Ruthenium(II)- and copper(I)-catalyzed synthesis of click-xylosides and assessment of their glycosaminoglycan priming activity

Article abstract of DOI:10.1016/j.bmcl.2017.10.004

Xylosides are small molecules that serve as primers of glycosaminoglycan biosynthesis. Xyloside mediated modulation of biological functions depends on the extent of priming activity and fine structures of primed GAG chains. In earlier studies, copper (Cu)

Full text of DOI:10.1016/j.bmcl.2017.10.004

Accepted Manuscript
Ruthenium(II)- and copper(I)- catalyzed synthesis of click-xylosides and as-
sessment of their glycosaminoglycan priming activity
Caitlin P. Mencio, Dinesh R. Garud, Yosuke Doi, Yiling Bi, Hariprasad
Vankayalapati, Mamoru Koketsu, Balagurunathan Kuberan
PII:
S0960-894X(17)30978-2
https://doi.org/10.1016/j.bmcl.2017.10.004
BMCL 25333
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
31 August 2017
30 September 2017
1 October 2017
Please cite this article as: Mencio, C.P., Garud, D.R., Doi, Y., Bi, Y., Vankayalapati, H., Koketsu, M., Kuberan, B.,
Ruthenium(II)- and copper(I)- catalyzed synthesis of click-xylosides and assessment of their glycosaminoglycan
priming activity, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.
2017.10.004
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Bioorganic & Medicinal Chemistry Letters
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Ruthenium(II)- and copper(I)- catalyzed synthesis of click-xylosides and
assessment of their glycosaminoglycan priming activity
Caitlin P Mencioa,b, Dinesh R Garudc,1, Yosuke Doid, Yiling Bib, Hariprasad Vankayalapatie, Mamoru
Koketsud,f,* and Balagurunathan Kuberana,b,g,∗
aInterdepartmental program in Neuroscience, University of Utah, Salt Lake City, Utah 84112 USA
bDepartment of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112 USA
c Department of Chemistry, Sir Parashurambhau College, Tilak road, Pune 411030 India
d Department of Materials Science and Technology, Faculty of Engineering, Gifu University, Gifu 501-1193, Japan
e Division of Oncology of School of Medicine and Center for Investigational Therapeutics (CIT) at Huntsman Cancer Institute, University of Utah, 2000 Circle of
Hope, Salt Lake City, Utah 84112 USA
f Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, Gifu 501-1193 Japan
g Department of Biology, University of Utah, Salt Lake City, Utah 84112 USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Xylosides are small molecules that serve as primers of glycosaminoglycan biosynthesis.
Xyloside mediated modulation of biological functions depends on the extent of priming activity
and fine structures of primed GAG chains. In earlier studies, copper (Cu) catalyzed synthesis of
click-xylosides and their priming activity were extensively documented. In the current study,
ruthenium (Ru) mediated catalysis was employed to synthesize xylosides with a 1,5-linkage
between the xylose and the triazole ring instead of a 1,4-linkage as found in Cu-catalyzed click-
xyloside synthesis. Mono- and bis- click-xylosides were synthesized using each catalytic method
and their glycosaminoglycan priming activity was assessed in vitro using a cellular system. Ru-
catalyzed click-xylosides showed a higher priming activity as measured by incorporation of
radioactive sulfate into primed glycosaminoglycan chains. This study demonstrates that altering
the linkage of the aglycone to the triazole ring changes the priming activity. Computational
modeling provides a molecular rationale for higher priming ability of Ru-mediated click-
xylosides. Higher GAG priming activity is attributed to the formation of more stable interactions
between the 1,5-linked xylosides and β-1,4-galactosyltransferase 7 (β4GalT7).
Revised
Accepted
Available online
Keywords:
Glycosaminoglycan;
Click Chemistry;
Xyloside
2017 Elsevier Ltd. All rights reserved
.
———
* Corresponding authors - e-mail: Prof. Mamoru Koketsu (koketsu@gifu-u.ac.jp); B. Kuberan (kuby.balagurunathan@utah.edu)
1 Affiliated to Savitribai Phule Pune University, Pune 411007 India (formerly University of Pune).

xylosides initiated GAG production but there were distinct
differences in priming ability between 1,4- and 1,5-xylosides
(Fig. 1). 1,5-Xylosides 4a-4g, synthesized using Ru-click
chemistry, showed higher priming activity than the
corresponding 1,4-xylosides 3a-3g, synthesized using Cu-click
chemistry. Also in comparing the dimeric xylosides, it should be
noted that the length of the linker between two xylose residues
dramatically affects the priming activity.
Proteoglycans (PGs) consist of a core protein and one or more
glycosaminoglycan (GAG) side chains. Biosynthesis of PG
occurs in the golgi and requires a number of enzymatic
modifications. Xylosylation of certain serine residues is the first
and essential step in the formation of GAG chains such as
heparan sulfate and chondroitin sulfate onto PGs.1,2 Previous
studies have found that xylosides can induce the formation of
GAG chains without the presence of a core protein.3-9 Several
monomeric and dimeric xylosides have been investigated in
earlier studies.9 Multimeric xylosides, which contain two or more
xylose residues attached to the aglycone, prime multiple GAG
chains on the same scaffold, closely mimic endogenous PGs.
Endogenous PGs often have two or more GAG chains attached to
the core protein and it is predicted that many of the biological
interactions between PGs and other proteins or factors are due to
the multivalent GAG chains. The nature of aglycone attached to
the xylose can affect not only the priming activity but also the
type, sulfation pattern and molecular weight of the primed
GAGs.4-6 Click-xylosides, a promising class of xylosides, were
primarily synthesized using copper [Cu(I)] catalyzed click10
reactions. The triazole linkage affords these molecules metabolic
stability in culture and in vivo.7-9 Earlier studies have examined
how these click-xylosides induce GAG formation and how
alteration of the aglycone can change the fine structures of
primed GAG chains.7-9
HO
HO
O
OH
AcO
AcO
AcO
O
N3
HO
1
2
i) cat. CuSO4,
i) 5 mol% Cp*RuCl(PPh3)2
THF, reflux, 1 to 2 days
ii) NaOMe, DCM:MeOH
(1:1), rt, 3 h
cat. Sodium Ascorbate,
DMF:H2O (3:1), r.t.,2 days
ii) NaOMe, DCM:MeOH
(1:1), rt, 3 h
N
O
N
N
HO
HO
N
N
HO
HO
O
N
HO
HO
( )n
HO
HO
O
N
( )n
HO
N
N
HO
HO
O
N
In this report, Ru-mediated11 1,5-linked click-xylosides were
prepared for the first time and their priming activity was analyzed
in comparison to corresponding 1,4-linked click-xylosides
prepared by Cu-mediated catalysis.12-14 Specifically, this work
investigates the impact of xyloside linkage size and orientation as
it relates to GAG priming activity.
HO
N
N
3a (n = 1, 61%)
3b (n = 2, 75%)
3c (n = 3, 71%)
4a (n = 1, 46%)
4b (n = 2, 49%)
4c (n = 3, 47%)
HO
HO
O
N
N
N
O
N
N
O
HO
HO
N
HO
D-Xylose served as a starting material to prepare β-D-
xylopyranosyl azide 1 in two consecutive steps.7-9 The treatment
of xylopyranosylazide 1 with 1,6-heptadiyne in the presence of
cat. CuSO4 and sodium ascorbate in DMF: H2O (3:1) followed by
deprotection of acetate groups using sodium methoxide readily
afforded dimeric xyloside 3a in overall 61% yield (Scheme 1).
HO
R
O
R
3d (R = H, 68%)
3e (R = F, 79%)
3f (R = NO2, 58%)
4d (R = H, 74%)
4e (R = F, 74%)
4f (R = NO2, 59%)
1
The structure of the compound 3a was confirmed by H-, 13C-
NMR and mass spectrometry. Under the similar Cu-catalyzed
reactions conditions, 1,7-octadiyne and 1,8-nonadiyene were
reacted with 1 to prepare corresponding dimeric xylosides 3b and
3c in 75% and 71% yields, respectively (Scheme 1). Next, the
monomeric xylosides 3d-3g were synthesized under similar
reaction conditions in good yields (Scheme 1) for the
comparative GAG priming activity studies.
HO
HO
O
N
N
N
N
N
HO
HO
O
N
HO
HO
O
O
3g (57%)
4g (80%)
Next, efforts were directed toward preparing 1,5- linked
xylosides. In this case, the reaction of xylopyranosylazide 1 with
1,6-heptadiyne in the presence of 5 mol% of (Cp*RuCl(PPh3)2 in
THF at reflux followed by sodium methoxide mediated
deprotection of acetate groups readily afforded the dimeric
xyloside 4a in overall 46% yield (Scheme 1). The structure of the
compound 4a was confirmed by the spectral data. Similarly, the
Ru-catalyzed reactions of 1 with a number of alkynes followed
by deprotection of acetate groups afforded corresponding 1,5-bis-
and mono- xylosides 4b-4g in good yields (Scheme 1).
Scheme 1. Synthesis of xylosides via Cu- or Ru-click chemistry.
There appears to be a negative correlation between the length
of the linker and the priming activity of the bis-xyloside. In both
Cu-click and Ru-click bis-xylosides, the compound with the three
and four carbon linker (3a, 4a, 3b, 4b) show similar priming
activity whereas bis-xylosides (3c,4c) containing the five carbon
linker, -[CH2]5- have lower priming activity. The length of the
carbon chain linker in dimeric xylosides influences priming in
both the 1,4- bis-xylosides and 1,5- bis-xylosides. However, even
in the situations of lower priming ability, the data clearly shows
that 1,5-linked xylosides have higher level of GAG priming
activity as measured by radioactive sulfate incorporation.
Four sets of monomeric (3d-3g, 4d-4g) and three sets of
dimeric xylosides (3a-3c, 4a-4c) were then assessed for their
GAG priming activity in vitro using pgsA-745 CHO cells6, which
are unable to synthesize GAG chains as they lack
xylosyltransferase, at
a concentration of 100 µM, using
previously described methods.7,8 As determined by 35S-sulfate
incorporation into primed GAG chains, both Cu- and Ru- click-

After assessing the priming activity of the xylosides, the
primed GAGs were analyzed for their chain length, sulfation
density and disaccharide composition. We found that all
xylosides primed primarily highly sulfated GAG chains. These
primed GAGs elute at similar retention times in weak-anion
exchange DEAE HPLC (S1.1), regardless of 1,5- and 1,4- linked
xyloside primers. It is interesting to note that all dimeric
xylosides produced a narrow peak within the HPLC profile while
the monomeric xylosides produced a much broader peak. After
determining sulfation density, the size of the primed GAGs was
assessed. The size differences of primed GAGs between 1,4- and
1,5- linked xylosides were found to be less significant.
residues of β4GalT7 enzyme. These docking experiments suggest
that higher priming activity of 1,5-xylosides is attributed to the
mode of binding of the phenoxymethyl moiety, its rigidity at
position 5, and stable π-stacking interactions with Tyr194,
Tyr196, Tyr199, and Trp224 residues. Our modeling studies and
docking results are consistent with earlier mutagenesis studies in
which Tyr196 was found to directly interact with aromatic
aglycone moiety,17 and that a highly hydrophobic environment,
consisting of Tyr194, Tyr196, and Tyr199, was critical for
binding of the xyloside substrates.18
The noticeable difference in priming activity indicates that
triazole linkage modulates GAG biosynthesis. In comparison to
the 1,4- linkage, the1,5- linkage may facilitate xyloside entry into
the cell and golgi. Additionally, the length of the carbon linker in
bis-xyloside substrates affects the priming capability of both 1,4-
and 1,5-linked xylosides, indicating that for dimeric xylosides
both the linkage and the linker length are important for priming
activity. Computational modeling suggests that the ability of the
Ru-click xylosides to form more stable interactions with β4GalT7
enzyme involved in GAG biosynthesis may provide insights into
the determination of priming capabilities of xylosides with
distinct aglycones. Further explorations of how modifications to
the triazole alter priming activity are necessary to fully
understand how the linkage between xylose and aglycone may
alter the priming activity.
Figure 1. Comparison of the incorporation of radioactive sulfate in GAG chains
primed by Cu-xylosides (3a-g) with Ru-xylosides (4a-g) as measured by 35S
scintillation count from three independent experiments (n=3, mean SD,**<0.01,
*** <0.001, ****<0.0001) .
Finally, the disaccharide compositional analysis of the primed
GAGs was undertaken. All xylosides produced primarily
chondroitin sulfate (CS) type GAG chains. After digestion of CS
chains with chondroitinase ABC, the disaccharide analyses
showed that the major disaccharide was found to be a
monosulfated disaccharide (S1.2). These results showed that
while the priming activity was dependent on the 1,5- versus 1,4-
linkage of the xylose to the triazole ring, the structural
characteristics of the primed GAGs did not change significantly
in terms of disaccharide composition and chain length.
To further understand the differences observed in the priming
ability of these synthetic click-xylosides, we conducted
computational docking experiments using a molecular modeling
software, Sybyl-X 2.1.15 We investigated the potential binding
position and orientation of low energy poses of all the
monomeric xylosides (3d-3g, 4d-4g) and their interaction with β-
1,4-galactosyltransferase 7 (β4GalT7), an enzyme involved in
GAG biosynthesis. The binding of 1,4-xylosides (3d-3g) and 1,5-
xylosides (4d-4g) showed distinct binding modes within the
β4GalT7 pocket (PDB ID: 4IRQ16). The binding mode of the
phenoxymethyl moiety of 1,4-linked click-xylosides is much
more non-predictive due to less sterically spaced aryl aglycone
substitutions, causing two different modes whereas the
substituted phenoxymethyl moiety of 1,5-xylosides exhibits
predictive single binding mode due to rigidity at the 5th position.
The key features of 1,5- xylosides responsible for higher priming
is attributed to the π-stacking interaction either through edge-face
and sandwich stacking interactions (Tyr194, Tyr196, Tyr199,
Trp224) as shown in Figure 2a. Therefore, by shifting the
aglycone from 1,4- (Figure 2b) to 1,5- linkage, the aromatic ring
was positioned to interact directly with aromatic amino acid
Figure 2. Docking results of a) 1,5-xylosides (ligand 4e shown in green stick)
and b) 1,4-xylosides (ligand 3e shown in orange stick) on β4GalT7 binding
pocket. While aglycone moiety adopts flexible orientation in 1,4-xylosides, the
aglycone aromatic rings in 1,5-xylosides form consistent π-stacking interactions
with Y194, Y196 and Y199.
Additionally, the ability to computationally predict the
interaction between varying xyloside structures and GAG
biosynthetic enzymes, especially those involved in the initiation
of GAG biosynthesis, may lead to possible target molecules for
more precise modulation of primed GAG chains. Examination of

different aglycone structures for Ru-click dimeric xylosides may
also shed light on the prominence of aglycone in the regulation of
GAG priming and may lead to the discovery of more potent
xylosides. Though Cu-click chemistry has been an efficient route
for synthesis of many xylosides, computational modeling shows
that Cu-click chemistry may not provide the most desirable
interaction between the enzyme and xyloside substrates.
Coupled with the evidence of increased priming activity of the
Ru-click xylosides as compared to their Cu-click counterparts, it
becomes clear that other forms of chemistry should be explored
to discover more potent xylosides carrying distinct aglycones to
fine tune GAG priming activity and the resulting GAG fine
structures.
Acknowledgments
This work was supported by the NHLBI sponsored Programs
of Excellence in Glycosciences Grant HL107152 (to BK) and by
the Japan Ministry of Education grant 20590005 (to MK).
Supplementary Material
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/
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Graphical
Abstract
Ruthenium(II)- and copper(I)-
catalyzed synthesis of click-xylosides
and assessment of their
Leave this area blank for abstract info.
glycosaminoglycan priming activity
Caitlin P Mencioa,b,# , Dinesh R Garudc, ,#, Yosuke Doid, Yiling Bib, Hariprasad Vankayalapatie, Mamoru Koketsud,f,* and
Balagurunathan Kuberana,b,g,∗
3 (1,4-linked xylose)
4 (1,5-linked xy
Golgi