Rules for priming and inhibition of glycosaminoglycan biosynthesis; Probing the β4GalT7 active site

Article abstract of DOI:10.1039/c4sc01244e

β-1,4-Galactosyltransferase 7 (β4GalT7) is an essential enzyme in the biosynthesis of glycosaminoglycan (GAG) chains of proteoglycans (PGs). Mammalian cells produce PGs, which are involved in biological processes such as cell growth and differentiation. T

Full text of DOI:10.1039/c4sc01244e

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Rules for priming and inhibition of
glycosaminoglycan biosynthesis; probing the
Cite this: Chem. Sci., 2014, 5, 3501
b4GalT7 active site†
Anna Siegbahn,a Sophie Manner,‡a Andrea Persson,‡ab Emil Tykesson,‡b
Karin Holmqvist,a Agata Ochocinska,a Jerk Ronnols,c Anders Sundin,a Katrin Mani,b
¨
b
c
a
*
Gunilla Westergren-Thorsson, Goran Widmalm and Ulf Ellervik
¨
b-1,4-Galactosyltransferase 7 (b4GalT7) is an essential enzyme in the biosynthesis of glycosaminoglycan
(GAG) chains of proteoglycans (PGs). Mammalian cells produce PGs, which are involved in biological
processes such as cell growth and differentiation. The PGs consist of a core protein, with one or several
GAG chains attached. Both the structure of the PGs and the GAG chains, and the expression of the
enzymes involved in their biosynthesis and degradation, vary between normal cells and tumor cells. The
biosynthesis of GAG chains is initiated by xylosylation of a serine residue of the core protein, followed by
galactosylation by b4GalT7. The biosynthesis can also be initiated by exogenously added b-D-
xylopyranosides with hydrophobic aglycons, which thus can act as acceptor substrates for b4GalT7. To
determine the structural requirements for b4GalT7 activity, we have cloned and expressed the enzyme
and designed a focused library of 2-naphthyl b-D-xylopyranosides with modifications of the xylose
moiety. Based on enzymatic studies, that is galactosylation and its inhibition, conformational analysis and
molecular modeling using the crystal structure, we propose that the binding pocket of b4GalT7 is very
narrow, with a precise set of important hydrogen bonds. Xylose appears to be the optimal acceptor
substrate for galactosylation by b4GalT7. However, we show that modifications of the xylose moiety of
the b-D-xylopyranosides can render inhibitors of galactosylation. Such compounds will be valuable tools
for the exploration of GAG and PG biosynthesis and a starting point for development of anti-tumor agents.
Received 30th April 2014
Accepted 11th June 2014
DOI: 10.1039/c4sc01244e
www.rsc.org/chemicalscience
invasion, and metastasis.1–3 Both the structure of the PGs and
the GAG chains, and the expression of enzymes involved in their
Introduction
biosynthesis and degradation vary between normal cells and
tumor cells. For example, Sasisekharan and co-workers
successfully used GAG fragments, released from tumor cell
surfaces, to inhibit tumor growth and colonization in vivo.4 The
enzymes involved in the biosynthesis of GAG chains are thus
promising targets for drug development.
The biosynthesis of HS and CS/DS is initiated by xylosylation
of a serine residue of the core protein by xylosyltransferase
(XT).5 The xylosylated protein is then galactosylated in a step-
wise manner by two galactosyltransferases (b4GalT7, also
known as GalTI,6 and b3GalT6, also known as GalTII) and
subsequently glucuronated by glucuronyltransferase I (GlcAT-I)
to form a linker tetrasaccharide that is the branching point for
the biosynthesis of HS and CS/DS (Fig. 1a). During the poly-
merization of HS and CS/DS, posttranslational modications,
such as O- and N-sulfation, result in extensive structural diver-
sity of the chains.
b-1,4-Galactosyltransferase 7 (b4GalT7) is a key enzyme in the
biosynthesis of heparan sulfate (HS) and chondroitin sulfate/
dermatan sulfate (CS/DS) glycosaminoglycan (GAG) chains. The
GAG chains are linear polysaccharides of alternating uronic
acids and aminosugars, which are most oen linked to a core
protein to form proteoglycans (PGs). PGs interact with growth
factors and are important for a diverse set of biological
processes, such as cell growth and differentiation. In addition,
the PG/GAG expression is critical in the pathobiology of all
stages of cancer progression, such as proliferation, tumor
aCentre for Analysis and Synthesis, Centre 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 Experimental Medical Science, Lund University, BMC A13, SE-221 00
Lund, Sweden
cDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-
106 91 Stockholm, Sweden
The biosynthesis of GAG chains can also be initiated by
xylosides carrying hydrophobic aglycons, such as 2-naphthyl
b-D-xylopyranoside (XylNap, 1a, Fig. 1b and Chart 1).7,8 The
xylosides can, when exogenously supplied to cells, act as
† Electronic supplementary information (ESI) available: Experimental parts for
congurational and conformational analysis, biological testing, and synthesis.
See DOI: 10.1039/c4sc01244e
‡ These authors contributed equally.
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analogs with modications in positions 2, 3, and 4 of the cor-
responding benzyl b-D-xylopyranosides.22 Interestingly, the
authors also reported GAG priming by the 4-keto compound 6f
(Chart 1), at similar concentrations as XylOBn (1b), and by
benzyl 4-C-methyl-b-D-xylopyranoside (6a) at approximately 100-
fold higher concentrations of 6a compared to 1b.
In 2013, Qasba and co-workers published the crystal struc-
ture of a substrate-enzyme complex of human b4GalT7, as well
as that of the similar enzyme from Drosophila melanogaster.23,24
The two enzymes show a signicant overlap in the catalytic
pocket, and the Drosophila variety was used in co-crystallization
with UDP-galactose (UDP-Gal) and xylobiose (i.e., Xylb1-4Xyl).
To suppress decomposition of the UDP-Gal by the enzyme, the
authors used a mutated D211N-b4GalT7. They proposed that
the catalytic reactions are initiated by binding of UDP-Gal and a
manganese ion to b4GalT7. This binding initiates a conforma-
tional reorganization from an open form to a closed confor-
mation, with simultaneous formation of a xylose-binding
pocket. The xylose moiety is then perfectly positioned for
formation of the glycosidic bond and the authors proposed an
SN2-like mechanism, similar to the catalytic mechanism shown
by b4GalT1.25,26 Furthermore, aromatic residues at the entrance
of the catalytic pocket could explain the strong GAG priming
shown by some xylosides with aromatic aglycons.
We hypothesize that b4GalT7 does not tolerate any modi-
cations of the xylose moiety, and that it is possible to design
effective inhibitors of GAG biosynthesis by modifying xylose. To
pinpoint the requirements and limitations for priming and
inhibition of GAG chains we synthesized, and unequivocally
characterized, a collection of xyloside analogs (Chart 1). To rule
out any effects due to cellular uptake of these compounds, we
cloned and expressed b4GalT7, the enzyme that catalyzes the
second step of the formation of the linker tetrasaccharide, and
tested xyloside analogs in a cell-free assay.
Fig. 1 (a) Biosynthesis of the linker tetrasaccharide of HS and CS/DS
proteoglycans. (b) Galactosylation of XylNap to form GalXylNap.
acceptors in the rst galactosylation step by b4GalT7, and thus
provide a simplied model system for GAG biosynthesis, and
serve as a starting point for development of anti-tumor drugs.
Up until the cloning of b4GalT7, the exploration of the
biosynthesis of GAG chains has been limited to synthesis and
testing of xylosides and xyloside analogs in cell assays. These
studies have established the structural importance of the
aglycon for the amount of GAG priming as well as for the type of
chain primed (i.e., HS or CS/DS).9–18 Much less is known
regarding modications of the xylose moiety. In 2011, we
reported the synthesis of a series of modied 2-naphthyl b-D-
xylopyranosides, where each hydroxyl group was epimerized or
replaced by a hydrogen atom, a uorine atom, or a methoxy
group (Chart 1, compounds 2c–f, 3c–f, and 4c–f).19 The amount
of GAG priming induced on these compounds was investigated
and showed that treatment with XylNap initiated the expected
priming of GAG chains, whereas none of the tested analogs
showed priming of GAG chains. However, we observed a small
inhibitory effect on the endogenous glycosylation of PG for the
4-uoro and the 4-deoxy analogs (4d, 33%, 4e, 24%). These
results were in accordance with ndings from Kuberan and co-
workers, who presented GAG inhibitors based on 4-deoxy-4-
uoro-xylopyranosides with up to 90% inhibition.20,21 However,
some of our results were contradictory to earlier investigations
by Esko and co-workers who reported GAG priming by xyloside
Results
Synthesis
Benzylation of XylNap (1a) under phase transfer conditions
generated a separable mixture of dibenzylated 7, 8, and 9
(Scheme 1).19 Next, the methyl group was introduced using a
Dess–Martin oxidation–Grignard addition sequence, which
resulted in stereoisomeric mixtures, separable by column
chromatography. Finally, to avoid saturation of the naphthyl
moiety, debenzylation was performed using catalytic hydroge-
nation in HCl/DMF,27 which gave the expected methylated 2-
naphthyl b-D-xylosides 2a, 2b, 3a, 3b, 4a, and 4b. A similar
procedure, using isopropylidene protection, gave the corre-
sponding compound 4g, carrying an ethyl group in position 4.
Compound 5a was synthesized from per-O-acetylated L-idose
(18)28 in a standard aromatic glycosylation, while compound 5b
was synthesized from the commercially available 2-naphthyl
b-D-glucopyranoside (5c) by a selective tosylation of position 6
followed by reduction using LiAlH4 (Scheme 2, top). Compound
5d is commercially available.
In addition, we intended to synthesize 6a, previously
synthesized by Esko,22 due to its structural similarities with 4a,
Chart 1 Structures of xylosides and xyloside analogs.
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56% yield. Conformational analysis, vide infra, unequivocally
conrmed an equatorial orientation of the 4-C-methyl group. It
is reasonable to believe that the structure of the compound
synthesized and tested by Esko and co-workers was 6b rather
than 6a.
Congurational and conformational analysis
The stereochemical assignments of the endocyclic tertiary
carbon atoms are based on 1H,1H nuclear Overhauser effects
(NOEs), 3JC,H and 3JH,H coupling constants, 13C and 1H chemical
shis. With these at hand, the extent of the major conformation
of each compound was assessed. The 1H and 13C chemical shis
1
of the xylose residues were assigned by 1D H and 13C experi-
1
1
1
ments in conjunction with a few 2D H, H- and H, 13C-corre-
lated experiments (ESI†).
Axially oriented methyl groups are in close proximity to axial
hydrogen atoms on g-carbons as well as vicinal hydrogen
atoms, whereas equatorial methyl groups are proximate only to
Scheme 1 Synthesis of compounds modified in positions 2, 3, and 4.
Reagents and conditions: (i) BnBr, TBAI (cat.), KOH (10% aq), CH2Cl2,
r.t., 16 h, 7 (6%), 8 (66%), 9 (14%); (ii) (a) 15% Dess–Martin periodinane in
CH2Cl2, CH2Cl2, r.t., N2, 2 h; (b) 3 M MeMgBr in Et2O, r.t., N2, 1.5 h, 10
(57%), 11 (10%), 12 (19%), 13 (68%); (iii) (a) 15% Dess–Martin periodinane
in CH2Cl2, CH2Cl2, r.t., N2, 2 h; (b) 3 M MeMgBr in Et2O, 0 ꢀC to r.t., N2,
1.5 h, 14 (42%), 15 (42%); (iv) 10% Pd/C (14%), HCl (22 eq.), DMF, H2, r.t.,
3 h, 2a (67%), 2b (44%), 3a (76%), 3b (49%), 4a (70%), 4b (54%); (v)
2-methoxy propene, CSA (cat.), DMF, r.t., 1.5 h, (51%); (vi) (a) 2 M oxalyl
chloride in CH2Cl2, DMSO, CH2Cl2, À78 ꢀC, 2 h; (b) 3 M EtMgBr in Et2O,
THF, r.t., 12 h, (38%); (vii) 70% AcOH, 1 h, (56%).
4
vicinal hydrogen atoms (in the C1 conformation). Employing
1D 1H,1H-DPFGSE-NOESY experiments,30 selective excitation of
the methyl groups in compounds 2a, 2b, 3a, 3b, 4a, and 4b
resulted in the NMR spectra displayed in Fig. 2. The 2-(R)-
conguration (i.e., axial orientation of methyl group) of
compound 2a was assigned via a prominent NOE-correlation
from the methyl protons to H4 (Fig. 2). For compound 2b, NOE-
correlations were only observed to H1 and H3, which supports
an equatorially oriented methyl group and hence a 2-(S)-
conguration. The 3-(S)-conguration of compound 3a was
assigned via NOE-correlations from the methyl protons to H1
and H5pro-S, whereas these were essentially absent in
compound 3b. The stereochemical assignment at C4 of
compound 4a was partly obstructed due to overlap of the H2
and H3 resonances in the 1H NMR spectrum. However, a
stronger correlation between the methyl group and H5pro-R
than between the methyl group and H5pro-S was observed,
which indicates an axial orientation and consequently the 4-(R)-
Scheme
2 Synthesis of compounds modified in position 5 and
synthesis of compound 6b. Reagents and conditions: (i) BF3$OEt2 (2.5
eq.), Et3N (0.5 eq.), 2-naphthol (1.5 eq.), CH2Cl2, 0 ꢀC, 2 h, (78%); (ii) 1 M
NaOMe (0.1 eq.), MeOH, r.t., 2 h, (81%); (iii) TsCl (1.5 eq.), ZnBr2 (1.1 eq.),
pyridine, À25 ꢀC, 1 h, (86%); (iv) LiAlH4 (6 eq.), THF, 30 ꢀC, 22 h, (61%).
(v) 2-Methoxy propene (5.8 eq.), TFA (cat.), DMF, r.t., 6 h, (47%); (vi) (a) 2
M oxalyl chloride in CH2Cl2 (3 eq.), DMSO (6 eq.), CH2Cl2, À78 ꢀC, 2 h
(b) 3 M MeMgBr in Et2O (3 eq.), THF, r.t., 12 h, (55%); (vii) Dowex 50Wx4,
MeOH, r.t., 4 h, (56%).
following the original procedure (Scheme 2, bottom). Starting
from benzyl b-D-xylopyranoside (1b),29 selective protection of 2-
OH and 3-OH, using 2-methoxy propene and CSA in DMF, gave
21 in 47% yield with an additional 12% of the corresponding
3,4-protected analog. Next, the methyl group was introduced in
21, using a Swern oxidation–Grignard addition sequence. To
our surprise, compound 22 was the only isolated product.
Finally, deprotection of 22 with acidic ion exchange resin in
methanol, gave benzyl 4-methyl-b-D-arabinopyranoside (6b) in
Fig. 2 Selected regions of 1D 1H,1H-NOESY spectra of compounds
2a–b, 3a–b, and 4a–b (top to bottom) in methanol-d4 as solvent.
Selective excitation was carried out on the proton resonance from the
C-methyl group using a smix of 0.41 s in the experiment. An asterisk
marks residual CD3OH solvent peak.
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conguration. For compound 4b, NOEs of similar magnitudes 2a, by placing the methyl group in pseudo-equatorial orienta-
were observed for the signals of H3, H5pro-R, and H5pro-S, tion and the anomeric substituent in pseudo-axial orientation.
1
2
when irradiating the methyl resonance, which clearly indicates For compound 3a, the S5 and/or SO conformations would be
an equatorial orientation of the methyl group and thus the 4-(S)- feasible non-4C1 states. The 1C4 conformation would relieve
conguration. Selective excitation of the methyl group in steric strain in compounds 2a, 3a, and 4a by orienting the
compound 6b gave a nearly identical NOE result to that respective methyl groups equatorially, and benet from the
observed for compound 4b.
anomeric effect by placing the exo-anomeric oxygen in an axial
3
According to Karplus-type equations,31–33 the JCH coupling orientation. Estimates based on the available 3JHH data suggest
constants from the 13C nuclei of axially oriented methyl groups that up to about 20% of the conformational space for
to axial vicinal protons should be larger compared to those from compounds 2a and 3a are occupied by non-4C1 conformations,
3
equatorially oriented methyl groups. Selected JCH coupling whereas up to 10% is the suggested gure for compound 4a and
constants were measured via the 1DLR experiment,34,35 that <10% for compounds 2b, 3b, 4b, and 6b.
relies on selective excitation of 13C resonances followed by
Employing the above described methodology to additional
evolution of the heteronuclear coupling and detection on the 1H xyloside compounds resulted in >90% of the C1 conformation
nuclei. Heteronuclear coupling constants were determined by for 4f, solely the 4C1 conformation for 4g, an equilibrium
modied J doubling in the frequency domain.36 In all cases of between the 4C1, 2SO, and 1C4 conformations being ꢁ1/4, ꢁ1/4,
the pairwise comparisons for the diastereomeric pairs, 2a and and ꢁ1/2, respectively, for 5a where the 2SO conformation is
4
3
2b, 3a and 3b, as well as 4a and 4b, we observed larger JCH supported by a conspicuous NOE between the H5 and H2
coupling constants for 2a, 3a, and 4a, compared to 2b, 3b, and protons,39 and solely the 4C1 conformation for 5b.
4b. This is consistent with an antiperiplanar arrangement in the
To summarize, we have unequivocally determined the
3
former set of molecules. The observed JCH coupling constants congurations of each stereocenter in the synthesized target
for compound 6b were similar to those observed for compound compounds described above (Schemes 1 and 2). Furthermore,
4b, verifying the 4-(S)-conguration of 6b.22
we observed that the majority of our xyloside analogs prefer-
In all diastereomeric pairs, the 13C chemical shis of axial entially adopts the 4C1 conformation. Compounds bearing axial
methyl groups were shied upeld compared to those of methyl groups are exceptions, and for compound 5a, the 1C4
equatorial methyl groups,37 with inter-pair differences ranging conformation seems to be most populated.
from 2–6 ppm. The 1H chemical shis of axial protons in
g-position to the methyl groups were shied downeld when
the methyl groups were oriented equatorially, hydroxyl groups
b4GalT7 assay
thus being axial, in compounds 2b, 3b, and 4b. This is in A truncated version of b4GalT7 fused with glutathione S-trans-
accordance with what is seen for dH5 of hexapyranosides with ferase (GST) was expressed in E. coli, yielding 12 mg of puried
respect to a change in anomeric conguration.38 Typically, a (>95% by SDS PAGE) protein per liter of growth medium. As
downeld chemical shi displacement DdH of 0.2 ppm was previously reported,40 the truncated E. coli expression product of
observed upon a 1,3-syn-axial interaction with an oxygen atom. b4GalT7 fused to GST does not substantially affect substrate
4
Deviations from the C1 conformation can be observed and binding and catalytic properties of b4GalT7, compared to the
3
quantied to some extent by JHH coupling constant analysis. membrane-bound mammalian product. This enabled us to use
The compounds bearing axial methyl groups, 2a, 3a, and 4a, the bacterial product as a model for the mammalian enzyme.
3
have smaller JHH coupling constants than their respective
XylNap (1a) is known to be a primer of GAG chains, and is
thereby a substrate for b4GalT7 (Fig. 1b). The activity of b4GalT7
diastereoisomers. The maximum values of JH1,H2, ,
3JH2,H3
3
3JH3,H4, and JH4,H5pro-S, correspond to the 4C1 conformation; was thus determined using XylNap as acceptor substrate and
thus, the observed coupling differences arise due to population UDP-Gal as donor substrate. The reaction time and the amount
contributions of non-4C1 conformers for these compounds. This of enzyme were chosen so that the production of disaccharide,
is in agreement with the expected larger 1,3-diaxial steric that is GalXylNap, showed rst-order dependence of these two
repulsion of a methyl group compared to a hydroxyl group. The parameters. The reaction progress was analyzed by HPLC using
larger coupling constant deviations observed for compound 3a, uorescence detection. We thus observed a new peak with a
compared to all other compounds reect, with respect to steric shorter retention time than XylNap and with a mass spectra
repulsion, the signicance of the number of 1,3-diaxial corresponding to GalXylNap (Fig. 3a). The recombinant b4GalT7
hydrogen atoms in relation to the axial methyl substituent, two showed Michaelis–Menten kinetics for XylNap with a KM value of
3
for 3a and one for 2a and 4a.
0.78 mM (Fig. 3b). In accordance with the literature,41 the
Plausibly populated non-4C1 conformers are the skew enzyme showed excess-substrate inhibition from UDP-Gal, and
conformers 1S5 and 2SO, and the 1C4 conformation. The 2SO the KM value for UDP-Gal (0.35 mM) was comparable to previ-
conformation has previously been shown to be present in xylose ously reported data.40–42 The solubilities of the xylosides in
derivatives.19 In compound 4a, the methyl group would be DMSO limited the maximum concentration of substrate that
oriented pseudo-equatorially in the 2SO conformation. In could be added in the assay to approximately 2.5 mM.
2
compound 2a, however, the SO conformation would increase
To determine if our synthesized xyloside analogs could serve
steric interactions between the methyl group and H5pro-R. as substrates for b4GalT7 (i.e., if the analogs are galactosylated),
Rather, the 1S5 conformation seems benecial for compound each compound was added as the only acceptor substrate in the
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Fig. 4 Galactosylation and inhibition of galactosylation by b4GalT7.
White bars indicate amount of galactosylation (% of the effect shown
by XylNap). Black filled bars show inhibition of galactosylation of Xyl-
Nap (% of control without added xyloside analog; 0.5 mM of XylNap
and 2.0 mM of xyloside analog). The y-axis displays compound
number in bold face (the substituent in equatorial orientation, axial
orientation). Compound 2e is displayed by two white bars. This
compound is probably galactosylated at two different positions.
Fig. 3 Galactosylation and inhibition of galactosylation by b4GalT7. (a)
HPLC chromatograms for galactosylation experiments with XylNap.
The GalXylNap peak is increasing with increasing concentrations of
XylNap; 0.25 mM (dotted line), 1.0 mM (dashed line), and 2.0 mM (solid
line). (b) Michelis–Menten representation of the activity of b4GalT7 (V)
as a function of the XylNap concentration. (c) HPLC chromatograms
for inhibition experiments with 4f. The GalXylNap peak is decreasing
with increasing concentrations of 4f; 0 mM (dotted line), 0.5 mM
(dashed line), and 1.5 mM (solid line). (d) Relative inhibition of GalXyl-
Nap plotted versus the concentration of 4f. The areas of the GalXylNap
peaks, upon addition of 4f, are compared to the areas of the GalXylNap
peaks without inhibitor.
Effects on GAG priming in cells
As previously mentioned, the GAG priming abilities of XylNap
(1a), as well as of compounds 2c–f, 3c–f, and 4c–f have been
investigated earlier in HCC70 cells and CCD-1095Sk cells.19
Here, we investigated the GAG priming abilities of XylNap and
compounds 2a, 2b, 3a, 3b, 4a, 4b, 4g, 5a, 5b, and 6b using XT
decient pgsA-745 cells. Since these cells are incapable of
synthesizing complete PGs, they produce very small amounts of
endogenous GAG chains. This make them suitable for investi-
gations of xyloside priming. The cells were incubated with
the xyloside analogs in low sulfate medium supplemented with
[35S]sulfate. Aer 5 h of incubation, the GAG chains were iso-
lated from the culture media and the amount of radioactivity in
each sample was counted. Except for XylNap, no xyloside analog
induced signicant priming of GAG chains (data not shown).
To investigate if the inhibitory effects observed in the
b4GalT7 assay were transferable to cell culture studies, HCC70
cells were treated with either XylNap alone or with equimolar
amounts (0.1 mM) of compounds 4b or 4d. Aer 24 h of incu-
bation in low-sulfate medium, supplemented with [35S]sulfate
and xyloside analogs, the GAG chains were isolated from the
culture media. The samples were run on a Superose 6 column
and the radioactivity was measured (Fig. 5). The addition of
either 4b or 4d resulted in a decrease of GAG chains primed
from XylNap, corresponding to 50% and 25%, respectively. To
conclude, this indicates that the results from the enzymatic
investigations of b4GalT7 (that is galactosylation and its inhi-
bition) also can be observed in cellular systems.
b4GalT7 assay (Fig. 3c and d). As expected, most of these xylo-
side analogs failed to act as substrates for b4GalT7, with
exceptions for compounds 2a, 2d, 2e, and 5a that showed weak
galactosylation, compared to XylNap (Fig. 4, white bars). In each
case of galactosylation, the mass spectra for the corresponding
galactosylated products were determined, and were in all cases
found to be accurate.
Previous studies have shown GAG inhibitory effects of
specic xyloside analogs.19–21 To determine the inhibitory
activity of our compounds, XylNap was used as mimic of the
natural substrate (i.e., the xylosylated PG core protein). A xed
amount of XylNap was added, together with different concen-
trations of the xyloside analogs, and the amount of formed
GalXylNap was measured. A decrease in GalXylNap formation,
compared to the control, indicates that the compound is an
inhibitor of the enzyme (Fig. 3c and d). Interestingly, we
observed a weak decrease in GalXylNap formation in the pres-
ence of compounds 2c, 2d, 3d, 4e, and 4g, and signicant
reduction of GalXylNap formation for compounds 2e, 4b, 4d,
and 4f. The results are shown in Fig. 4 (black bars) and dis-
cussed below (detailed data in ESI†). To conclude, any modi-
cation of the xylose moiety rendered analogs unable to function
as efficient substrates for b4GalT7, and several compounds
showed effective inhibition of b4GalT7.
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Fig. 6 Proposed binding pattern of xylosides and UDP-Gal in the
binding pocket of b4GalT7.
Fig. 5 Superose 6-profiles for [35S]sulfate-labeled material isolated
from HCC70 cells after treatment with XylNap (solid line), XylNap and
4d (dashed line), and XylNap and 4b (dotted line).
order to understand these results, we performed molecular
modeling based on X-ray diffraction data of the Drosophila
¨
b4GalT7 mutant D211N (PDB ID 4M4K), using Schrodinger
2013 soware suite, with the OPLS-2005 force eld and GB/SA
solvation for water. The xylose moiety of XylNap adopts a very
similar orientation and binding mode as the xylobiose in the
crystal structure (Fig. 7a).
Discussion
By combining our structure-activity relationship studies with
the known crystal structure of b4GalT7, we can now provide a
detailed model for binding of xylosides and xyloside analogs to
b4GalT7.
From our conformational analysis vide supra, we conclude
that compound 4b is locked to a 4C1 conformation. Interestingly,
in this conformation, the axial 4-OH group is positioned in an
orientation suitable for nucleophilic attack with ample space for
the equatorial methyl group (Fig. 7b). However, in the axial
orientation it is not possible to form the important hydrogen
bond from 4-OH to Asp211, which results in diminished nucle-
ophilicity of O4 of xylose moiety (Fig. 6). Consequently, this
compound acts as an inhibitor of galactosylation rather than a
substrate for galactosylation. On the contrary, the axial methyl
group in compound 4a clearly eliminates the possibility for
nucleophilic substitution due to steric hindrance, and this
compound showed no galactosylation or inhibition (Fig. 7c).
As previously mentioned, Esko and co-workers reported a
study of substituted benzyl xylosides and the amount of GAG
priming on these compounds in pgsA-745 cells.22 As expected,
most modications of the 4-hydroxyl group, such as methyla-
tion, deoxygenation, or epimerization (Chart 1, compounds 6c–
e), rendered inactive compounds. On the contrary, methylation
of positions 2 or 3 gave analogs that initiated GAG priming,
although at higher concentrations compared to XylOBn (1b).
The 3-deoxy-3-uoro analog induced priming at approximately
10-fold higher concentrations compared to the parent
compound. Interestingly, the authors reported priming of GAG
chains by benzyl 4-C-methyl-b-D-xylopyranoside (6a) at approx-
imately 100-fold higher concentrations compared to XylOBn.
Our attempts to synthesize compound 6a, using the same
method as Esko and co-workers, resulted in the epimeric 6b, a
compound very difficult to distinguish from 6a. As mentioned,
when investigating the structurally similar compounds 4a and
4b, no signicant galactosylation could be observed. Instead,
compounds 4b and 6b acted as inhibitors of b4GalT7 and
compound 6b showed 43% inhibition (ESI†).
Most modications of position 2 in xylose, rendered analogs
less prone to galactosylation, compared to XylNap (1a).
However, we observed weak galactosylation of compound 2a,
carrying an axial methyl group, in contrast to 2b. The 2-uoro
analog 2d showed galactosylation and inhibition, whereas the 2-
methoxy analog 2c showed inhibition similar to 2d and very
weak galactosylation. This indicates that the hydroxyl group in
position 2 might acts as a hydrogen bond acceptor. Interest-
ingly, the 2-deoxy analog 2e showed formation of two different
compounds. Compound 2e lacks substituents in both C2 and
C5 of the xylose moiety, and thus shows pseudo-rotational
symmetry. It is reasonable that it ts the active site in the
enzyme in two different orientations and can be galactosylated
at both position 3 and 4. Compound 2e, also showed very effi-
cient inhibition of galactosylation (64%), possibly explained by
the exibility in binding orientation.
Modications of position 3 resulted in inactive compounds,
with the exception of the 3-deoxy-3-uoro analog 3d. The uoro
substituent is similar in size as a hydroxyl group and can
probably accept a hydrogen bond from Asp211, but with a
reversed hydrogen bond pattern (Fig. 6). From inspection of the
crystal structure,23 it is obvious that the binding pocket is
narrow and compound 3c, carrying a sterically more demanding
methoxy group, does not t, and is thus inactive. The impor-
tance of the 3-OH is further validated by the deoxy compound
3e, which showed no galactosylation or inhibition.
From the perspective of inhibitor design, our results indicate
that position 4 is the most interesting. The addition of an axial
methyl group, compound 4a, rendered an inactive compound
while the epimeric analog 4b, carrying an equatorial methyl
group and an axial hydroxyl group, showed strong inhibition. In
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we observed 50% inhibition of XylNap induced GAG synthesis
in HCC70 cells by compound 4b and 25% by compound 4d. As
previously mentioned, Kuberan and co-workers reported a
4-uoro analog with up to 90% inhibition in CHO cells.20,21
The methyl analog 5a showed very weak galactosylation and
no inhibition. Interestingly, this analog adopts the unusual 2SO
conformation. It is reasonable to assume that the addition of an
axial methyl group results in a compound that is tolerated by
the enzyme, due to a conformational change, but that this
analog is a less preferred substrate for b4GalT7. Any equatorial
groups in position 5, compounds 5b–d gave complete loss of
activity. These analogs are conformationally rigid and locked to
the 4C1 conformation, in which the group in position 5 induces
strong steric hindrance. From the crystal structure, it is
apparent that both O3 and O4 of the xylose moiety form
hydrogen bonds to the side chain of Asp211, whereas the
hydrophobic moiety of Tyr177 interacts with C5 of xylose, and
thus determines the specicity. Consequently, it was shown
that a mutation of Tyr177 to Gly changes the specicity from
xylose to glucose.23,24
Conclusions
We have synthesized a number of xyloside analogs for explo-
ration of the b4GalT7 active site. The identity and conformation
of these compounds have been determined by several NMR
techniques in combination with molecular modeling. The
abilities of these compounds to act as substrates or inhibitors of
b4GalT7 have been investigated in a cell-free assay and further
validated by cell studies.
We conclude that the binding pocket of b4GalT7 is very narrow
and decorated with a precise set of hydrogen bond acceptors.
From our data, we propose that xylose is the optimal substrate for
galactosylation. Every single hydrogen bond is necessary for effi-
cient galactosylation of xylosides, and any modication renders
less active or inactive compounds. Consequently, we anticipate
that it is difficult to synthesize better substrates by modication of
the xylose moiety. However, modications of the aglycon could
provide more efficient substrates.
In contrast to the limited options for design of GAG primers,
we open up for development of very tight binders that can
function as inhibitors of galactosylation, and presumably,
depending on cell permeability, act as strong inhibitors of the
GAG biosynthesis. Such compounds would be valuable tools for
the exploration of GAG and PG biosynthesis in normal cells and
tumor cells, and we speculate in that such inhibitors may be a
starting point for development of pharmaceuticals. The main
target for modications of the xylose moiety is position 4, where
a number of structural analogs show strong inhibition. Of
special interest is epimerization of the 4-OH group, which
results in potent inhibitors and consequently absence of
galactosylation.
Fig. 7 Molecular modeling of xylosides in b4GalT7. (a) O4 of the xylose
moiety in XylNap (1a) is oriented for nucleophilic substitution on C1 of
UDP-Gal. The xylose moiety adopts a very similar orientation and
binding mode as the original xylobiose in the crystal structure. (b) In
compound 4b, the axial 4-OH is situated in an orientation suitable for
nucleophilic substitution, similar to XylNap. However, the loss of one
hydrogen bond results in diminished nucleophilicity. (c) In 4a, the
possibility for nucleophilic substitution is eliminated due to steric
hindrance from the axial methyl group.
It is worth to notice that the galactosylation of XylNap is very
efficient and in our hands, using pgsA-745 cells, we observed
maximum GAG priming on XylNap at concentrations of 0.1
mM. In our initial studies, we did observe signicant priming
for compounds 2b, 4b, and 6b at 1 mM in pgsA-745 cells, very
similar to the results of Esko and co-workers. However, upon
close inspection using uorescence detection, we realized that
these samples were contaminated with trace amounts of Xyl-
Nap. This was enough to give false dose response curves in our
studies. Aer careful HPLC purication, we observed no
signicant GAG priming from these compounds (see ESI† for an
elaborate discussion).
The absence of an equatorial methyl group, as in compound
4f, resulted in an even stronger inhibition (52%) whereas the
addition of a sterically demanding ethyl group resulted in a
diminished inhibitory effect (4g, 13%), compared to 4b (41%).
We propose that xyloside analogs with axial hydroxyl groups in
position 4 can act as strong inhibitors of galactosylation and
that the corresponding equatorial groups induce steric
hindrance and diminished inhibition.
As expected, in the absence of a hydroxyl group, the deoxy
compound 4e and the 4-uoro analog 4d did not show any
galactosylation. However, 4d showed strong inhibition of gal-
actosylation. These results were transferable to cell studies and
Acknowledgements
¨
This work was supported by grants from the Alfred Osterlund
Foundation, the Crafoord Foundation, the Evy and Gunnar
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3508 | Chem. Sci., 2014, 5, 3501–3508
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