan
Proteoglycan
Inhibitor
Xyloside
Click chemistry
Ó 2010 Elsevier Ltd. All rights reserved.
Proteoglycans are composed of a core protein and glycosamino-
glycan (GAG) side chains such as heparan sulfate (HS) and chondroi-
tin sulfate (CS). The GAG side chains are attached to core protein at
certain serine residues through a common linkage tetrasaccharide,
GlcAb(1?3)Galb(1?3)Galb(1?4)Xylb(1-O-Ser).1,2 Interactions of
the sulfated GAG side chains with a wide array of proteins, including
proteases, growth factors, morphogens, cytokines, and extracellular
matrix structuralproteins regulatenumerous biologicalfunctions.3–6
Assembly of GAG chain involves the following events: xylosylation
of certain serine amino acids located on the core proteins, formation
of the linkage tetrasaccharide, elongation by alternate addition of
glucuronyl residues and hexosamine residues (GlcNAc for HS and
GalNAc for CS), and maturation of GAG side chains with various sul-
fation/epimerization events along the GAG chains.7,8
biosynthesis as they do not have an acceptor hydroxyl group at
C-4 position for subsequent sugar attachment and elongation.13
Furthermore, these modified xylosides could affect galactosyltrans-
ferase-1, an enzyme involved in assembly of the linkage region, and
Various xylosides carrying hydrophobic aglycone groups have
been shown to induce free GAG chains in several cellular systems
and model organisms for nearly four decades.9–12 We have earlier
shown that a number of 4-deoxy-4-fluoroxylosides perturb GAG
⇑
Corresponding author. Tel./fax: +81 58 293 2619.
E-mail address: koketsu@gifu-u.ac.jp (M. Koketsu).
Figure 1. 4-Deoxy-4-fluoroxylosides are inhibitors of glycosaminoglycan/proteo-
These authors contributed equally to this work.
glycan biosynthesis.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.

7270
Y. Tsuzuki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7269–7273
OMe
OMe
O
OH
HO
O
, TsOH
BzCl/Pyr
0oC to rt, 2 h
O
O
O
O
O
DMF, rt, 1 h
56%
OH
OH
BzO
OH
OH
OBz
1
2
3
79%
OH
HO
OH
BzCl/Pyr
-20oC,1 h, 0oC,2 h,
rt,15 h
AcOH/H2O(3:2)
95oC, 25 min
DAST
O
O
BzO
dry CH2Cl2,
BzO
BzO
OBz
OBz
-40oC to rt ,3 h
5
4
67%
53%
32%
O
F
O
F
TMS-Azide, SnCl4
Dry DCM
BzO
N3
BzO
BzO
OBz
OBz
7
6
70%
Scheme 1. Synthesis of 4-deoxy-4-fluoroxylopyranosyl azide derivative 7.
Table 1
Synthesis of various 1-(4-deoxy-4-fluoro-D-xylopyranosyl)-1,2,3-triazoles
CuSO4,
N
N
N
O
F
Sodium L-ascorbate
O
R
F
N3
BzO
+
R
BzO
DMF/H2O=3/1
OBz
OBz
11
7
10
N
N
N
NaOCH3
O
F
R
HO
CH3OH/CH2Cl2=1/1
OH
12
Entry
R
11
12
Reaction time
16 h
Yield (%)
Ratio (
a:b)
Reaction time (h)
3
Yield (%)
Ratio (
a:b)
O
F
1
2
3
4
5
93 (11a)
7:10
70 (12a)
75 (12b)
68 (12c)
71 (12d)
81 (12e)
3:5
O
Cl
18 h
87 (11b)
91 (11c)
95(11d)
80 (11e)
3:5
3:5
7:10
4:5
3
3
3
3
7:10
3:5
O
O
O
Br
I
18 h
17 h
3:5
NO2
2 days
7:10
6
7
8
9
2 days
4 days
4 days
1 day
54 (11f)
82 (11g)
68 (11h)
89 (11i)
2:1
4:5
5:2
1:2
3
8
3
3
58 (12f)
60 (12g)
56 (12h)
63 (12i)
5:2
3:5
5:2
3:5
O
O
O
S
Br

Y. Tsuzuki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7269–7273
7271
Table 1 (continued)
Entry
R
11
12
Reaction time
3 days
Yield (%)
Ratio (
a:b)
Reaction time (h)
3
Yield (%)
Ratio (
a:b)
O
10
45 (11j)
10:3
80 (12j)
5:2
11
12
2 days
7 days
87 (11k)
75 (111)
1:2
1:2
3
3
40 (12k)
70 (121)
7:10
2:5
O
Ratio [azide 7:alkyne 10 = 1:1.5].
thereby inhibit proteoglycan (PG) biosynthesis (Fig. 1). In our efforts
to develop new approaches to selectively block PG biosynthesis, we
synthesized additional library of 4-deoxy-4-fluoroxylosides using
click chemistry and examined them for their ability to modulate
GAG biosynthesis in endothelial cells. We identified a number of
4-deoxy-4-fluoroxylosides that efficiently inhibit PG biosynthesis
in a selective manner. We predict that these GAG biosynthetic inhib-
itors are valuable chemicalbiology agents to define the biologicalac-
tions of GAG chains in model organisms. We describe here the
der dry solvent conditions as shown in Scheme 1 (see Supplemen-
tary data for more detail).
The xylopyranosyl azide derivative 7 was then treated with a
wide variety of triple bond-containing hydrophobic agents 10 in
the presence of Cu2+ salt/ascorbic acid. The products of click reac-
tion, 1,2,3-triazole derivatives11, were obtained in moderateto high
yields as a mixture of a:b-isomers (Table 1). After click chemistry,
benzoylated derivatives 11 were deprotected under Zemplén condi-
tions to obtain the4-deoxy-4-fluoroxyloside derivatives 12 (Table 1)
syntheses of various 1-(4-deoxy-4-fluoro-b-
D
-xylopyranosyl)-
(see Supplementary data for more detail). The a:b-isomers of com-
1,2,3-triazole derivatives and their biological evaluation as GAG bio-
synthetic inhibitors in endothelial cells.
pound 12 could not be separated by conventional silica gel chroma-
***
tography. A mixture of the
a:b-isomers of 12
were completely
The synthesis of GAG biosynthetic inhibitors was begun with
arabinopyranose 1. We employed protection of C-3 and C-4 hydroxy
groups as isopropylidene acetal of -arabinopyranose 1 in the first
step to obtain 3,4-O-isopropylidene- -arabinopyranose 2. Com-
L
-
separated by reverse phase chromatography of HPLC system (see
Supplementary data). The structures of obtained products, 12a
12l
and 12ab–12lb were confirmed by 1H, 13C, 19F NMR, and MS
spectra.
a–
L
a
L
pound 2 was then subjected to 1,2-di-O-benzylation, removal of
the 3,4-O-isopropylidene acetal and 3-O-benzylation to give com-
pound 5 as shown in Scheme 1.14,15 Deoxyfluorocarbohydrate ana-
logs, which contain a fluorine atom instead of a hydroxyl group,
are extensively used as mechanistic probes or inhibitors of various
enzymes involved in glycosylation. Therefore, we predicted that
replacement of the C-4 hydroxyl group with a fluorine atom in
xylose might block the transfer of the galactose residue from
Gal-UDP catalyzed by galactosyltransferase-1. The treatment of
compound 5 carrying unprotected hydroxyl group at C-4 with dieth-
ylaminosulfur trifluoride should lead to nucleophilic displacement
of the hydroxyl group by fluoride with concomitant inversion of con-
figuration.16 Thus, the decisive step in our synthetic strategy is the
inversion of the axial hydroxyl group at C-4 of the partially benzoy-
Bovine lung microvascular endothelial cells (BLMVEC) were
examined for viability in the presence of 4-deoxy-4-fluoroxylosides
(12a
reagent.21 Cell viability assay identified seven 4-deoxy-4-fluoroxy-
losides, 12a , 12ab, 12e , 12eb, 12k , 12kb, and 12l , which were
not toxic to the cells at 300 M or lower concentrations. Based
on these results and our earlier findings that most 4-deoxy-4-fluo-
roxylsoides inhibit cellular GAG biosynthesis efficiently at 300
a/b–12la
/b) at various concentrations using CellTiter-BlueÒ
a
a
a
a
l
l
M
lated L-arabinose derivative 5 with diethylaminosulfur trifluoride
(DAST) to obtain the 4-deoxy-4-fluoro-xylopyranoside derivative
6.17 Compound 6 was then appended with various hydrophobic
groups (aglycone) at the anomeric carbon. We and other groups
have shown previously that the nature of the hydrophobic aglycone
dramatically influences the priming ability of xylosides, as different
hydrophobic aglycones are differentially recognized by the biosyn-
thetic enzymes involved in the assembly of GAG chains.18,19 We
identified a number of hydrophobic groups that were important
for optimal priming of GAG chains by click-xylosides in our previous
studies.19,20 On the basis of those findings, we have selectively cho-
sen a set of promising hydrophobic groups to be appended at the
anomeric carbon of 4-deoxy-4-fluoroxylopyranosyl azide. We uti-
lized the robust click chemical methodology in the present study,
as we successfully employed such an approach in our earlier studies,
to prepare various 4-deoxy-4-fluoroxylosides as potential biosyn-
thetic inhibitors. We synthesized the xylopyranosyl azide derivative
7 by treating compound 6 with tetramethylsilyl azide and SnCl4 un-
Figure 2. Inhibitory effect of 4-deoxy-4-fluoroxylosides on GAG biosynthesis in
BLMVEC. Cells were incubated with 300 lM of 4-deoxy-4-fluoroxylosides for 24 h
in the presence of [3H]-glucosamine. Control cells were grown in the absence of
compounds under identical conditions for comparison. GAG chains were then
purified and radioactivity was measured using a liquid scintillation counter as
described in ‘References and Notes’.

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Y. Tsuzuki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7269–7273
Figure 3. 4-Deoxy-4-fluoroxylosides affect GAG biosynthesis in BLMVEC. 3H-labeled-GAG chains were isolated from control and 4-deoxy-4-fluoroxylosides treated cells as
described in the experimental section and were loaded onto a DEAE HPLC column. Elution profiles of GAG chains were analyzed using in-line radiomatic detector coupled to
HPLC. The bound GAG chains were eluted with a linear gradient of 1 M NaCl. The chromatographic profiles of GAG chains isolated from control cells that were treated with
DMSO (gray trace) or cells that were treated (black trace) with 4-deoxy-4-fluoroxylosides (12aa, 12ea, 12ka, 12la, 12ab, 12eb, and 12kb) are shown here. HS: heparan sulfate,
CS: chondroitin sulfate.
or higher concentrations, we decided to screen these promising
seven xyloside derivatives at 300 M for their ability to inhibit PG
biosynthesis in BLMVEC cells. Cells were treated with 300 M of
4-fluoroxylosides, 12a , 12ab, 12e , 12eb, 12k , 12kb, and 12l
to participate at various stages of diseases progression through
regulating GAG-extracellular matrix and cell–cell interactions.
l
l
Acknowledgments
a
a
a
a,
in the presence of 3H-glucosamine for 24 h. GAG chains were then
purified and quantified by measuring radioactivity using a liquid
scintillation counter as described in the experimental section. The
results indicated that all of these 4-deoxy-4-fluoroxylosides, except
12kb, inhibit PG biosynthesis to various degrees in BLMVEC
(Fig. 2).22 Furthermore, we utilized anion-exchange HPLC to analyze
the inhibitory activity of these seven xylosides on cellular GAG bio-
synthesis. HS andCSappeared in theanion-exchange chromatogram
as two independent peaks between 20 and 50 min (Fig. 3). It is
known that HS elutes earlier than CS in the linear salt gradient
because HS carries overall less negative charge density.20 Cells that
aretreatedwithDMSOalonehavenoeffects ontheirGAGbiosynthe-
sis and thus used as control. Among the 4-deoxy-4-fluoroxylosides
This work was supported by grants from Japan Ministry of
Education 20590005 (to M.K.), Human Frontier Science Program
and NIH grants, GM075168 (to B.K.). We acknowledge a graduate
fellowship support from Vietnam Education Foundation (to T.N.).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.10.085.
References and notes
1. Muir, H. Biochem. J. 1958, 69, 195.
screened, 12ea and 12eb were the best inhibitors of PG biosynthesis
2. Lindahl, U.; Roden, L. J. Biol. Chem. 1966, 241, 2113.
3. Götting, C.; Kuhn, J.; Kleesiek, K. S. Cell. Mol. Life Sci. 2007, 64, 1498.
4. Schwartz, N. B.; Domowicz, M. Glycoconjugate J. 2004, 21, 329.
5. Salmivirta, M.; Lidholt, K.; Lindahl, U. FASEB J. 1996, 10, 1270.
6. Raman, K.; Kuberan, B. Curr. Chem. Biol. 2010, 4, 20.
7. Habuchi, H.; Habuchi, O.; Kimata, K. Glycoconjugate J. 2004, 21, 47.
8. Prydz, K.; Dalen, K. T. J. Cell Sci. 2000, 113, 193.
9. Okayama, M.; Kimata, K.; Suzuki, S. J. Biochem. 1973, 74, 1069.
10. Levitt, D.; Dorfman, A. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 2201.
11. Raman, K.; Kuberan, B. Mol. Biosyst. 2010, 6, 1800.
12. Rapraeger, A. J. Cell Biol. 1989, 109, 2509.
13. Garud, D. R.; Tran, V. M.; Victor, X. V.; Koketsu, M.; Kuberan, B. J. Biol. Chem.
2008, 283, 28881.
14. Batey, J. F.; Bullock, C.; O’Brien, E.; Williams, J. M. Carbohydr. Res. 1975, 43, 43.
15. Ibatullin, F. M.; Shabalin, K. A.; Jänis, J. V.; Selivanov, S. I. Tetrahedron Lett. 2001,
42, 4565.
and these inhibitors were able to reduce GAG production by ꢀ80%
(Figs. 2 and 3) without affecting cell viability.23
In conclusion, we have synthesized various 1-(4-deoxy-4-flu-
oro-b-
their ability to inhibit PG biosynthesis and identified two deriva-
tives, 12e and 12eb, as best inhibitors of GAG biosynthesis. Future
D-xylopyranosyl)-1,2,3-triazole derivatives 12, screened for
a
studies will be directed toward investigating the utility of these
biosynthetic inhibitors in defining the structural basis for the
biological actions of glycosaminoglycan glycome and advancing
our knowledge of structural and functional heparanomics. These
derivatives can also be used as therapeutic agents in various
diseases including cancer where glycosaminoglycans are known

Y. Tsuzuki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7269–7273
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16. Dax, K.; Albert, M.; Ortner, J.; Paul, B. J. Carbohydr. Res. 2000, 327, 47.
17. Wicki, J.; Schloegl, J.; Tarling, C. A.; Withers, S. G. Biochemistry 2007, 46, 6996.
18. Fritz, T. A.; Lugemwa, F. N.; Sarkar, A. K.; Esko, J. D. J. Biol. Chem. 1994, 269, 300.
19. Kuberan, B.; Ethirajan, M.; Victor, X. V.; Tran, V.; Nguyen, K.; Do, A.
ChemBioChem 2008, 9, 198.
cells/well in 1 ml of Ham’s F-12 medium with 10% fetal bovine serum
(Hyclone). Seeded cells were incubated for 24 h at 37 °C. Each well was
subsequently washed and supplemented with 890
1 mM glucose and 10% dialyzed fetal bovine serum before treated with 10
DMSO (vehicle, control) or 10 l of stock solution containing 4-fluoroxylosides
(30 mM in DMSO) for 24 h. One hundred microliter of 3H-glucosamine (Perkin–
Elmer, 100 Ci in deionized water) was added to each well to radiolabel the
l
l of DMEM medium with
ll of
l
20. Victor, X. V.; Nguyen, K. T.; Ethirajan, M.; Tran, V.; Nguyen, K.; Kuberan, B. J.
Biol. Chem. 2009, 284, 25842.
l
21. Cytotoxicity assay: BLMVEC were examined for viability in the presence of 4-
deoxy-4-fluoroxylosides derivatives using CellTiter-BlueÒ reagent (Promega).
Cells were seeded into duplicate wells of a 96-well plate at a density of
GAG chains. After 24 h, pronase solution was added to each well and incubated
at 37 °C overnight to harvest the GAG chains. The treated samples were
centrifuged at 16,000g for 5 min and the supernatant was diluted with half-a-
volume of 0.016% Triton X-100 and loaded on a 0.2 ml DEAE-sepharose column
(Amersham Biosciences). The column was first pre-equilibrated with 2 ml of
wash buffer (20 mM NaOAc, 0.1 M NaCl and 0.01% Triton X-100, pH 6.0) and
then washed with 6 ml of wash buffer. The bound GAGs were eluted with
1.2 ml of elution buffer (20 mM NaOAc, 1 M NaCl, pH 6.0). Fifty microliter of
2.5 Â 104 cells/well in 125
ll of Ham’s F-12 medium with 10% fetal bovine
serum (Hyclone). Cells were treated with DMSO (control) or 4-fluoroxylosides
at different concentrations (0.1 mM, 0.3 and 1.0 mM) for 24 h at 37 °C. Cells
were also treated with 3% H2O2 as a negative control. CellTiter-Blue reagent
(25
ll) was added to each well and incubated for 2 h. The reaction was then
stopped by the addition of 50
l
l of 3% SDS solution. The fluorescence was
eluate from each well were analyzed for radioactivity using
scintillation counter.
a liquid
measured using a Spectra-Max M5 microplate reader (Molecular Devices,
Sunnyvale, CA) at an excitation wavelength of 560 nm and an emission
wavelength of 590 nm.
23. Anion exchange chromatography: The extent of inhibition of GAG biosynthesis
was determined by analyzing the elution profile of the GAG chains on an anion
exchange column coupled with an in-line radiometric detector. Radiolabeled
GAGs were loaded onto a DEAE-HPLC column and then eluted for 80 min with
a linear gradient of 0.2–1 M NaCl.
22. Screening
of
4-deoxy-4-fluoroxylosides
in
BLMVEC:
Inhibition
of
glycosaminoglycan biosynthesis by 4-deoxy-4-fluoroxylosides was examined
using BLMVEC. Cells were seeded in a 12-well plate at a density of 2 Â 105