Exploration of the active site of β4GalT7: Modifications of the aglycon of aromatic xylosides

Article abstract of DOI:10.1039/c4ob02632b

Proteoglycans (PGs) are macromolecules that consist of long linear polysaccharides, glycosaminoglycan (GAG) chains, covalently attached to a core protein by the carbohydrate xylose. The biosynthesis of GAG chains is initiated by xylosylation of the core protein followed by galactosylation by the galactosyltransferase β4GalT7. Some β-d-xylosides, such as 2-naphthyl β-d-xylopyranoside, can induce GAG synthesis by serving as acceptor substrates for β4GalT7 and by that also compete with the GAG synthesis on core proteins. Here we present structure-activity relationships for β4GalT7 and xylosides with modifications of the aromatic aglycon, using enzymatic assays, cell studies, and molecular docking simulations. The results show that the aglycons reside on the outside of the active site of the enzyme and that quite bulky aglycons are accepted. By separating the aromatic aglycon from the xylose moiety by linkers, a trend towards increased galactosylation with increased linker length is observed. The galactosylation is influenced by the identity and position of substituents in the aromatic framework, and generally, only xylosides with β-glycosidic linkages function as good substrates for β4GalT7. We also show that the galactosylation ability of a xyloside is increased by replacing the anomeric oxygen with sulfur, but decreased by replacing it with carbon. Finally, we propose that reaction kinetics of galactosylation by β4GalT7 is dependent on subtle differences in orientation of the xylose moiety. This journal is

Full text of DOI:10.1039/c4ob02632b

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
Exploration of the active site of β4GalT7:
modifications of the aglycon of aromatic
xylosides†
Cite this: Org. Biomol. Chem., 2015,
13, 3351
Anna Siegbahn,‡a Karin Thorsheim,‡a Jonas Ståhle,b Sophie Manner,a
Christoffer Hamark,b Andrea Persson,c Emil Tykesson,c Katrin Mani,c
Gunilla Westergren-Thorsson,c Göran Widmalmb and Ulf Ellervik*a
Proteoglycans (PGs) are macromolecules that consist of long linear polysaccharides, glycosaminoglycan
(GAG) chains, covalently attached to a core protein by the carbohydrate xylose. The biosynthesis of GAG
chains is initiated by xylosylation of the core protein followed by galactosylation by the galactosyltransfer-
ase β4GalT7. Some β-D-xylosides, such as 2-naphthyl β-D-xylopyranoside, can induce GAG synthesis by
serving as acceptor substrates for β4GalT7 and by that also compete with the GAG synthesis on core pro-
teins. Here we present structure–activity relationships for β4GalT7 and xylosides with modifications of the
aromatic aglycon, using enzymatic assays, cell studies, and molecular docking simulations. The results
show that the aglycons reside on the outside of the active site of the enzyme and that quite bulky agly-
cons are accepted. By separating the aromatic aglycon from the xylose moiety by linkers, a trend towards
increased galactosylation with increased linker length is observed. The galactosylation is influenced by the
identity and position of substituents in the aromatic framework, and generally, only xylosides with β-gly-
cosidic linkages function as good substrates for β4GalT7. We also show that the galactosylation ability of a
xyloside is increased by replacing the anomeric oxygen with sulfur, but decreased by replacing it with
carbon. Finally, we propose that reaction kinetics of galactosylation by β4GalT7 is dependent on subtle
differences in orientation of the xylose moiety.
Received 19th December 2014,
Accepted 28th January 2015
DOI: 10.1039/c4ob02632b
www.rsc.org/obc
(GalT-II),2 followed by addition of one glucuronic acid unit, by
a glucuronyltransferase (GlcAT-I), to form a tetrasaccharide
1 Introduction
Proteoglycans (PGs) are highly anionic macromolecules that linker, i.e. GlcA(β1→3)Gal(β1→3)Gal(β1→4)Xylβ-PG (Fig. 1a).
consist of one or several long linear polysaccharides, glyco- This linker is then polymerized from repeating disaccharide
saminoglycan chains (GAGs), covalently attached to a core units. Furthermore, during and after the polymerization
protein. Two classes of GAG chains, chondroitin sulfate (CS)/ of the GAG chains, modifications, such as epimerization, O-
dermatan sulfate (DS) and heparin/heparan sulfate (HS), are and N-sulfation, and N-deacetylation, result in an extensive
linked to the core protein by the carbohydrate xylose. The bio- structural diversity of the GAG chains.
synthesis of these are initiated by xylosylation of the core
β-D-Xylosides, such as 2-naphthyl β-D-xylopyranoside
protein by xylosyltransferases (XT). Two galactose residues are (1, XylNap), serve as substrates for β4GalT7 and can thereby
then added stepwise to the xylosylated protein by two different initiate formation of soluble GAG chains (Fig. 1b), and at the
galactosyltransferases, β4GalT7 (GalT-I)1 and β3GalT6
aCenter for Analysis and Synthesis, Center for Chemistry and Chemical Engineering,
Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
E-mail: ulf.ellervik@chem.lu.se
bDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University,
SE-106 91 Stockholm, Sweden
cDepartment of Experimental Medical Science, Lund University, BMC, SE-221 00
Lund, Sweden
†Electronic supplementary information (ESI) available: 1H-NMR, 13C-NMR, MS
of disaccharides from β4GalT7 assay. See DOI: 10.1039/c4ob02632b
‡These authors contributed equally to this work.
Fig. 1 (a) Biosynthesis of the linker tetrasaccharide of HS and CS/DS
PGs. (b) Galactosylation of XylNap to form GalXylNap.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 3351–3362 | 3351

View Article Online
Paper
Organic & Biomolecular Chemistry
Chart 1 Investigated glycosides.
same time compete with GAG synthesis on core proteins
forming PGs.
In our recent study4 we also observed strong inhibition of
β4GalT7 by 2-naphthyl α-L-arabinoside, a xyloside analog epi-
All enzymes that are known to be involved in the bio- merized at position 4. Thus, in addition to xylosides, we have
synthesis of the tetrasaccharide linker have been cloned,3 and included arabinosides to investigate inhibition of β4GalT7.
we have recently reported assays for measurement of galactosy-
lation by, and inhibition of, the enzyme β4GalT7.4 We investi-
gated the structure–activity relationships for modifications of
the xylose moiety and found that the binding pocket of
β4GalT7 is narrow, with a precise set of important hydrogen
bonds. We have also shown that xylose appears to be the
2 Results
2.1 Synthesis
Compounds 1,10 2,11 9,12 12–15,13 16,7 17,14 18,15 21,16 and
2217 have been reported previously, and compounds 2 and 20
optimal acceptor substrate for galactosylation by the enzyme
and that modifications on the xylose moiety of the β-D-xylo-
pyranosides instead can render inhibitors of galactosylation.
The biological functions of PGs are mainly due to inter-
actions of GAG chains with different protein ligands and regu-
latory factors, which make them important for several normal
cellular processes, but also for cancer growth, invasion, and
metastasis.5 A possible therapeutic strategy may thus be to
target key enzymes in the GAG biosynthesis. For example,
tumor growth has been blocked by treatment of a HSPG-inhi-
biting xyloside in combination with an inhibitor of the for-
mation of polyamines,6 and we have earlier shown xyloside-
induced, selective inhibition of the growth of tumor cells
in vitro as well as in vivo.7,8
We envision that inhibitors of β4GalT7 will be valuable
tools for the exploration of GAG biosynthesis and a starting
point for development of anti-tumor agents. Galactosyltrans-
ferase inhibitors are often based on donor substrate analogs,
i.e. UDP-Gal.9 By instead using inhibitors based on the accep-
tor substrate, in this case xylose, an increased selectivity can
be obtained. To broaden the knowledge of the structural
requirements for β4GalT7, we now present data for modifi-
cations of the aglycon according to Chart 1.
are commercially available. Compounds 3, 4, 5, and 7 have
been published before,10 however, full physical characteri-
zation has not been reported, hence, the syntheses and the ana-
lyses are described herein. The majority of the xylosides were
synthesized through glycosylation of peracetylated β-D-xylopyra-
nose 27 with different aromatic aglycons (phenol, 1-naphthol,
4-phenylphenol, 6-cyano-2-naphthol, and 2-naphthalenethiol)
in CH2Cl2 with BF3·Et2O and Et3N (Scheme 1). The naphthyl
thioxyloside 32 was smoothly oxidized to the corresponding
β-linked sulfone 33 in excellent yield using mCPBA. To form
the α-anomer of 1, the glycosylation reaction was performed in
MeCN with BF3·Et2O and 2-naphthol without addition of Et3N.
Subsequent de-O-acetylation using standard Zemplén con-
ditions generated 3, 4, 5, 7, 8, 11, and 19 in good yields.
2-Naphthyl β-L-xylopyranoside 10 was synthesized according to
the method described above for the xylosides with β-linkage,
starting from peracetylated β-L-xylopyranose 34 (Scheme 1).
(9-Anthracene)-methyl xyloside 6 could not be synthesized
from peracetylated xylose 27, but was instead synthesized from
peracetylated β-xylosyl bromide 36 via a Koenigs–Knorr reac-
tion using Ag2O followed by standard Zemplén deprotection
(Scheme 1).
3352 | Org. Biomol. Chem., 2015, 13, 3351–3362
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
42 from 13% to 30%, calculated from L-arabinose 41
(Scheme 1). Thus, acetylation of L-arabinose 41 with KOAc in
Ac2O generated an α/β-mixture (approximately 5.4 : 1), and gly-
cosylation of that mixture with 2-(2-chloroethoxy)–ethanol in
the presence of BF3·Et2O formed the arabinoside 42. Treat-
ment of 42 with 2-naphthol, K2CO3, and 18-crown-6 produced
43 in 40% and deacetylation generated arabinoside 26 in good
yield.
2.2 β4GalT7 assay
We have expressed a truncated version of β4GalT7 fused with
glutathione S-transferase in E. coli and used it in enzymatic
assays to measure the galactosylation by, and inhibition of,
β4GalT7.4 To determine if the glycosides could serve as accep-
tor substrates for β4GalT7, various concentrations of the glyco-
sides were incubated with the recombinant β4GalT7 and the
donor substrate, UDP-Gal, for 30 minutes at 37 °C. The reac-
tion progress was analyzed using HPLC with fluorescence
detection. We found that none of the arabinosides served as
substrate for β4GalT7 (data not shown), whereas all of the syn-
thesized xylosides were galactosylated, except L-XylNap 10 and
α-XylNap 11 (Table 1). The identities of galactosylated products
were verified by LCMS analysis (ESI, Table S1†). The kinetic
parameters, summarized in Table 1, were calculated using the
nonlinear regression to the Michaelis–Menten model. We
observed excess-substrate inhibition of the enzyme by 5–8, 16,
and 18 (Fig. 2), at the studied substrate concentrations. For
Table 1 Galactosylation of xylosides by β4GalT7
Km
(mM)
Vmax
kcat
kcat/Km
Compound
(pmol s−1
)
(s−1
)
(mM−1 s−1
)
Part A: variation of aglycon size
1a
4
0.58
0.57
0.17
0.12
1.1
1.3
0.72
0.43
1.5
1.8
1.0
2.6
3.2
6.0
5.1
Scheme 1 Reagents and conditions: (i) BF3·Et2O, 1-naphthol, Et3N,
CH2Cl2; BF3·Et2O, 4-phenylphenol, Et3N, CH2Cl2; BF3·Et2O, 6-cyano-2-
naphthol, Et3N, CH2Cl2; BF3·Et2O, 2-naphthalenethiol, CH2Cl2; BF3·Et2O,
2-naphthol, Et3N, CH2Cl2; (ii) NaOMe (1 M), MeOH; (iii) mCPBA, CH2Cl2,
(iv) BF3·Et2O, 2-naphthol, MeCN; (v) 9-anthracenemethanol, Ag2O, 4 Å
MS, CH2Cl2; (vi) 2-naphthalenethiol, TBAHS, Na2CO3 (1 M), EtOAc; (vii)
5b
6b
0.60
Part B: variation at the anomeric center or of absolute configuration
1a
7b
0.58
0.25
0.066
2.4
1.1
1.7
1.7
1.8
—
1.5
2.3
2.4
2.5
—
2.6
9.4
37
1.0
—
2-naphthalenemethanol, Ag2O, 4 Å MS, CH2Cl2; (viii) KOAc, Ac2O; (ix) 8b
9
10
11
BF3·Et2O, 2-(2-chloroethoxy)–ethanol, CH2Cl2; (x) 2-naphthol, K2CO3,
18-crown-6, DMF, 80 °C.
—
—
—
—
—
1a
1.7
2.6
2.7
1.3
3.3
2.2
5.5
4.6
14
Peracetylated β-arabinopyranosyl bromide 3718 was used as
starting point for the synthesis of arabinosides. Phase transfer
catalysis, a mild procedure with excellent anomeric stereo-
selectivity, was used to synthesize naphthyl thioarabinoside 38
(Scheme 1). To obtain the arabinosyl sulfone 24, thioarabino-
side 38 was oxidized using the same procedure as for com-
pound 8, generating the desired sulfone in 96% yield. Using
arabinosyl bromide 37 as a donor in a Koenigs–Knorr reaction
with 2-naphthalenemethanol, in the presence of Ag2O, gener-
ated 40. Deacetylation of 38–40 yielded the corresponding ara-
binosides 23–25, respectively. The Koenigs–Knorr glycosylation
reaction could also be used for the synthesis of 26. However,
the route via peracetylated L-arabinose increased the yield of
13
Part D: variation of electronic properties
1a
0.58
0.58
0.31
0.31
0.60
1.1
1.6
1.2
1.1
2.0
1.5
2.2
1.6
1.5
2.8
2.6
3.8
5.2
4.8
4.7
16b
17
18b
19
a The assay was carried out with various batches of enzyme, and
slightly different kinetic parameters for were obtained. The
compounds in each part were investigated with the same batch.
Hence, comparisons can be made within the same part. b Apparent
kinetic parameters were obtained for concentrations up to the highest
observed reaction rate.
1
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 3351–3362 | 3353

View Article Online
Paper
Organic & Biomolecular Chemistry
activity to various extents (Table 2), but none as efficiently as
the previously reported compound 22.
2.3 GAG priming in cells
Compound 8 showed the highest activity (kcat/Km) according to
the β4GalT7 assay (Table 1). To investigate if this result was
transferable to the GAG priming ability in cell culture, the
GAG priming of compound 8 in HCC70 cells was determined,
using compound 1 and 7 as reference compounds.7,14 HCC70
cells were incubated with the xyloside analogs in low sulfate
medium supplemented with [35S]sulfate. After 24 h of incu-
bation at 37 °C, [35S]sulfate-labeled material was isolated from
the culture media, separated based on size by HPLC, and the
amount of radioactivity was measured to estimate the amount
of GAG priming. Compounds 1 and 7 were found to initiate
the formation of GAG priming to a similar extent, whereas
compound 8 primed 3.4 times as much GAG chains as 1
(Fig. 3). This correlates well with data from the enzymatic
assay.
Fig. 2 Michaelis–Menten representation of the activity of β4GalT7 (V)
as a function of the concentration of compound 8.
Table 2 Inhibition of GalXylNap formation by β4GalT7; 0.5 mM XylNap
and 2.0 mM of glycoside were added in the assay in each experiment.
The decrease of formed GalXylNap in presence of glycoside is expressed
in % of the amount GalXylNap formed without added glycoside
2.4 Molecular docking simulations of the Michaelis complex
To gain further insight into the galactosylation reaction, the
xyloside acceptor substrates 1, 5, 7, 8, and 12–15 were studied
in silico in complex with β4GalT7. Molecular docking simu-
lations were performed with the Autodock VINA software and
the acceptor ligands were docked to the protein in the closed
conformation in the presence of a rigid UDP donor as well as
manganese (based on coordinates from PDB IDs 4M4K and
Inhibition
(% of control)
Compound
Xylosides
2
3
7
8
10
21 13
44
3
80 0.3
93
0
1
11
0
Arabinosides
20
21
22
23
24
25
26
11
28
63
22
38
0
3
8
5
3
3
10 0.3
these compounds, the apparent kinetic parameters were calcu-
lated using the concentrations up to the highest observed reac-
tion rate.
To determine the potential inhibitory effect of selected gly-
cosides, XylNap, which is known to be a substrate for β4GalT7,
was used as a mimic of the natural substrate (i.e. the xylosy-
lated core protein). A fixed amount of XylNap was added,
together with different concentrations of glycosides, and the
amount of formed GalXylNap was measured. A decrease in for-
mation of GalXylNap, compared to control, indicates that the
tested glycoside compete with XylNap for the binding site of
β4GalT7. The decrease in formation of GalXylNap, was used to
indirectly determine the galactosylation efficiency of the
methyl and phenyl xylosides (compounds 2 and 3), since fluo-
rescence could not be used for detection of these. As expected,
compounds 2 and 3, showed less decrease of galactosylation of
XylNap, than, for example, compound 7 (Table 2). Several ara-
binose analogs were investigated and found to inhibit β4GalT7
Fig. 3 Xyloside-induced priming of GAG chains (approx. fractions
35–50) isolated from HCC70 cells. The cells were incubated with
XylNap (1, solid grey line), 7 (dashed line), and 8 (dotted line) and
secreted polyanionic material was subjected to size exclusion chrom-
atography on a Superose 6 column. The solid black line shows the result
for untreated cells.
3354 | Org. Biomol. Chem., 2015, 13, 3351–3362
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
4LW6). Dockings were performed in both presence and
absence of water molecules19 in the crystal structure but it was
concluded that additional insights were not gained when
including water. In addition, protein interactions to the
naphthyl moiety were hindered by water in the crystal struc-
ture. Thus, water molecules were excluded from the docking
simulations.
An initial docking was performed with acceptor ligand 1 in
presence of UDP in the donor site (PDB ID 4LW6). The nine
highest ranked poses showed a non-uniform distribution of
the ligand throughout the binding pocket of the protein,
including several cases with the xylose residues positioned in
the donor substrate site. These findings indicate that the
acceptor substrate competes for this site. It also highlights the
importance of a galactose residue to form the acceptor sub-
strate binding site (vide infra). Subsequent dockings were per-
formed to mimic the Michaelis complex, i.e. with UDP-
galactose as the donor (PDB ID 4M4K). From the generated
binding poses of the docked ligands (1, 5, 7, 8, and 12–15), a
similar positioning of the xylose moiety was observed. The
acceptor binding site is a shallow pocket, which is defined by
acidic residues as well as the donor substrate. The pocket
encloses xylose whereas the aglycon moiety extends from the
binding site, interacting with the exterior of the enzyme, con-
sisting mainly of aromatic residues (Fig. 4a and b).
One geometry optimized complex for each ligand, selected
through cluster analyses of the docked poses, was analyzed in
more detail. From these results we suggest that the xylose
moiety is oriented in the binding site with a beneficial CH–π
interaction between H5 and the aromatic ring of Y177. All xylo-
side hydroxyl groups act as hydrogen bond donors, whereas
O3 also acts as a hydrogen bond acceptor for L209. The hydro-
gen-bonding pattern changes, depending on the coupling to
the aromatic moiety. For ligand 1 and the sulfone containing
ligand 8, the xylose moiety is tilted, compared to the other
compounds, resulting in hydrogen bonds from both O2 and
O3 to D212 and from O4 to N211 (Fig. 4b). Compounds 5, 7,
and 12–15 show a more pronounced CH–π interaction from H5
to Y177 and O2, O3, and O4 hydrogen bond to D212, N211,
and either O4 in UDP-Gal or N211, respectively (Fig. 4a). This
latter orientation closely resembles that of xylobiose from the
crystal structure.20 Furthermore, the naphthyl group generally
adopts a face-to-face stacking to the aromatic ring of Y179.
Fig. 4 Active site region of β4GalT7 mutant D211N complex with UDP-
D-Gal, manganese and (a) ligand 12 or (b) ligand 8. Residues W207,
G208, K259, and R263 have been hidden to increase visibility of the
active site.
the outside of the pocket. Apart from compounds 10 and 11,
all of the other studied xylosides (Chart 1) were galactosylated
by β4GalT7, and several of the studied xylosides showed sub-
strate inhibition. Even if the mechanism for substrate inhi-
bition is unclear, it is possible that it originates in substrate
binding to the enzyme without binding of UDP-Gal. Further-
more, our molecular modeling indicates that the acceptor sub-
strate competes for the same site as the donor substrates,
suggesting that the acceptor may act as an inhibitor by
binding to the β4GalT7-UDP complex.
3 Discussion
We have synthesized a number of glycosides and investigated
their interactions with β4GalT7 using enzymatic assays, cell
studies, and molecular docking simulations. From the compu-
tational studies, we observe that the acceptor binding site of
β4GalT7 is a shallow pocket enclosing the xylose moiety where
the aglycon extends out to the surface of the enzyme. The xylo-
side moiety displays only subtle differences in binding orien-
3.1 Part A: variation of aglycon size
tation between the different ligands. The naphthyl moiety Several xylosides carrying relatively small aglycons have shown
tends to interact with a face-to-face π-stacking with Y179 on GAG priming ability in earlier cell studies and enzymatic
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 3351–3362 | 3355

View Article Online
Paper
Organic & Biomolecular Chemistry
assays. In an early study by Robinson et al.,21 it was shown that where 1, 4, and 5 were found to initiate GAG synthesis. The
D-xylose initiated GAG priming to a small extent, whereas 2 kinetic parameters for galactosylation of 6 were similar to 5. It
was about four times, and 44 and 45 about twelve times, more is reasonable that the aglycon, which extends out of the
efficient. In 1991, Lugemwa and Esko studied xylosides with binding pocket, provides stronger binding by favorable π–π
different aglycon size.22 They found that xylosides carrying aro- interactions, but that the effect is limited and not favored by
matic aglycons were more efficient GAG primers than aliphatic even larger aromatic moieties.
analogs. For example, GAG chains primed by 46, were shown
to incorporate 14 times more [35S] sulfate compared to 47.
Later, Esko and co-workers23 found that both 48 and 49
3.2 Part B: variation at the anomeric center or of absolute
configuration
primed GAG chains in the same range as 46 and 1. The disac-
charide 50 did not initiate GAG synthesis but when three of
the hydroxyl groups were methylated, the priming of GAG
chains was comparable to that by 1.24 The natural product 51
was also found to initiate GAG synthesis.25 In 2005, we syn-
thesized compound 52, a fluorescently labeled analog to 17,
which was taken up by cells but did not initiate priming of
GAG chains.26 Apart from O-linked xylosides with large agly-
cons, Kuberan and co-workers27,28 have made triazole-linked
xylosides of which some primed GAG chains, like 53, while
others did not.
The results from our β4GalT7 assay were in agreement with
results from earlier investigations. According to the molecular
modeling, there is a π–π interaction between the naphthyl moi-
eties of 1 and 7 and the side-chain of Y179. It can be assumed
that the π–π interaction between Y179 and phenyl xyloside 3 is
weaker since the distance between the two residues is larger.
Methyl xyloside 2, lacking an aromatic aglycon, cannot interact
in such a favorable manner.
It has long been known that α-xylosides, such as 54, 55, 56,
and 57 (Chart 2), do not initiate GAG synthesis, and these com-
pounds have therefore often been included in studies as nega-
tive controls.21,29–32 However, there are studies in which some
GAG priming by α-xylosides have been observed, e.g. 5633 and
some triazole-linked xylosides.34
Xylosides with S-xylosidic bonds generally initiate GAG
priming comparable to the corresponding O-xylosides.21,32,33,35
For example, we have earlier investigated a series of hydroxy-
naphthyl 1-thio-β-D-xylopyranosides that all showed strong
priming of relatively short GAG chains.36 C- and N-xylosides
have been reported to be less efficient as GAG primers com-
pared to the corresponding O- or S-xylosides.33,35,37 Kuberan
and co-workers27,28,34,38–40 have made several series of amide-
and triazole-linked xylosides of which the GAG priming ability
varied with variations in the aglycon moiety.
As expected, the L-xyloside 10 and the α-xyloside 11 were
not found to be galactosylated by β4GalT7. The C-xyloside 9
was galactosylated to a significantly smaller extent than 1, in
contrast to the sulfur analog 7 that was galactosylated more
efficiently. Interestingly, the sulfone analog 8 was found to be
galactosylated by β4GalT7 several times more efficiently than
1. The very efficient galactosylation of 8 by β4GalT7 was
Compounds 1 and 4, which differ in the binding orien-
tation of the naphthyl moiety, were galactosylated by β4GalT7
to a similar extent, whereas compound 5, with a biphenyl
aglycon, showed a slightly increased galactosylation. These
results correlate with the study by Esko and co-workers23
Chart 2 Discussed glycosides with various biological activities (literature data).
3356 | Org. Biomol. Chem., 2015, 13, 3351–3362
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
In this study, the galactosylation of compounds 12–15 was
investigated in the β4GalT7 assay. There is a trend towards
increased galactosylation with increased xylose–aglycon dis-
tance (Fig. 5). However, the galactosylation does not perfectly
correlate with the GAG priming for either cancer cells or
normal cells. This leads us to speculate that the amount of
xyloside-induced GAG priming is influenced not only by the
galactosylation ability of the corresponding xylosides, but also
other factors. For example, differences between cancer cells
and normal cells regarding xyloside uptake, intracellular trans-
port, or glycosyltransferases in the GAG biosynthesis.
3.4 Part D: variation of electronic properties
p-Nitrophenyl β-D-xylopyranoside 61 was one of the first xylo-
sides for which GAG priming ability was observed, and it has
been used as a control in several cell studies and enzymatic
studies.29,42–44 Xylosides with a carboxyl group attached to the
aromatic ring in the aglycon have been observed to be less
efficient as GAG primers compared to the corresponding
methyl ester or non-substituted compound.15,32,35 In solution,
these xylosides are most probably charged, which limit the
uptake and the cellular transportation. Furthermore, all 14
possible hydroxynaphthyl β-D-xylopyranosides have been evalu-
ated and were shown to initiate synthesis of GAG chains with
different proportions of HS chains.45 Moreover, the methoxy
ether 16 was reported to initiate synthesis of GAG chains,7 and
the methoxy ether of 62 was galactosylated in an enzymatic
assay.46
Fig. 5 GAG priming and galactosylation plotted against xylose–aglycon
distance (C1Xyl–C2Nap). The proportion of GAG priming is given as the
integrated value of fractions from cells treated with xyloside, divided by
the integrated values for fractions from untreated cells.13 The xylose-
aglycon distances are measured in nm from the anomeric carbon to the
aromatic carbon coupled to the linker. Circles (blue) indicate GAG
priming in CCD-1095Sk cells, diamonds (red) indicate GAG priming in
HCC70 cells, and squares (black) indicate kcat/Km values (s−1, mM−1).
Compound numbers in parenthesis.
Since compound 17 has shown interesting properties, both
in priming of GAG chains and in selective growth inhibition of
tumor cells, we decided to investigate compounds modified in
position 6 of the naphthyl moiety. Therefore, groups with
electron donating or electron withdrawing properties were
introduced in this position. The kinetic parameters for galacto-
sylation by β4GalT7 of these compounds were found to be in
the same range. Hence, the electron density of the aromatic
residue does not seem to affect the binding to the active site to
any greater extent.
accompanied by efficient priming of GAG chains in cell
studies.
The relationship between the nucleophile and the electro-
phile in a chemical reaction has been shown to be highly
dependent on the distance and angle between the nucleophile
and the electrophile.41 In this case one may speculate that the
reaction kinetics of galactosylation by β4GalT7 is dependent
on the orientation of O4 of the xylose moiety in relation to C1
of the galactose moiety, and that subtle differences in orien-
tation might give rise to large effects in reaction efficiency.
3.5 Part E: inhibitors
Finally, to investigate if the galactosylation efficiency can be
used for design of inhibitors of β4GalT7, we included a few
arabinose analogs with various aglycons and investigated their
ability to inhibit β4GalT7 activity. However, we observed that
the corresponding arabinoside of an efficiently galactosylated
xyloside is not necessarily an efficient inhibitor. Since the
inhibitors do not participate in the reaction, the inhibitory
effects shown by the arabinosides obviously reflect other steps
in the binding sequence and thus deviate from the trends
shown for galactosylation substrates.
3.3 Part C: variation of linker length
In 1994, Esko and co-workers23 noted that the GAG priming
ability was not affected when the distance between the xylose
residue and the naphthyl moiety in XylNap was increased by a
linker. It has also been reported that a triazole-linker, com-
pound 58, increased the GAG priming compared to 59.28
Recently, we made a systematic investigation of the effects
of the xylose–aglycon distance on GAG priming ability.13
A
series of xylosides, where the xylose residue was separated
from the naphthyl aglycon with linkers of different lengths,
was synthesized (compounds 12–15), and the GAG priming
abilities were investigated in cells. The results varied between
different cell lines, but an enhancement of the GAG priming
4 Conclusions
ability was generally observed when the xylose–naphthyl dis- From structure–activity relationships combined with molecular
tance increased (Fig. 5).
docking simulations, we conclude that xylose should be conju-
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 3351–3362 | 3357

View Article Online
Paper
Organic & Biomolecular Chemistry
gated to an aromatic aglycon in order to be efficiently galacto- solvent under reduced pressure and the crude residues were
sylated by β4GalT7, and subsequently initiate GAG synthesis in purified with column chromatography using EtOAc–heptane,
cells. Since the aglycons reside on the outside of the active site EtOAc–toluene, or Et2O–petroleum ether as eluent.
of the enzyme, quite bulky aglycons seem to be accepted.
5.1.2 Typical procedure for glycosylation using Ag2O
Furthermore, the aromatic aglycon can be separated from the (method B). Ag2O (1 eq.) was added to a stirred solution of
xylose moiety by linkers and retain, or increase, the amount of bromide donor (1 eq.), alcohol (2 eq.), and 4 Å MS in CH2Cl2
galactosylation. Generally, only xylosides with β-glycosidic link- (0.2 M) at 0 °C. After 1 h, the temperature was let to reach rt
ages are good substrates for β4GalT7. We also show that the and the reaction mixture was stirred for 18 h before filtration
galactosylation ability of a xyloside is increased by replacing through SiO2 and removal of solvent under reduced pressure.
the anomeric oxygen with sulfur, but decreased by replacing it The crude residues were purified with column chromatography
with carbon. Substituents on the aromatic aglycon might alter using EtOAc–heptane as eluent.
the GAG priming ability depending on identity and position in
5.1.3 Typical procedure for sulfone synthesis. mCPBA
the aromatic framework. Finally, we propose that reaction (4 eq.) was added in portions over 5 minutes to a stirred solu-
kinetics of galactosylation by β4GalT7 is dependent on subtle tion of thioglycoside (1 eq.) in CH2Cl2 (0.1 M) at 0 °C. After
differences in orientation of the xylose moiety. Thus, a good 2 hours, CH2Cl2 was added and the organic phase was washed
substrate for galactosylation is not necessarily a good starting with satd aq. NaHCO3, brine, and water. The organic phase
point for design of inhibitors.
was dried before removal of solvent under reduced pressure
and the crude residues were purified with column chromato-
graphy using EtOAc–heptane as eluent.
5.1.4 Typical procedure for de-O-acetylation (Zemplén con-
ditions). Glycoside (1 eq.) was stirred in MeOH (0.3 M) con-
taining NaOMe (0.05 M) at rt. After 4 h, glacial AcOH was
5 Experimental part
5.1 Synthesis
All moisture- and air-sensitive reactions were carried out under added until neutral pH before removal of solvent under
an atmosphere of dry nitrogen using oven-dried glassware. All reduced pressure. The crude residues were purified with
solvents were dried prior to use unless otherwise stated. column chromatography using MeOH–CH2Cl2 as eluent.
5.1.4.1 Phenyl β-D-xylopyranoside (3). Compound
3 was
Purchased reagents were used without further purification.
Organic phases were dried using Biotage ISOLUTE Phase
separators. Chromatographic separations were performed on
Matrex silica gel (25–70 μm), or on a Biotage Isolera One flash
purification system using Biotage SNAP KP-Sil silica cartridges.
Thin-layer chromatography was performed on precoated TLC
alumina plates coated with silica gel 60 F254 0.25 mm (Merck).
Spots were visualized with UV light or by staining with para-
anisaldehyde. NMR spectra were recorded at ambient tempera-
tures with a Bruker Avance II 400 MHz spectrometer at
400 MHz (1H) and at 100 MHz (13C). 1H NMR spectra were
assigned using COSY (2D homonuclear shift correlation) with
a gradient selection and NOESY (nuclear Overhauser effect
spectroscopy). Chemical shifts are given in ppm, with refer-
ence to residual internals CDCl3 (δ 7.26), CD3OD (δ 3.31),
(CD3)2SO (δ 2.50), or C6D6 (δ 7.16). Coupling constant values
are given in Hz. Mass spectra were recorded on Micromass
Q-Tof micro™. Optical rotations were measured on Perkin
Elmer instrument, Model 341 polarimeter and are given in
10−1 deg cm2 g−1. A Waters 600 Series HPLC system equipped
with a Waters Symmetry C18 column (5 μm, 19 × 100 mm) was
used for purification. Synthesis and physical characterization
of compounds 1,10 2,11 9,12 12–15,13 16,7 17,14 18,15 21,16 and
2217 have been reported previously and compounds 2, 20, 27,
28, 34, 36, and 41 are commercially available.
obtained from 28 following the de-O-acetylation method as a
1
white solid (94%). [α]D20 −51 (c 1, MeOH), H-NMR (CD3OD) δ
7.25–7.30 (m, 2H, ArH), 7.03–7.07 (m, 2H, ArH), 6.98–7.02 (m,
1H, ArH), 4.85–4.87 (m, 1H, H-1), 3.92 (dd, 1H, J 5.4, 11.4 Hz,
H-5e), 3.55–3.61 (m, 1H, H-4), 3.40–3.47 (m, 2H, H-2, H-3),
3.35 (dd, 1H, J 10.4, 11.2 Hz, H-5a); 13C-NMR (CD3OD) δ 158.9,
130.4, 123.4, 117.8, 102.8, 77.7, 74.7, 71.0, 66.9; HRMS calcd
for C11H14O5Na+ [M + Na]: 249.0733; found: 249.0736.
5.1.4.2 1-Naphthyl β-D-xylopyranoside (4). Compound 4 was
obtained from 29 following the de-O-acetylation method as a
1
white solid (69%). [α]D20 −73 (c 2, MeOH), H-NMR (CD3OD) δ
8.36–8.39 (m, 1H, ArH), 7.77–7.80 (m, 1H, ArH), 7.50 (d, 1H, J
8.4 Hz, ArH), 7.43–7.47 (m, 2H, ArH), 7.36 (t, 1H, J 8.0 Hz,
ArH), 7.13 (d, 1H, J 8.0 Hz, ArH), 5.07 (d, 1H, J 7.6 Hz, H-1),
3.97 (dd, 1H, J 5.2, 11.6 Hz, H-5e), 3.63–3.69 (m, 2H, H-2, H-4),
3.51 (t, 1H, J 8.8 Hz, H-3), 3.40 (t, 1H, J 11.2 Hz, H-5a);
13C-NMR (CD3OD) δ 154.5, 136.1, 128.4, 127.5, 127.3, 126.8,
126.3, 123.3, 123.1, 110.5, 103.3, 77.8, 74.9, 71.1, 67.0;
HRMS calcd for C15H16O5Na+ [M + Na]: 299.0895; found:
299.0895.
5.1.4.3 4-Phenylphenyl β-D-xylopyranoside (5). Compound 5
was obtained from 30 following the de-O-acetylation method
as a white solid (88%). [α]2D0 −21 (c 0.7, MeOH), 1H-NMR
(CD3OD) δ 7.53–7.58 (m, 4H, ArH), 7.38–7.42 (m, 2H, ArH),
7.27–7.31 (m, 1H, ArH), 7.11–7.15 (m, 2H, ArH), 4.91 (d, 1H, J
7.2 Hz, H-1), 3.94 (dd, 1H, J 5.6, 11.6 Hz, H-5e), 3.58–3.62 (m,
1H, H-4), 3.43–3.48 (m, 2H, H-2, H-3), 3.38 (dd, 1H, J 10.2, 11.5
Hz, H-5a); 13C-NMR (CD3OD) δ 158.5, 142.0, 136.8, 129.8,
129.0, 127.9, 127.7, 118.1, 102.9, 77.7, 74.8, 71.0, 67.0; HRMS
calcd for C17H18O5Na+ [M + Na]: 325.1052; found: 325.1052.
5.1.1 Typical procedure for glycosylation using BF3·Et2O
(method A). BF3·OEt2 (2.5 eq.) was added to a stirred solution
of Et3N (0.5 eq.), alcohol (1.5 eq.), and per-O-acetylated donor
(1 eq.) in CH2Cl2 (0.2 M) at rt. After 6 h, satd aq. NaHCO3 was
added and the reaction mixture was extracted with CH2Cl2.
The combined organic phases were dried before removal of
3358 | Org. Biomol. Chem., 2015, 13, 3351–3362
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
5.1.4.4 (9-Anthracene)-methyl β-D-xylopyranoside (6). Com-
5.1.4.9 2-(6-Cyano-naphthyl) β-D-xylopyranoside (19). Com-
pound 6 was obtained from 36 using glycosylation method B pound 19 was obtained from 31 following the de-O-acetylation
followed by de-O-acetylation of crude (9-anthracene)-methyl method as a white solid (67%). [α]2D0 −30 (c 0.9, MeOH),
2,3,4-tri-O-acetyl β-D-xylopyranoside as a light yellow solid 1H-NMR (CD3OD) δ 8.28 (s, 1H, ArH), 7.93 (d, 1H, J 2.0 Hz,
1
(2%). [α]2D0 −7 (c 0.6, MeOH), H-NMR (CD3OD) δ 8.53 (s, 1H, ArH), 7.91 (s, 1H, ArH), 7.60 (dd, 1H, J 1.6, 8.4 Hz, ArH), 7.50
ArH), 8.49 (d, 2H, J 8.8 Hz, ArH), 8.05 (d, 2H, J 8.4 Hz, ArH), (d, 1H, J 2.0 Hz, ArH), 7.39 (dd, 1H, J 2.4, 9.2 Hz, ArH), 5.10 (d,
7.54–7.58 (m, 2H, ArH), 7.46–7.50 (m, 2H, ArH), 5.83 (d, 1H, J 1H, J 7.2 Hz, H-1), 3.98 (dd, 1H, J 5.2, 11.2 Hz, H-5e), 3.59–3.62
11.2 Hz, CH2Ar), 5.67 (d, 1H, J 11.2 Hz, CH2Ar), 4.45 (d, 1H, J (m, 1H, H-4), 3.46–3.55 (m, 3H, H-2, H-3, H-5a); 13C-NMR
7.6 Hz, H-1), 4.22 (dd, 1H, J 5.2, 11.2 Hz, H-5e), 3.49–3.60 (m, (CD3OD) δ 159.1, 137.6, 135.0, 131.3, 129.9, 129.6, 127.9, 121.8,
1H, H-4), 3.20–3.29 (m, 3H, H-2, H-3, H-5a); 13C-NMR (CD3OD) 120.3, 111.6, 108.3, 102.4, 77.6, 74.7, 71.0, 67.0; HRMS calcd
δ 132.9, 132.6, 129.9, 129.6, 129.0, 127.3, 126.1, 125.6, 104.0, for C16H15NO5Na+ [M + Na]: 324.0848; found: 324.0849.
77.9, 74.8, 71.3, 67.2, 63.8; HRMS calcd for C20H20O5Na+
5.1.4.10 2-Naphthyl 1-thio-α-L-arabinopyranoside (23). Com-
pound 23 was obtained from 38 following the de-O-acetylation
[M + Na]: 363.1208; found: 363.1207.
5.1.4.5 2-Naphthyl 1-thio-β-D-xylopyranoside (7). Compound method as a white solid (95%). [α]2D1 −48 (c 1, MeOH); 1H NMR
7 was obtained from 32 following the de-O-acetylation method (CD3OD): δ 8.01 (d, 1H, J 1.2 Hz, ArH), 7.78–7.83 (m, 3H, ArH),
as a white solid (83%). [α]2D0 −50 (c 0.7, MeOH), 1H-NMR 7.60 (dd, 1H, J 1.8, 8.6 Hz, ArH), 7.43–7.50 (m, 2H, ArH), 4.77
(CD3OD) δ 8.01 (d, 1H, J 0.8 Hz, ArH), 7.79–7.84 (m, 2H, ArH), (d, 1H, J 8.4 Hz, H-1), 4.02 (dd, 1H, J 3.6, 12.4 Hz, H-5a),
7.80 (d, 1H, J 8.8 Hz, ArH), 7.59 (dd, 1H, J 1.6, 8.8 Hz, ArH), 3.90–3.92 (m, 1H, H-4), 3.75 (t, 1H, J 8.2 Hz, H-2), 3.60–3.65
7.45–7.51 (m, 2H, ArH), 4.67 (d, 1H, J 9.2 Hz, H-1), 3.97 (dd, (m, 2H, H-5e, H-3); 13C NMR (CD3OD): δ 135.1, 133.8, 133.6,
1H, J 4.8, 11.2 Hz, H-5e), 3.46–3.52 (m, 1H, H-4), 3.37 (t, 1H, J 130.9, 130.0, 129.3, 128.7, 128.4, 127.5, 127.1, 90.2, 75.0, 71.6,
8.4 Hz, H-3), 3.23–3.31 (m, 2H, H-2, H-5a); 13C-NMR (CD3OD) δ 69.6, 69.5; HRMS calcd for C15H16O4SNa+ [M + Na]: 315.0667;
135.1, 134.0, 132.2, 131.8, 130.5, 129.3, 128.7, 128.5, 127.6, found: 315.0659.
5.1.4.11 α-L-Arabinopyranosyl 2-naphthyl sulfone (24). Com-
pound 24 was obtained from 39 following the de-O-acetylation
127.3, 90.0, 79.2, 73.8, 70.9, 70.5; HRMS calcd for
C15H16O4SNa+ [M + Na]: 315.0667; found: 315.0667.
5.1.4.6 2-Naphthyl β-D-xylopyranosyl sulfone (8). Compound method as a white solid (45%). [α]2D1 +2 (c 0.9, DMSO); 1H NMR
8 was obtained from 33 following the de-O-acetylation method ((CD3)2SO): δ 8.54 (s, 1H, ArH), 8.20 (d, 1H, J 8.0 Hz, ArH), 8.14
as a white solid (69%). [α]2D1 −35 (c 0.5, MeOH); 1H NMR (d, 1H, J 8.8 Hz, ArH), 8.08 (d, 1H, J 8.0 Hz, ArH), 7.87 (dd, 1H,
(CD3OD): δ 8.54 (s, 1H, ArH), 8.07–8.11 (m, 2H, ArH), 8.02 (d, J 1.6, 8.4 Hz, ArH), 7.73–7.77 (m, 1H, ArH), 7.68–7.71 (m, 1H,
1H, J 8.4 Hz, ArH), 7.90 (dd, 1H, J 1.6, 8.8 Hz, ArH), 7.66–7.75 ArH), 5.31 (d, 1H, J 6.0 Hz, OH), 4.97 (d, 1H, J 6.0 Hz, OH),
(m, 2H, ArH), 4.44 (d, 1H, J 9.2 Hz, H-1), 3.87 (dd, 1H, J 4.8, 4.64 (d, 1H, J 2.0 Hz, OH), 4.44 (d, 1H, J 8.8 Hz, H-1), 3.76–3.82
11.2 Hz, H-5e), 3.63–3.67 (m, 1H, H-2), 3.35–3.39 (m, 2H, H-3, (m, 1H, H-2), 3.72 (dd, 1H, J 2.0, 12 Hz, H-5a), 3.62 (s, 1H,
H-4), 3.16–3.20 (m, 1H, H-5a); 13C NMR (CD3OD): δ 137.0, H-4), 3.49 (d, 1H, J 11.6 Hz, H-5e) 3.41–3.43 (m, 1H, H-3); 13C
135.3, 133.5, 132.6, 130.63, 130.59, 130.0, 129.1, 128.8, 125.2, NMR ((CD3)2SO): δ 135.0, 134.8, 131.6, 130.6, 129.5, 129.3,
93.7, 79.1, 71.2, 71.0, 70.2; HRMS calcd for C15H16O6SNa+ 128.7, 127.9, 127.6, 124.1, 92.6, 73.2, 70.5, 67.7, 66.7; HRMS
[M + Na]: 347.0565; found: 347.0557.
calcd for C15H16O6SNa+ [M + Na]: 347.0565; found: 347.0557.
5.1.4.7 2-Naphthyl β-L-xylopyranoside (10). Compound 10
5.1.4.12 (2-Naphthyl)-methyl α-L-arabinopyranoside (25).
was obtained from 35 following the de-O-acetylation method Compound 25 was obtained from 40 following the de-O-acety-
as a white solid (91%). [α]2D1 +38 (c 0.7, MeOH); 1H NMR lation method as a white solid (90%). [α]2D1 −20 (c 0.7, MeOH);
(CD3OD): δ 7.76–7.80 (m, 3H, ArH), 7.41–7.45 (m, 2H, ArH), 1H NMR (CD3OD): δ 7.88 (s, 1H, ArH), 7.82–7.85 (m, 3H, ArH),
7.35–7.37 (m, 1H, ArH), 7.27 (dd, 1H, J 2.4, 8.8 Hz, ArH), 5.03 7.54 (dd, 1H, J 1.6, 8.4 Hz, ArH), 7.44–7.49 (m, 2H, ArH), 5.02
(d, 1H, J 7.2 Hz, H-1), 3.97 (dd, 1H, J 5.2, 11.2 Hz, H-5e), (d, 1H, J 12.4 Hz, CH2), 4.79 (d, 1H, J 12.0 Hz, CH2), 4.35 (d,
3.58–3.64 (m, 1H, H-4), 4.43–4.53 (m, 3H, H-2, H-3, H-5a); 13C 1H, J 6.8 Hz, H-1), 3.92 (dd, 1H, J 3.2, 12.4 Hz, H-5a), 3.82–3.84
NMR (CD3OD): δ 156.7, 135.8, 131.3, 130.4, 128.6, 128.1, 127.4, (m, 1H, H-4), 3.66 (dd, 1H, J 6.8, 8.8 Hz, H-2), 3.57 (dd, 1H, J
125.3, 120.0, 111.9, 102.9, 77.8, 74.8, 71.1, 67.0; HRMS calcd 1.6, 12.4 Hz, H-5e), 3.53 (dd, 1H, J 3.4, 9 Hz, H-3); 13C NMR
for C15H16O5Na+ [M + Na]: 299.0895; found: 299.0892.
(CD3OD): δ 136.6, 134.8, 134.5, 129.0, 128.9, 128.7, 127.7,
127.13, 127.09, 126.9, 103.9, 74.3, 72.5, 71.6, 69.7, 66.9; HRMS
calcd for C16H18O5Na+ [M + Na]: 313.1052; found: 313.1054.
5.1.4.13 5-(2-Naphthoxy)-3-oxo-1-pentyl α-L-arabinopyrano-
side (26). Compound 26 was obtained from 43 following the
de-O-acetylation method as a white solid (83%). [α]2D1 +9 (c 0.9,
MeOH); 1H NMR (CD3OD): δ 7.74–7.76 (m, 3H, ArH), 7.39–7.43
(m, 1H, ArH), 7.29–7.33 (m, 1H, ArH), 7.24 (d, 1H, J 2.8 Hz,
ArH), 7.16 (dd, 1H, J 2.4, 8.8 Hz, ArH), 4.24–4.26 (m, 3H, H-1,
CH2), 3.96–4.01 (m, 1H, CH2), 3.92–3.95 (m, 2H, CH2), 3.86
(dd, 1H, J 2.8, 12.4 Hz, H-5a), 3.72–3.81 (m, 4H, H-4, CH2),
3.58 (dd, 1H, J 6.8, 8.8 Hz, H-2), 4.50–4.54 (m, 2H, H-5e, H-3);
5.1.4.8 2-Naphthyl α-D-xylopyranoside (11). Compound 11
was obtained from 27 and 2-naphthol using glycosylation
method A followed by de-O-acetylation of crude 2-naphthyl
2,3,4-tri-O-acetyl α-D-xylopyranoside as a white solid (33%).
[α]2D2 +181 (c 0.2, MeOH). 1H-NMR (CD3OD) δ 7.73–7.880 (m,
3H, ArH), 7.50 (d, 1H, J 2.4 Hz, ArH), 7.40–7.44 (m, 1H, ArH),
7.30–7.37 (m, 2H, ArH), 5.62 (d, 1H, J 3.6 Hz, H-1), 3.84–3.88
(m, 1H H-3), 3.59–3.67 (m, 4H, H-2, H-4, H-5a, H-5e); 13C NMR
(CD3OD) δ 156.1, 135.9, 131.2, 130.4, 128.6, 128.1, 127.4, 125.2,
120.1, 111.9, 99.1, 75.2, 73.4, 71.4, 63.7; HRMS calcd for
C15H16O5Na+ [M + Na]: 299.0895; found: 299.0887.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 3351–3362 | 3359

View Article Online
Paper
Organic & Biomolecular Chemistry
13C NMR (CD3OD): δ 158.1, 136.1, 130.6, 130.4, 128.6, 127.9,
127.3, 124.7, 119.8, 107.7, 105.0, 74.2, 72.4, 71.7, 70.9, 69.6,
69.4, 68.5, 66.9; HRMS calcd for C19H24O7Na+ [M + Na]:
387.1420; found: 387.1415.
5.1.4.18 2-Naphthyl
2,3,4-tri-O-acetyl
β-D-xylopyranosyl
sulfone (33). Compound 33 was obtained from 32 following
the oxidation method as a white solid (91%). [α]2D1 −81 (c 0.8,
CHCl3); 1H NMR (CDCl3): δ 8.50 (s, 1H, ArH), 8.05 (d, 1H, J
5.1.4.14 1-Naphthyl 2,3,4-tri-O-acetyl-β-D-xylopyranoside (29). 8 Hz, ArH), 8.01 (d, 1H, J 8.8 Hz, ArH), 7.96 (d, 1H, J 8 Hz,
Compound 29 was obtained from 27 and 1-naphthol following ArH), 7.87 (dd, 1H, J 1.8, 8.6 Hz, ArH), 7.72 (dt, 1H, J 1.2,
glycosylation method A as a white solid (75%). 1H-NMR 7.6 Hz, ArH), 7.66 (dt, 1H, J 1.4, 7.6 Hz, ArH), 5.30 (m, 1H,
(CDCl3) δ 8.13–8.15 (m, 1H, ArH), 7.81–7.83 (m, 1H, ArH), 7.55 H-2), 5.25 (t, 1H, J 8.8 Hz, H-3), 4.77–4.83 (m, 1H, H-4), 4.55 (d,
(d, 1H, J 8.0 Hz, ArH), 7.46–7.52 (m, 2H, ArH), 7.38 (t, 1H, J 1H, J 9.2 Hz, H-1), 4.21 (dd, 1H, J 5.2, 11.6 Hz, H-5e), 3.37 (dd,
7.6 Hz, ArH), 7.07 (d, 1H, J 7.6 Hz, ArH), 5.29–5.41 (m, 2H, 1H, J 9.6, 11.6 Hz, H-5a), 2.13, 2.01, 1.99 (s, 3H each, OAc); 13
C
H-1, H-2), 5.31 (t, 1H, J 7.2 Hz, H-3), 5.04–5.09 (m, 1H, H-4), NMR (CDCl3): δ 170.2, 169.8, 169.6, 135.9, 132.8, 132.1, 132.0,
4.28 (dd, 1H, J 4.8, 12.4 Hz, H-5e), 3.60 (dd, 1H, J 7.6, 12.0 Hz, 130.0, 129.9, 129.3, 128.1, 127.8, 124.5, 90.0, 72.7, 68.3, 67.0,
H-5a), 2.15, 2.11, 2.08 (s, 3H each, OAc); 13C-NMR (CDCl3) 66.9, 20.9, 20.7; HRMS calcd for C21H22O9SNa+ [M + Na]:
δ 170.0, 170.0, 169.7, 152.5, 134.6, 127.7, 126.7, 126.0, 125.9, 473.0882; found: 473.0879.
5.1.4.19 2-Naphthyl 2,3,4-tri-O-acetyl β-L-xylopyranoside
(35). Compound 35 was obtained from 34 and 2-naphthol fol-
lowing glycosylation method A as a white solid (78%). [α]2D1 +28
(c 0.9, CHCl3); 1H NMR (CDCl3): δ 7.73–7.80 (m, 3H, ArH),
125.8, 122.8, 121.8, 108.9, 98.8, 70.6, 70.1, 68.6, 62.0, 21.0,
21.0, 20.9; HRMS calcd for C21H22O8Na+ [M + Na]: 425.1212;
found: 425.1211.
5.1.4.15 4-Phenylphenyl 2,3,4-tri-O-acetyl-β-D-xylopyranoside
(30). Compound 30 was obtained from 27 and 4-phenylphenol 7.44–7.48 (m, 1H, ArH), 7.36–7.41 (m, 2H, ArH), 7.18 (dd, 1H, J
following glycosylation method A as a white solid (72%). 2.4, 8.8 Hz, ArH), 5.34 (d, 1H, J 6.0 Hz, H-1), 5.22–5.35 (m, 2H,
1H-NMR (CDCl3) δ 7.51–7.55 (m, 4H, ArH), 7.42 (t, 2H, J H-3, H-2), 5.02–5.07 (m, 1H, H-4), 4.28 (dd, 1H, J 4.6, 12.2 Hz,
7.2 Hz, ArH), 7.32 (t, 1H, J 7.4 Hz, ArH), 7.06–7.10 (m, 2H, H-5e), 3.60 (dd, 1H, J 7.6, 12 Hz, H-5a), 2.113, 2.105, 2.10 (s,
ArH), 5.21–5.29 (m, 3H, H-1, H-2, H-3), 5.02–5.07 (m, 1H, H-4), 3H each, OAc); 13C NMR (CDCl3): δ 170.1, 170.0, 169.6, 154.5,
4.25 (dd, 1H, J 4.4, 12.4 Hz, H-5e), 3.55 (dd, 1H, J 7.6, 12.0 Hz, 134.3, 130.2, 129.8, 127.8, 127.3, 126.7, 124.8, 118.9, 111.4,
H-5a), 2.10, 2.10, 2.09 (s, 3H each, OAc); 13C-NMR (CDCl3) δ 98.7, 70.7, 70.2, 68.6, 62.0, 21.0, 20.93, 20.90; HRMS calcd for
170.0, 169.9, 169.4, 156.1, 140.5, 136.2, 128.8, 128.3, 127.1, C21H22O8Na+ [M + Na]: 425.1212; found: 425.1202.
5.1.4.20 2-Naphthyl 2,3,4-tri-O-acetyl 1-thio-α-L-arabinopyra-
noside (38). Arabinopyranosyl bromide 3718 (330 mg) was dis-
solved in EtOAc (3.3 mL) and 2-naphthalenethiol (334 mg,
126.9, 117.1, 98.5, 70.8, 70.2, 68.5, 61.9, 20.8, 20.8, 20.7; HRMS
calcd for C23H24O8Na+ [M + Na]: 451.1369; found: 451.1367.
5.1.4.16 2-(6-Cyano-naphthyl) 2,3,4-tri-O-acetyl-β-D-xylopyra-
noside (31). Compound 31 was obtained from 27 and 6-cyano- 2.08 mmol), TBAHS (tetrabutylammonium hydrogen sulfate)
2-naphthol following glycosylation method A as a white solid (532 mg, 1.57 mmol), and aqueous Na2CO3 (1 M, 3.3 mL) were
(33%). 1H-NMR (CDCl3) δ 8.15 (s, 1H, ArH), 7.82 (d, 1H, J added. After 12 h, the reaction mixture was diluted with EtOAc
13.6 Hz, ArH), 7.80 (d, 1H, J 13.2 Hz, ArH), 7.57 (dd, 1H, J 1.6, (15 mL) and washed with satd aq. Na2CO3 (2 × 20 mL), brine
8.4 Hz, ArH), 7.37 (d, 1H, J 2.4 Hz, ArH), 7.28 (dd, 1H, J 2.8, (20 mL), and water (20 mL). The organic phase was dried
9.2 Hz, ArH), 5.40 (d, 1H, J 5.2 Hz, H-1), 5.21–5.28 (m, 2H, H-2, before removal of solvent under reduced pressure. Column
H-3), 5.01–5.04 (m, 1H, H-4), 4.27 (dd, 1H, J 4.4, 12.0 Hz, chromatography (SiO2, 10 → 30% EtOAc in heptane) gave 38 as
H-5e), 3.49 (dd, 1H, J 6.8, 12.4 Hz, H-5a), 2.11, 2.10, 2.10 (s, 3H a white solid (262 mg, 39% over 3 steps from L-arabinose).
1
each, OAc); 13C-NMR (CDCl3) δ 169.9, 169.9, 169.5, 156.6, [α]2D1 +5 (c 1, CHCl3); H NMR (CDCl3): δ 8.01 (d, 1H, J 2 Hz,
136.0, 133.9, 130.5, 128.7, 128.4, 127.3, 120.6, 119.4, 110.9, ArH), 7.78–7.83 (m, 3H, ArH), 7.56 (dd, 1H, J 1.8, 8.6 Hz, ArH),
108.0, 97.9, 70.1, 69.7, 68.3, 61.9, 20.9, 20.9, 20.8; HRMS calcd 7.47–7.52 (m, 2H, ArH), 5.28–5.32 (m, 2H, H-2, H-4), 5.14 (dd,
for C22H21NO8Na+ [M + Na]: 450.1165; found: 450.1163.
1H, J 3.4, 8.2 Hz, H-3), 4.92 (d, 1H, J 7.6 Hz, H-1), 4.20 (dd, 1H,
J 4.4, 12.4 Hz, H-5a), 3.71 (dd, 1H, J 2.0, 12.8 Hz, H-5e), 2.13,
2.09, 2.07 (s, 3H each, OAc); 13C NMR (CDCl3): δ 170.4, 170.1,
169.6, 133.6, 132.8, 131.7, 130.6, 129.6, 128.7, 127.9, 127.8,
126.8, 126.7, 87.1, 70.6, 68.7, 67.6, 65.4, 21.0, 20.9; HRMS
calcd for C21H22O7SNa+ [M + Na]: 441.0984; found: 441.0977.
5.1.4.21 2-Naphthyl 2,3,4-tri-O-acetyl α-L-arabinopyranosyl
5.1.4.17 2-Naphthyl 2,3,4-tri-O-acetyl-1-thio-β-D-xylopyrano-
side (32). BF3·OEt2 (1.0 mL, 7.9 mmol) was added to a stirred
solution of 2-naphthalenethiol (766 mg, 4.78 mmol) and
1,2,3,4-tetra-O-acetyl-β-D-xylopyranose 27 (1.01 g, 3.19 mmol) in
CH2Cl2 (3 mL) at rt. The reaction was performed as for glycosy-
lation method A yielding 32 as a white solid (1.05 g, 79%).
1H-NMR (CDCl3) δ 7.98 (d, 1H, J 1.6 Hz, ArH), 7.78–7.83 (m, sulfone (39). Compound 39 was obtained from 38 following
3H, ArH), 7.54 (dd, 1H, J 2.0, 8.8 Hz, ArH), 7.47–7.51 (m, 2H, the oxidation method as a white solid (96%). [α]2D1 −44 (c 1,
ArH), 5.20 (t, 1H, J 8.0 Hz, H-3), 5.00 (t, 1H, J 8.0 Hz, H-2), DMSO); 1H NMR (CDCl3): δ 8.56 (s, 1H, ArH), 8.06 (d, 1H, J 8.0
4.90–4.96 (m, 2H, H-1, H-4), 4.32 (dd, 1H, J 5.2, 12.0 Hz, H-5e), Hz, ArH), 8.01 (d, 1H, J 8.8 Hz, ArH), 7.93 (m, 2H, ArH), 7.72
3.45 (dd, 1H, J 8.8, 12.0 Hz, H-5a), 2.12, 2.06, 2.05 (s, 3H each, (dt, 1H, J 1.3, 7.5 Hz, ArH), 7.64 (dt, 1H, J 1.3, 7.5 Hz, ArH),
OAc); 13C-NMR (CDCl3) δ 170.0, 169.9, 169.5, 133.6, 132.9, 5.53 (t, 1H, J 9.4 Hz, H-2), 5.13–5.14 (m, 1H, H-4), 5.05 (dd, 1H,
132.2, 129.9, 129.6, 128.8, 127.8, 126.8, 86.4, 71.9, 70.0, 68.5, J 3.2, 9.6 Hz, H-3), 4.55 (d, 1H, J 9.2 Hz, H-1), 4.10 (dd, 1H, J
65.2, 21.0, 20.9; HRMS calcd for C21H22O7SNa+ [M + Na]: 2.4, 13.2 Hz, H-5a), 3.68 (dd, 1H, J 1.0, 13 Hz, H-5e), 2.53, 1.99,
441.0984; found: 441.0980.
1.64 (s, 3H each, OAc); 13C NMR (CDCl3): δ 170.2, 170.1, 169.6,
3360 | Org. Biomol. Chem., 2015, 13, 3351–3362
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
135.8, 132.8, 132.2, 132.1, 129.9, 129.8, 128.9, 128.0, 127.7, H-1), 4.21–4.23 (m, 2H, CH2), 3.96–4.03 (m, 2H, H-5a, CH2),
124.9, 90.1, 71.0, 67.7, 67.6, 64.6, 21.0, 20.8, 20.4; HRMS calcd 3.88–3.91 (m, 2H, CH2), 3.73–3.76 (m, 3H, CH2), 3.58 (dd, 1H, J
for C21H22O9SNa+ [M + Na]: 473.0882; found: 473.0877.
1.8, 13.0 Hz, H-5e), 2.11, 2.05, 2.01 (s, 3H each, OAc); 13C NMR
(CDCl3): δ 170.4, 170.3, 169.6, 156.8, 134.5, 129.5, 129.1, 127.7,
5.1.4.22 (2-Naphthyl)-methyl 2,3,4-tri-O-acetyl α-L-arabino-
pyranoside (40). Compound 40 was obtained from 3718 and 126.8, 126.4, 123.8, 119.0, 106.7, 101.1, 70.6, 70.2, 69.9, 69.2,
2-naphthalenemethanol following glycosylation method B as a 68.9, 67.7, 67.5, 63.2, 21.0, 20.8, 20.7; HRMS calcd for
white solid (20% over 3 steps from L-arabinose). 1H NMR C25H30O10Na+ [M + Na]: 513.1737; found: 513.1736.
(CDCl3): δ 7.79–7.83 (m, 3H, ArH), 7.75 (s, 1H, ArH), 7.44–7.49
5.2 β4GalT7 assay
(m, 2H, ArH), 7.41 (dd, 1H, J 1.6, 8.4 Hz, ArH), 5.25–5.31 (m,
2H, H-2, H-4), 4.99–5.05 (m, 2H, H-3, CH2), 4.75 (d, 1H, J
12.4 Hz, CH2), 4.52 (d, 1H, J 6.8 Hz, H-1), 4.06 (dd, 1H, J 3.4,
13.0 Hz, H-5a), 3.61 (dd, 1H, J 1.6, 12.8 Hz, H-5e), 2.13, 2.03,
2.00 (s, 3H each, OAc); 13C NMR (CDCl3): δ 170.3, 170.1, 169.4,
134.4, 133.1, 133.0, 128.2, 127.8, 127.7, 126.5, 126.3, 126.1,
125.5, 99.4, 70.4, 70.1, 69.2, 67.6, 63.0, 20.9, 20.8, 20.7; HRMS
calcd for C22H24O8Na+ [M + Na]: 439.1369; found: 439.1361.
5.1.4.23 5-Chloro-3-oxo-1-pentyl 2,3,4-tri-O-acetyl-α-L-arabi-
nopyranoside (42). KOAc (7.90 g, 80.5 mmol) was suspended in
acetic anhydride (110 mL) and heated to 130 °C. L-Arabinose
41 (10.0 g, 66.9 mmol) was added in portions over 5 minutes
and after 1.5 hours of heating at 130 °C, the reaction mixture
was allowed to reach rt before poured onto ice. The aqueous
phase was extracted with CH2Cl2 (3 × 500 mL) and the com-
bined organic layers were washed with satd aq. NaHCO3 (1 L),
dried, and stirred with activated charcoal. After 30 minutes,
the mixture was filtered through silica and concentrated to
yield 1,2,3,4-tetra-O-acetyl-L-arabinopyranose as a yellow oil
(23.2 g) that was used without further purification. BF3·Et2O
(1.10 mL, 8.68 mmol) was added to a stirred solution of crude
peracetylated L-arabinose (1.09 g) and 2-(2-chloroethoxy)-
ethanol (540 μL, 5.12 mmol) in CH2Cl2 (10 mL) at 0 °C. The
reaction was performed as for glycosylation method A yielding
42 as a white solid (30% over 2 steps from L-arabinose). 1H
NMR (CDCl3): δ 5.24–5.26 (m, 1H, H-4), 5.19 (dd, 1H, J 7.2, 9.2
Hz, H-2), 5.03 (dd, 1H, J 3.6, 9.2 Hz, H-3), 4.52 (d, 1H, J 7.2 Hz,
H-1), 4.03 (dd, 1H, J 3.3, 13.2 Hz, H-5a), 3.92–3.97 (m, 1H,
CH2), 3.59–3.75 (m, 8H, CH2, H-5e), 2.13, 2.07, 2.01 (s, 3H
each, OAc); 13C NMR (CDCl3): δ 170.5, 170.3, 169.7, 101.1, 71.6,
70.5, 70.3, 69.2, 68.8, 67.8, 63.3, 43.0, 21.1, 21.0, 20.8; HRMS
calcd for C15H23ClO9Na+ [M + Na]: 405.0928; found: 405.0919.
5.1.4.24 5-(2-Naphthoxy)-3-oxo-1-pentyl 2,3,4-tri-O-acetyl-α-L-
arabinopyranoside (43). 2-Naphthol (127 mg, 0.879 mmol),
K2CO3 (70 mg, 0.51 mmol), and 18-crown-6 (1.21 g,
4.57 mmol) were stirred in DMF (2 mL) for 1 hour before 42
(166 mg, 0.434 mmol), dissolved in DMF (3 mL), was added.
The β4GalT7 assay has been described in detail previously.4
5.3 GAG priming in cells
The procedures for cell culture, radiolabeling, and isolation of
xyloside-primed [35S]sulfate-labeled polyanionic material have
been described in detail previously.14
5.4 Molecular docking simulations of the Michaelis complex
For molecular docking simulations, the protein crystal struc-
ture of Drosophila β-1,4-Galactosyltransferase 7 mutant D211N
in complex with xylobiose, manganese, UDP-galactose, or UDP
(PDB ID: 4M4K and 4LW6 respectively) was prepared using
Autodock Tools (ADT).47 The xylobiose molecule was removed
and water molecules were either included or excluded in the
docking (vide supra). The ligands were built using the gview
program (GaussView Version 5, Dennington et al., Semichem
Inc., Shawnee Mission, KS, 2009.) and geometry-optimized
using the ORCA 2.9.1 package48 with the B3LYP functional and
a 6-31G* basis set.
The docking simulations were performed using the Auto-
dock VINA 1.1.2 package49 using a cubic 24 Å grid, an exhaus-
tiveness of 64 and a random starting seed. Residues deemed to
have possible interactions with the naphthyl group, K176, H178,
and Y179, were treated as flexible residues. The nine top-scored
poses from the docking simulations were analyzed and grouped
based on interaction similarities. Subsequently, one representa-
tive structure from the major cluster was chosen for each ligand
(among the three highest ranked poses in all cases). The
selected structures were H-bond optimized and energy mini-
mized employing a 0.6 Å heavy atom restraint (OPLS-2005 force
field) with the Protein preparation wizard within the Maestro
software.50 The resulting geometries were then evaluated.
Acknowledgements
The reaction mixture was heated to 80 °C for 40 hours before it This work was supported by grants from the Alfred Österlund
was allowed to reach rt and water (10 mL) was added. The Foundation, the Crafoord Foundation, the Evy and Gunnar
aqueous phase was extracted with EtOAc (3 × 10 mL) and the Sandberg Foundation, Greta and John Kock, the Gunnar
combined organic phases were washed with brine (50 mL) and Nilsson Cancer Foundation, the Heart & Lung Foundation, the
dried before removal of solvent under reduced pressure. Knut and Alice Wallenberg Foundation, the Koch Foundation,
Column chromatography (SiO2, 20 → 40% EtOAc in heptane) Lars Hiertas Memorial Foundation, the Magnus Bergwall
1
gave 43 as a light brown solid (85 mg, 40%). H NMR (CDCl3): Foundation, the Medical Faculty of Lund University, Lund
δ 7.71–7.77 (m, 3H, ArH), 7.41–7.45 (m, 1H, ArH), 7.31–7.35 University, the Royal Physiographic Society in Lund, Syskonen
(m, 1H, ArH), 7.17 (dd, 1H, J 2.4, 8.8 Hz, ArH), 7.14 (d, 1H, J Svenssons Foundation for Medical Research, the Swedish
2.4 Hz, ArH), 5.23–5.25 (m, 1H, H-4), 5.21 (dd, 1H, J 7.0, 9.4 Cancer Society, the Swedish Medical Research Council (11550),
Hz, H-2), 5.02 (dd, 1H, J 3.6, 9.2 Hz, H-3), 4.50 (d, 1H, J 6.8 Hz, and the Swedish Research Council.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 3351–3362 | 3361

View Article Online
Paper
Organic & Biomolecular Chemistry
24 A. K. Sarkar, T. A. Fritz, W. H. Taylor and J. D. Esko, Proc.
Natl. Acad. Sci. U. S. A., 1995, 92, 3323.
Notes and references
1 R. Almeida, S. B. Levery, U. Mandel, H. Kresse, 25 A. Sinha, W. H. Taylor, I. H. Khan, S. T. McDaniel and
T. Schwientek, E. P. Bennett and H. Clausen, J. Biol. Chem.,
1999, 274, 26165.
2 X. Bai, D. Zhou, J. R. Brown, B. E. Crawford, T. Hennet and
J. D. Esko, J. Biol. Chem., 2001, 276, 48189.
3 P. Carlsson and L. Kjellen, Handbook of Experimental
Pharmacology, 2012, vol. 207, p. 23.
4 A. Siegbahn, S. Manner, A. Persson, E. Tykesson,
J. D. Esko, J. Nat. Prod., 1999, 62, 1036.
26 R. Johnsson, K. Mani, F. Cheng and U. Ellervik, J. Org.
Chem., 2006, 71, 3444.
27 V. M. Tran, X. V. Victor, J. W. Yockman and B. Kuberan,
Glycoconjugate J., 2010, 27, 625.
28 V. M. Tran and B. Kuberan, Bioconjugate Chem., 2014, 25,
262.
K. Holmqvist, A. Ochocinska, J. Roennols, A. Sundin, 29 N. B. Schwartz, L. Galligani, P.-L. Ho and A. Dorfman, Proc.
K. Mani, G. Westergren-Thorsson, G. Widmalm and
U. Ellervik, Chem. Sci., 2014, 5, 3501.
5 M. M. Fuster and J. D. Esko, Nat. Rev. Cancer, 2005, 5, 526.
Natl. Acad. Sci. U. S. A., 1974, 71, 4047.
30 N. B. Schwartz and L. Roden, J. Biol. Chem., 1975, 250,
5200.
6 M. Belting, L. Borsig, M. M. Fuster, J. R. Brown, L. Persson, 31 Y. Fukunaga, M. Sobue, N. Suzuki, H. Kushida and
L.-A. Fransson and J. D. Esko, Proc. Natl. Acad. Sci. U. S. A.,
2002, 99, 371.
7 K. Mani, M. Belting, U. Ellervik, N. Falk, G. Svensson,
S. Suzuki, Biochim. Biophys. Acta, 1975, 381, 443.
32 J. A. Robinson and H. C. Robinson, Biochem. J., 1981, 194,
839.
S. Sandgren, F. Cheng and L.-A. Fransson, Glycobiology, 33 S. O. Kolset, K. Sakurai, I. Ivhed, A. Oevervatn and
2004, 14, 387. S. Suzuki, Biochem. J., 1990, 265, 637.
8 U. Nilsson, R. Johnsson, L.-A. Fransson, U. Ellervik and 34 B. Kuberan, M. Ethirajan, X. V. Victor, V. Tran, K. Nguyen
K. Mani, Cancer Res., 2010, 70, 3771. and A. Do, ChemBioChem, 2008, 9, 198.
9 K. Descroix and G. K. Wagner, Org. Biomol. Chem., 2011, 9, 35 M. Sobue, H. Habuchi, K. Ito, H. Yonekura, K. Oguri,
1855.
K. Sakurai, S. Kamohara, Y. Ueno, R. Noyori and S. Suzuki,
Biochem. J., 1987, 241, 591.
36 M. Jacobsson, K. Mani and U. Ellervik, Bioorg. Med. Chem.,
2007, 15, 5283.
37 J. Malmberg, K. Mani, E. Sawen, A. Wiren and U. Ellervik,
Bioorg. Med. Chem., 2006, 14, 6659.
38 X. V. Victor, T. K. N. Nguyen, M. Ethirajan, V. M. Tran,
K. V. Nguyen and B. Kuberan, J. Biol. Chem., 2009, 284,
25842.
10 T. A. Fritz, F. N. Lugemwa, A. K. Sarkar and J. D. Esko,
J. Biol. Chem., 1994, 269, 300.
11 C. T. Bishop and F. P. Cooper, Can. J. Chem., 1962, 40,
224.
12 J. Malmberg, K. Mani, E. Saewen, A. Wiren and U. Ellervik,
Bioorg. Med. Chem., 2006, 14, 6659.
13 K. Holmqvist, A. Persson, R. Johnsson, J. Lofgren, K. Mani
and U. Ellervik, Bioorg. Med. Chem., 2013, 21, 3310.
14 K. Mani, B. Havsmark, S. Persson, Y. Kaneda, 39 K. Raman and B. Kuberan, Mol. BioSyst., 2010, 6, 1800.
H. Yamamoto, K. Sakurai, S. Ashikari, H. Habuchi, 40 T. K. N. Nguyen, V. M. Tran, V. Sorna, I. Eriksson,
S. Suzuki, K. Kimata, A. Malmstrom, G. Westergren-
Thorsson and L.-A. Fransson, Cancer Res., 1998, 58, 1099.
15 C.-O. Abrahamsson, U. Ellervik, J. Eriksson-Bajtner,
A. Kojima, M. Koketsu, D. Loganathan, L. Kjellen,
R. I. Dorsky, C.-B. Chien and B. Kuberan, ACS Chem. Biol.,
2013, 8, 939.
M. Jacobsson and K. Mani, Carbohydr. Res., 2008, 343, 41 F. M. Menger, Adv. Chem. Ser., 1987, 215, 209.
1473.
42 M. Okayama, K. Kimata and S. Suzuki, J. Biochem., 1973,
74, 1069.
43 F. Daligault, S. Rahuel-clermont, S. Gulberti, M.-T. Cung,
G. Branlant, P. Netter, J. Magdalou and V. Lattard, Biochem.
J., 2009, 418, 605.
16 K. Higashi, H. Hori, T. Ishiyama, Y. Okimoto, I. Kusakabe
and T. Yasui, Agric. Biol. Chem., 1983, 47, 1123.
17 A. Siegbahn, U. Aili, A. Ochocinska, M. Olofsson,
J. Roennols, K. Mani, G. Widmalm and U. Ellervik, Bioorg.
Med. Chem., 2011, 19, 4114.
44 R. L. Stevens and K. F. Austen, J. Biol. Chem., 1982, 257, 253.
18 S. C. Timmons and D. L. Jakeman, Carbohydr. Res., 2008, 45 M. Jacobsson, U. Ellervik, M. Belting and K. Mani, J. Med.
343, 865. Chem., 2006, 49, 1932.
19 M. Lundborg, E. Ali and G. Widmalm, Carbohydr. Res., 46 S. Gulberti, V. Lattard, M. Fondeur, J.-C. Jacquinet,
2013, 378, 133.
G. Mulliert, P. Netter, J. Magdalou, M. Ouzzine and
S. Fournel-Gigleux, J. Biol. Chem., 2005, 280, 1417.
47 M. Morris Garrett, R. Huey, W. Lindstrom, F. Sanner
Michel, K. Belew Richard, S. Goodsell David and J. Olson
Arthur, J. Comput. Chem., 2009, 30, 2785.
20 Y. Tsutsui, B. Ramakrishnan and P. K. Qasba, J. Biol.
Chem., 2013, 288, 31963.
21 H. C. Robinson, M. J. Brett, P. J. Tralaggan, D. A. Lowther
and M. Okayama, Biochem. J., 1975, 148, 25.
22 F. N. Lugemwa and J. D. Esko, J. Biol. Chem., 1991, 266, 48 F. Neese, Wiley Interdiscip. Rev.: Comput. Mol. Sci., 2012, 2, 73.
6674. 49 O. Trott and A. J. Olson, J. Comput. Chem., 2010, 31, 455.
23 T. A. Fritz, F. N. Lugemwa, A. K. Sarkar and J. D. Esko, 50 Schrödinger Release 2014-3: Maestro, version 9.9, LLC, Schrö-
J. Biol. Chem., 1994, 269, 300.
dinger, New York, NY, 2014.
3362 | Org. Biomol. Chem., 2015, 13, 3351–3362
This journal is © The Royal Society of Chemistry 2015