Glycoclusters as lectin inhibitors: Comparative analysis on two plant agglutinins with different folding as a step towards rules for selectivity

Article abstract of DOI:10.1016/j.tet.2015.07.020

The emerging physiological significance of carbohydrate (glycan)-protein (lectin) recognition engenders the interest to design synthetic inhibitors with a high level of selectivity among natural sugar receptors. Plant agglutinins are common models to dete

Full text of DOI:10.1016/j.tet.2015.07.020

Tetrahedron xxx (2015) 1e14
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Glycoclusters as lectin inhibitors: comparative analysis on two plant
agglutinins with different folding as a step towards rules for
selectivity
,
b
a
b,
ꢀ a
*
*
Sabine Andre , Shane O’Sullivan , Hans-Joachim Gabius , Paul V. Murphy
a
€
Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Veterinarstr. 13, 80539 Munich, Germany
b School of Chemistry, National University of Ireland Galway, University Road, Galway, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
The emerging physiological significance of carbohydrate (glycan)eprotein (lectin) recognition engenders
the interest to design synthetic inhibitors with a high level of selectivity among natural sugar receptors.
Plant agglutinins are common models to determine structureeactivity relationships. Focussing on the
contribution of valency towards selectivity, copper-catalysed azide (sugar derivative)ealkyne (scaffold)
cycloaddition yielded a panel of 10 bi- to tetravalent glycoclusters with N-acetylglucosamine as the
bioactive headgroup. They were introduced into assays using (neo)glycoproteins and cell surfaces as
platforms to study carbohydrate-dependent lectin binding. The ability of the bivalent compounds, which
Received 25 April 2015
Received in revised form 19 June 2015
Accepted 6 July 2015
Available online xxx
Keywords:
Glycoprotein
Lectin
Modelling
N-Acetylglucosamine
Valency
ꢁ
exhibit a distance profile of the sugar headgroups of about 16e21 A, for intramolecular bridging of two
contact sites from the eight hevein domains of wheat germ agglutinin led to comparatively high en-
ꢁ
hancements of inhibitory potency relative to a tetrameric leguminous lectin (distance profile of 50e70 A
between sugar-specific sites), especially for a b-S-glycoside. The extent of inhibition at fixed concen-
trations of the sugar depended on the type of matrix used for the assay. Increases to tri- and tetravalency
played a less important role than the anomeric position to keep cross-reactivity low, these tested to-
pologies enabling cross-linking for both lectins. The potential for cis-interactions (intramolecular in-
teractions), with glycoclusters serving as molecular rulers, is suggested to help designing selective
blocking reagents.
Ó 2015 Published by Elsevier Ltd.
1. Introduction
constituent of such a network. Towards this end, glycoclusters
(glycosylated scaffolds with at least bivalency) with galactosides as
Synthetic chemistry substantially contributes to the progress in
unravelling structureeactivity relationships of carbohydrate
(glycan)eprotein (lectin) interactions, also referred to as cracking
the sugar code.1 A major question to be answered concerns the
origin of the apparent specificity and selectivity of carbohydrate-
binding proteins (lectins) for distinct cellular glycans presented
by specific glycoconjugates (counterreceptors), in structural and
topological terms. Growing insights into the natural occurrence of
diverse sugar receptors is facilitating to classify them into groups,
either formed by homologous family members or by non-related
proteins sharing at least monosaccharide specificity, for example,
headgroups have proven their applicability in the test case of
galectins, revealing routes towards selectivity among the three
types of structural design of these lectins and natural/engineered
variants (for examples, please see Ref. 3). Such data strengthen the
assumption that these synthetic products can be potent sensors of
the topological and/or spatial aspects of lectin binding and that
their design can achieve differential reactivity to various types of
receptors for compounds presenting the same carbohydrate epi-
tope. Herein, we test the concept on a further letter of the sugar
alphabet and two folding patterns of lectins.
N-Acetylglucosamine (GlcNAc), like galactose, can be spatially
accessible when positioned at branch termini of N- and mucin-
type O-glycans as well as on glycolipids. Physiologically, N-gly-
b
-galactoside-specific receptors defined as C-type lectins or galec-
tins.1f,2 Due to their functional cooperation in vivo, versatile tools
are needed to comparatively dissect recognition properties of each
cans with terminal b-GlcNAc are abundant in basophils and eo-
sinophils, and an increase in such GlcNAc-terminated structures in
several types of carcinoma intimates a value as tumour marker,
with potential functional significance.4 In fact,
a panel of
ꢀ
* Corresponding authors. E-mail addresses: sabine.andre@lmu.de (S. Andre),
paul.v.murphy@nuigalway.ie (P.V. Murphy).
http://dx.doi.org/10.1016/j.tet.2015.07.020
0040-4020/Ó 2015 Published by Elsevier Ltd.
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2
endogenous lectins is known with specificity for GlcNAc, among
them soluble host defence effectors (collectins, ficolins), trans-
membrane pattern recognition receptors such as the tandem-
repeat-type macrophage mannose receptor, the trimeric lan-
gerin, chitinase 3-like-1/2 (YKL-40/39) and also the trefoil factor
2.5 Truncated N-glycan structures are also presented by viral gly-
coproteins, turning them into docking sites for ensuing virus entry.
With this route defined, the liver/lymph node sinusoidal endo-
thelial cell C-type lectin (LSECtin, CLEC4G) with its micromolar
As proof-of-principle for a sensor activity of topological aspects,
we added a structurally different (tetrameric) leguminous lectin to
our test panel, i.e., Griffonia (Bandeiraea) simplicifolia agglutinin-II
(GSA-II). It prefers the a-anomeric linkage of GlcNAc to its b-iso-
meric form.8b,9 The common distance profile between sugar-
ꢁ
binding sites in leguminous lectins of 50e70 A makes intra-
molecular bridging impossible for the glycoclusters presented
herein. Integrating a specificity control with scaffold-presented N-
acetylgalactosamine
(GalNAc)
to
exclude
carbohydrate-
affinity to the GlcNAc
b
1,2Man disaccharide becomes a prominent
independent interactions to the linker and building on previous
experience with WGA and two bivalent compounds,7g we prepared
a panel of 10 bi- to tetravalent glycoclusters bearing GlcNAc (Fig. 1).
To ensure comparability, the nature of the linker was strictly kept
constant and only one parameter was altered. Structural variations
were introduced by changing anomeric configuration and valency,
and using either an O- or S-glycosidic linkage. The products were
assayed under identical conditions (e.g., aliquots of the same lectin
solution or cell suspensions per experimental series) to determine
their capacity to block binding of these two structurally dissimilar
model lectins to surface-immobilised (neo)glycoproteins and to
cells. The obtained data in the inhibition studies reveal glyco-
cluster- and lectin-type-dependent results, underscoring the suit-
ability to use glycoclusters as molecular rulers and as source of
selectivity.
potential target for therapeutic lectin blocking, as the chitin-
binding protein GbpA of Vibrio cholerae is, which acts as adher-
ence factor to intestinal mucins.6 These examples underscore the
emerging interest in developing bioactive and selective GlcNAc-
presenting glycoclusters. In this respect, previous research has
proven the particular usefulness of the plant lectin wheat germ
agglutinin (WGA) as a model.7 This sugar receptor with a total of
ꢁ
eight hevein domains (about 14 A apart) binds terminal GlcNAc
irrespective of its anomeric linkage and also interacts with the
innermost core GlcNAc moiety of complex-type N-glycans.8 In-
triguingly, bivalent (dumb-bell-like) compounds can be accom-
modated by two sites of the same protein, if linker length
(somehow) matches distance between contact sites, or cross-link
two proteins, in both cases saturating GlcNAc-binding sites.7
Fig. 1. Structure of glycoclusters 1e11.
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3
2. Results and discussion
2.2. Distance profiles of sugar headgroups by modelling
2.1. Synthesis of the test panel
Extending our previous study on GalNAc-presenting clusters, in
which we estimated average distances between the carbohydrate
ꢁ
The spacered precursors were selected to ensure identical linker
length and flexibility. Thus, thioglycoside 13 was obtained in two
steps from the thiol 12, prepared by a previously reported pro-
cedure (Scheme 1).10 In detail, the thiol 12 was first reacted with
1,2-dibromoethane in the presence of potassium carbonate in
acetone-water, and the resulting monobrominated product then
converted to the azide 13 in the presence of sodium azide and tetra-
N-butyl ammonium iodide in DMF. Next, the corresponding O-
glycosidic analogue 16 was generated. Glycosidation of 1411 using
2-chloroethanol in 1,2-dichloroethane with sulfuric acid on silica as
the promoter gave intermediate 15. Reaction of 15 with sodium
azide as for the preparation of 13 yielded 16. The preparation of 17
headgroups of 21e22 A (for bi- and trivalent compounds with the
ꢁ
ꢁ
ꢁ
aromatic core) and 14 A/27 A/30 A (latter across the diagonal) for
the tetravalent compound 7,7g the respective parameter was herein
calculated for both bi- and trivalent 9 and 10. The Maestro interface
(www.schrodinger.com) was used to build a conformer for 9,
ꢁ
implementing a distance constraint of 8.8 A from the anomeric
carbon of GlcNAc to the CH2 group bonded directly to the central
nitrogen atom; this constraint was chosen as it matches the cor-
responding distance in glycoclusters 1e8 and 11 (Fig. 2). Next, en-
ergy minimisations were carried out in Macromodel (OPLSAA force
field, gas phase; www.schrodinger.com). Under these conditions,
the two or three anomeric carbon atoms are separated by about
and 18 was carried out as previously described.6g Herewith,
and -S-derivatives were made available for the cluster design and
b-O/S-
16 A, in 10 adopting an equilateral triangular arrangement (Figs. 2
ꢁ
a
and 3). Owing to conformational flexibility this distance can be
expected to be a dynamic average. As with the other bi- to trivalent
glycoclusters, compounds 9 and 10 can apparently present their
synthesis, 18 with GalNAc that will later provide a glycocluster
serving as a specificity control.
Scheme 1.
The routine application of the copper-catalysed azideealkyne
cycloaddition (CuAAC) using pairs of azides (Scheme 1) and alkyne
derivatives (Scheme 2), followed by deacetylation, resulted in the
panel of the glycoclusters (Fig. 1). As listed, the propargyl de-
rivatives obtained from hydroquinone, resorcinol, trihydroxy-
benzene, tetraphenylethene and ammonia (19e23)12 were reacted
with the appropriate azide (12, 13, 15 or 18) in the presence of Cu(II)
SO4$5H2O and sodium ascorbate, in 1:1 THF/H2O, to lead to the
acetylated intermediates. The alkyne derivatives were generally
used as limiting reagents. In the case of the reaction of tripro-
pargylamine, the mono-, bi- and trivalent intermediates (24e26)
were all obtained after chromatography. Each compound, which
still had the sugar residue protected, was separated from the re-
agents by chromatography, and then the sugar residues de-O-
acetylated using a catalytic amount of a freshly prepared 1 M so-
dium methoxide solution in methanol. Bi- to tetravalent products
1e11 (Fig. 1) were obtained by this approach and purified in all
cases using reverse-phase column chromatography.
sugar headgroups in a special way to bridge adjacent binding sites
in hevein domains of WGA. In contrast, with binding sites in le-
guminous lectins about 50e70 A apart, such intramolecular
ꢁ
bridging will not occur for GSA-II.
In order to determine lectin reactivity of the glycoclusters we
performed inhibition assays, using incubation of a mixture of
a synthetic compound (or free sugar) with labelled lectin with
a (neo)glycoprotein matrix or with cells, and measuring the extent
of binding to glycan ligands.
2.3. Glycoclusters as inhibitors of GSA-II binding to a neo-
glycoprotein and cells
The leguminous lectin GSA-II is specific for non-reducing-end
GlcNAc residues. In the solid-phase assay, the labelled lectin did
not bind to complex-type N-glycans of asialofetuin (ASF). Removal
of the
treatment with
b
1,3/4-linked galactose moieties from these N-glycans by
-galactosidase led to a signal in binding assays.
b
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4
S. Andre et al. / Tetrahedron xxx (2015) 1e14
Scheme 2.
However, in relation, a neoglycoprotein presenting GlcNAc as a p-
isothiocyanato derivative on bovine serum albumin as carrier was
most reactive. Thus, this latter product was adsorbed to the plastic
surface of microtiter plate wells, and lectin binding was ascertained
to be saturable. It was dependent on the nature of the
monosaccharide and of the matrix, GalNAc-containing neo-
glycoprotein serving as specificity control, and inhibitable by cog-
nate sugar. The presence of the sugar diminished binding of GSA-II
to the neoglycoprotein to background level. Optimal sensitivity to
the presence of an inhibitor was achieved by using a lectin
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5
Fig. 2. Space filling models of 9 (two views).
Fig. 3. Schematic illustration of inter-residue distances in compounds 9 and 10 based on molecular modelling using Macromodel.
concentration, which just yielded a plateau level of optical density.
were seen with the three bivalent glycoclusters with the
a-S-gly-
Titrations with increasing quantities of free GlcNAc and GlcNAc
conjugated to various scaffolds (thus all concentrations are always
expressed as concentration of sugar) enabled to calculate the sugar
concentrations, at which the signal intensity of lectin binding was
reduced by 50% (IC50-value).
cosidic linkage (compounds 5, 6 and 9), with further increases in
valency leading to further stepwise improvements (Table 1). The
tetravalent 7 reached an approximately 200-fold enhancement of
relative inhibitory capacity. Considering the distance profiles in the
glycoclusters and in GSA-II, each glycocluster can be expected to
bind to a lectin molecule only via a single sugar, then with the
possibility for functional bi- to tetravalency, which will lead to
lectin cross-linking. As documented in detail for binding to up to
nine chains of the three complex-type N-glycans in ASF by galec-
tins, successive binding steps to the nonavalent ligand apparently
Reflecting the known preference for the a-anomer, the four b-
GlcNAc-containing divalent glycoclusters 1e4 were relatively weak
inhibitors of the interaction at the surface; this was irrespective of
the nature of the atom (S or O) at the anomeric centre (Table 1).
Respective 5e8-fold enhancements compared to compounds 1e4
Table 1
IC50-values of the di-to tetravalent compoundsa and the free monosaccharide in binding assays with biotinylated lectins and surface-immobilised (neo)glycoproteins (given in
m
M for the concentration of sugar)
Test compound
GSA-II (15
m
g/mL)/GlcNAc-BSA (100 ng)
WGA (20 ng/mL)/GlcNAc-BSA (20 ng)
WGA (20 mg/mL)/ASF (500 ng)
1
2
3
4
5
6
7
8
145 (4.1)
130 (4.6)
140 (4.3)
115 (5.2)
24 (25)
17 (35)
3 (200)
11 (55)
28 (21)
22 (27)
n.i.c
11 (2273)
8.5 (2941)
19 (1316)
15 (1667)
26 (962)
15 (1667)
2.8 (8929)
22 (1136)
46 (543)
39 (641)
n.i.c
1.6 (1000)
1.0 (1600)
2.1 (762)
1.6 (1000)
2.6b (615)
1.3b (1231)
12 (133)
44 (36)
9
10
11
13 (123)
78 (21)
n.i.c
GlcNAc
600
25,000
1600
Numbers in parentheses denote relative inhibitory potency set in relation to the free monosaccharide.
a
For structures, please see Fig. 1.
b
Tested in a previous study.7g
c
n.i.: not inhibitory (at least 1 mM).
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6
exhibit a gradient of decreasing binding constants.13 Kinetically, the
local vicinity of sugar headgroups in glycoclusters can have a posi-
tive impact on interfering with lectin binding to the ligand-
exposing surface in the solid-phase assay.
In order to address the question whether presentation of the
ligand on a cell surface may affect the inhibitory profile we selected
in Table 1). Interestingly, this concurs with the behaviour of cor-
respondingly compared glycocyclopeptides in a solid-phase assay,
although measurements of KD-values by isothermal titration calo-
rimetry were in favour of the O-linkage.8f Comparing relative po-
tencies of the bivalent compounds determined for GSA-II and WGA,
listed in Table 1, indicates an intramolecular binding of the glyco-
clusters to WGA, as is also likely for two sites of the tri- and tet-
ravalent compounds. In fact, conformational flexibility to allow
finding an optimal fit to two hevein domains in WGA has been
implied to explain the pronounced activity of a tris-(2-aminoethyl)
amine-based glycocluster.15 The low concentration of lectin used
argues against a trans-interaction. In contrast, precipitation anal-
ysis and detection of bridging between WGA molecules in a surface
plasmon resonance (SPR) assay documented the inherent capacity
of bi- and tetravalent glycoclusters also to engage in intermolecular
contacts.7c,d Of note, the details on assay condition and type must
thus always be considered when interpreting data on glycoclusters
as inhibitors, to avoid invalid extrapolations.
When WGA and the core region of complex-type N-glycans of
ASF are in contact and the WGA concentration needed to be higher
for adequate signal generation, the bivalent compounds remained
fairly active (Table 1). Cross-linking phenomena under these con-
ditions, as seen in SPR assays,7c,d may counterbalance lectin
blocking, hereby diminishing the direct inhibitory capacity. Also,
spatial constraints around the stem region of the glycan may reduce
probe accessibility especially for the bulky compounds. The CHO
cell panel enables to take these two constellations of glycan pre-
sentation for WGA binding to the level of cells.
a cell system with known expression of terminal b-GlcNAc moie-
ties, i.e., the Lec8 line from the panel of Chinese hamster ovary
(CHO) glycosylation mutants.14 In contrast to the wild-type (WT)
cells and the Lec2 mutant (reduced sialylation), these cells with
impaired galactosylation can be stained with labelled GSA-II in
a concentration-dependent and sugar-inhibitable manner. GlcNAc
(shown at 2 mM) drastically reduced lectin binding, and the impact
of the anomeric position in glycoclusters was as evident as in the
solid-phase assay (Fig. 4A). Also, increase in valency had a similar
impact, leading to strong reductions of cell staining with the tri-
valent compound 8 (Fig. 4B). Respective measurements with WGA
were next carried out to answer the question on relative potency of
the glycoclusters.
2.4. Glycoclusters as inhibitors of WGA binding to (neo)gly-
coproteins and cells
In the case of WGA, binding of glycoclusters is more complex
than for GSA-II: bi- and tetravalent compounds can engage in
trans(intermolecular)- and cis(intramolecular)-interactions.7bee
Also, the target selection of WGA is different from that of GSA-II.
WGA interacts with its cognate sugar when presented by the nat-
ural glycoprotein (ASF or fetuin), here invariably in contact with
both GlcNAc units of the N-glycan core (not the branch-end sialic
acid if N-glycans are sialylated as is the case for fetuin),8c and by the
neoglycoprotein (GlcNAc-BSA). This dual specificity by WGA to core
or terminal sugar units has facilitated study into the occurrence of
an effect of the matrix composition on the inhibition profile. In fact,
an influence of the nature of the scaffold for GlcNAc presentation
(porcine stomach mucin or covalently attached GlcNAc) had been
reported, with the relative inhibitory potency being larger when
testing the synthetic surface than the mucin.7b In our experiments
with the neoglycoprotein, the high-density of sugar per carrier
protein required a high concentration of cognate sugar to achieve
inhibition. The S-glycosidic linkage was more suited for inhibition
than its O-version for both surfaces (compare 1 with 3 and 2 with 4
The Lec8 mutant cells present b-GlcNAc at branch ends of N-
glycans, in addition to GlcNAc in the N-glycans’ core region. WGA
binding to the cells is efficiently blocked by the free mono-
saccharide, and the tetravalent 7 is highly potent (Fig. 5A). Grading
in solid-phase and cell assays appeared comparable, as shown for 1,
5 and 9 (Fig. 5B). The same holds true, when WT cells (GlcNAc
exclusively in the core of N-glycans) were processed (Fig. 6). In
relative terms, the results in both assays are in qualitative accor-
dance. However, grading can become different when comparing
inhibition patterns for homologous lectins with disparate display of
carbohydrate-binding sites to glycoproteins with three natural
forms of glycan branching.16 Using the same matrix (porcine
stomach mucin), two plant lectins with specificity either to GlcNAc
(WGA) or to N-acetyllactosamine (ECA, a leguminous lectin) and
Fig. 4. Fluorescent cell (CHO Lec8 glycosylation mutant) surface staining by labelled GSA-II (20
mg/mL). The control value (lectin-independent background) obtained by cell
processing without applying lectin is given as grey-shaded area, the 100%-value (lectin-dependent staining in the absence of a test compound) as thick black line. Numbers for
staining parameters (percentage of positive cells/mean fluorescence intensity) are presented for each scan, presented in semilogarithmic scaling. A: inhibition of lectin binding by 3
(at 200 mM), 5 (at 50 mM) and GlcNAc (at 2 mM). The list of numbers (from bottom to top) present the 100%-value and the results of the test compounds as given (100%-value,
staining in the presence of 3, 5 and GlcNAc). B: Inhibition of lectin binding (100%-value at bottom) by 10 and 8 (at 100 mM).
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7
Fig. 5. Fluorescent cell (CHO Lec8 glycosylation mutant) surface staining by labelled WGA (2
mg/mL) (for details on representation, please see legend to Fig. 4). A: Inhibition of lectin
binding by increasing concentrations of GlcNAc (2 mM, 10 mM, 20 mM) and by 7 (at 20 M). B: Inhibition of lectin binding by 9 (at 20
m
mM), 5 (at 20
m
M) and 1 (at 25 mM).
ꢁ
four
b
-sandwich monomers (with a 50e70 A distance profile;
GSA-II), molecular design by eight hevein domains (with about
ꢁ
14 A distance between contact sites for GlcNAc; WGA) makes
intramolecular bridging possible for WGA. Intermolecular cross-
linking, however, can occur in both cases. Relative inhibitory po-
tency of the glycoclusters depended on the type of matrix: marked
differences in impairing WGA binding were obtained, when either
the N-glycan core of a glycoprotein or a high-density GlcNAc
presentation of a neoglycoprotein was tested as ligand. Also con-
sidering the disparate sensitivity to inhibition by GlcNAc, selective
reactivity of a test compound between the two lectins (with up to
more than 100-fold difference in IC50-values) was achieved by
teaming up bivalency with an a-S-glycosidic linkage. Of note, the
tested tetravalent glycocluster was relatively more potent as an
inhibitor of WGA but the measured IC50-values were rather similar
for both lectins in the inhibition assays, due to the higher sus-
ceptibility of GSA-II binding to the presence of GlcNAc. In aggre-
gate, these results attest the merit of the concept to determine the
impact of each structural parameter of a glycocluster in a com-
parative setting. After all, the type of linker can also deliver con-
tributions to avidity, for WGA7f,15 or human lectins,3b,20 and
Fig. 6. Fluorescent cell (CHO WT line) surface staining by labelled WGA (8
details on representation, please see legend to Fig. 4). Inhibition of lectin binding by 9
(at 6 M), 5 (at 2 M) and 1 (2 M).
mg/mL) (for
m
m
m
possibly selectivity, deserving
a thorough systematic study.
lysine-based glycoclusters, the final IC50-values were similar for the
octavalent compound, with relative potency considerably higher in
the case of WGA.17 As a consequence, although the enhancement
factor may look impressive, tuning of selectivity would remain
a task in such a case. A similar phenomenon occurred in inhibition
of haemagglutination using persubstituted cyclodextrin, whereas
use of an octasilsesquioxane scaffold accounted for a more than
1000-fold difference in IC50-values between WGA and concanavalin
A in an inhibition setting.18 Evidently, increasing the probability for
cis-interactions, also referred to as ‘cooperative interactions’,19a as
in calix[4]arenes or carbosilane dendrimers, can enhance selectiv-
ity, for WGA and also shown among human galectins.19 In our case,
allowing reactivity of the same glycoclusters to two lectins, selec-
tivity for WGA can be attained at sites of low ligand density (N-
glycan core) with 5 and 6, combined with anomeric preference then
at any sites with bivalent 1e4.
Equally important, the realisation of the impact of the matrix gives
direction to take the inhibition assay to the level of tissues, the
actual site of bioactivity. Monitoring reduction of signal intensity
in lectin histochemistry when the sugar receptor is in contact with
in situ ligands (counterreceptors) is technically as feasible as a di-
rect binding assay using labelled glycoclusters.2c,21 The data on cell
assays documented herein encourage to extend assessment of
inhibitory glycocluster activity to tissue sections, giving our work
direction.
4. Experimental
4.1. General experimental
Unless otherwise noted, all commercially available compounds
were used as provided without further purification. Petroleum
ether with boiling point 40e60 ꢀC was used for column chroma-
tography and thin-layer chromatography (TLC). The NMR spectra
were recorded (25 ꢀC) at 500 MHz for 1H NMR and 126 MHz for 13C
3. Conclusions
Our model study characterises the impact of glycocluster
valency, along with anomeric presentation of GlcNAc and the
nature of the atom of the anomeric centre, on inhibitory potency
for two plant agglutinins with different folding. In contrast to the
NMR. Data are reported in the following order: chemical shift (d) in
parts per million (ppm); multiplicities indicated as s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet); coupling con-
stants (J) given in hertz (Hz). Chemical shifts are reported relative to
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S. Andre et al. / Tetrahedron xxx (2015) 1e14
8
internal standard Me4Si in CDCl3
25 ꢀC) for 1H and Me4Si in CDCl3 (
(
d
0.0) or HOD for D2O (
d
4.72,
4.3. 2-Chloroethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-
d
0.0) or CDCl3 (
d
77.0) for 13C. 1H
D-glucopyranoside 15
NMR signals were assigned with the aid of COSY, 13C NMR signals
using DEPT, gHSQCAD and/or gHMBCAD. Low- and high-resolution
mass spectra were in positive and/or negative mode as indicated in
each case. TLC was performed on aluminium sheets precoated with
silica gel and spots visualised by UV and charring with H2SO4/EtOH
(1:20) or cerium molybdate, unless otherwise stated. Chromatog-
raphy was carried out with silica gel 60 (0.040e0.630 mm) and
using a stepwise solvent polarity gradient correlated with TLC
mobility, unless otherwise stated. Reverse-phase silica used was
2-Acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-b-D-glucopyranose
(1.00 g, 2.57 mmol) was dissolved in 1,2-dichloroethane (5 mL) in
a microwave vial equipped with a stirring bar. To this mixture were
added H2SO4-silica (18 mg) and 2-chloroethanol (0.21 mL,
3.13 mmol). This mixture was then heated and stirred under mi-
crowave conditions at 110 ꢀC for 15 min. The reaction mixture was
then filtered through Celite and washed thoroughly with CH2Cl2.
The filtrate was then washed with saturated aqueous NaHCO3,
brine, then dried over NaSO4 and the solvent was removed. Column
chromatography (EtOAc/petroleum ether, 1:3 to 1:1 to 2:1 to 100%
EtOAc) gave the title compound (784 mg, 75%) as a white solid; 1H
ꢁ
a C18-reverse-phase silica gel (100 A pore size) available from
SigmaeAldrich (60756). CH2Cl2, MeOH, toluene and THF reaction
solvents were used as obtained from a Pure SolvÔ solvent purifi-
cation system.
NMR (500 MHz, CDCl3-d)
d
5.52 (d, J¼8.7 Hz, 1H, NH), 5.30 (dd,
J¼10.7, 9.3 Hz, 1H, H-3), 5.07 (t, J¼9.7 Hz, 1H, H-4), 4.77 (d, J¼8.4 Hz,
1H, H-1), 4.26 (dd, J¼12.3, 4.8 Hz, 1H, H-6a), 4.14 (dd, J¼12.3, 2.5 Hz,
1H, H-6b), 4.10 (dd, J¼10.7, 5.6 Hz, 1H, OCH2CH2Cl), 3.86 (dt, J¼10.6,
8.5 Hz, 1H, H-2), 3.78 (dt, J¼11.4, 6.4 Hz, 1H, OCH2CH2Cl), 3.71 (ddd,
J¼10.0, 4.8, 2.4 Hz,1H, H-5), 3.64 (dd, J¼6.3, 5.0 Hz, 2H, OCH2CH2Cl),
4.2. 2-Azidoethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-
thio-b-D-glucopyranoside 13
The thiol 14 (1.00 g, 2.75 mmol) was dissolved in acetone/H2O
(2:1,15 mL), and potassium carbonate (450 mg, 3.26 mmol) and 1,2-
dibromoethane (2 mL, 23.2 mmol) were then added. This mixture
was stirred at room temperature for 15 h, after which point it was
diluted with CH2Cl2. The layers were separated, with the aqueous
layer being re-extracted with further CH2Cl2. The combined organic
layers were dried over Na2SO4, and the solvent was removed.
Chromatography of the residue (CH2Cl2/MeOH, 95:5 to 93:7) gave
the mono-bromide precursor (898 mg, 69%) as a colourless solid;
2.09, 2.03, 2.03 (each 3H, each s, each OAc), 1.96 (s, 3H, NHAc); 13
C
NMR (126 MHz, CDCl3): d 170.81,170.66,170.40,169.36 (each C]O),
101.06 (C-1), 72.09 (C-3), 71.97 (C-5), 69.67 (OCH2CH2Cl), 68.48 (C-
4), 62.00 (C-6), 54.70 (C-2), 42.98 (OCH2CH2Cl), 23.35 (NHAc),
20.73, 20.67, 20.61 (each OAc); ES-HRMS calcd for C16H24ClNO9Na
432.1037, found m/z 432.1023 [MþNa]þ; IR (cmꢁ1): 1742, 1659,
1556, 1432, 1368, 1223, 1120, 1039, 907; Rf: 0.51 (1:19 MeOH/
20
CH2Cl2); [
a
]
ꢁ4.0 (c 0.10, CHCl3).
D
1H NMR (500 MHz, CDCl3):
d
5.67 (d, J¼9.2 Hz, 1H, NH), 5.18 (dd,
J¼10.3, 9.3 Hz, 1H, H-3), 5.06 (t, J¼9.7 Hz, 1H, H-4), 4.69 (d,
J¼10.4 Hz, 1H, H-1), 4.16 (d, J¼4.0 Hz, 2H, H-6a, H-6b), 4.06 (td,
J¼10.3, 9.1 Hz, 1H, H-2), 3.72 (dt, J¼10.0, 4.0 Hz, 1H, H-5), 3.60 (ddd,
J¼10.9, 9.8, 5.6 Hz, 1H, SCH2CH2Br), 3.51 (ddd, J¼10.9, 9.7, 5.6 Hz,
1H, SCH2CH2Br), 3.19 (ddd, J¼14.0,11.0, 5.6 Hz,1H, SCH2CH2Br), 2.99
(ddd, J¼14.0, 11.0, 5.6 Hz, 1H, SCH2CH2Br), 2.11, 2.03, 2.03 (each s,
each 3H, each OAc), 1.95 (s, 3H, NHAc); 13C NMR (126 MHz, CDCl3):
4.4. 2-Azidoethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
b-
D
-glucopyranoside 16
Chloride 15 (1.36 g, 3.32 mmol) was dissolved in DMF (22 mL),
and to this tetra-N-butylammonium iodide (1.29 g, 3.49 mmol) and
sodium azide (863 mg, 13.3 mmol) were added. The reaction mix-
ture was then heated to 80 ꢀC and was then stirred at this tem-
perature for 15 h. The reaction mixture was cooled, diluted with
CH2Cl2 and washed with water. The aqueous layer was re-extracted
with a portion of CH2Cl2. The organic portions were combined and
were then washed with water. The organic layer was then washed
with brine, dried over NaSO4 and the solvent then removed.
Chromatography (EtOAc/petroleum ether, 1:3 to 1:1 to 2:1 to 1:0)
gave the title compound (1.10 g, 80%) as a white solid; 1H NMR
d
171.06, 170.65, 170.14, 169.26 (each C]O), 85.11 (C-1), 76.02 (C-5),
73.40 (C-3), 68.30 (C-4), 62.29 (C-6), 53.40 (C-2), 32.90
(SCH2CH2Br), 30.95 (SCH2CH2Br), 23.24 (NHAc), 20.76, 20.65, 20.58
(each OAc); ES-HRMS calcd for C16H25NO8SBr 470.0484, found m/z
470.0471 [MþH]þ; IR (cmꢁ1): 3288, 1746, 1661, 1543, 1373, 1233,
20
1047, 946, 735; Rf: 0.46 (2:23 MeOH/CH2Cl2); [
a]
ꢁ42.5 (c 0.48,
D
CHCl3). This bromide intermediate (1.24 g, 2.64 mmol) was dis-
solved in DMF (20 mL), and to this were added tetrabutylammo-
nium iodide (1.12 g, 3.03 mmol) and sodium azide (705 mg,
10.8 mmol). The mixture was heated to 80 ꢀC and stirred for 15 h at
this temperature and then allowed to cool to room temperature,
after which it was diluted with CH2Cl2 and washed a number of
times with H2O, in an effort to remove DMF. The organic layer was
then dried over Na2SO4 and the solvent was removed. Chroma-
tography of the residue (EtOAc/petroleum ether, 1:3 to 1:1) gave
the title compound (934 mg, 82%) as a white solid; 1H NMR
(500 MHz, CDCl3-d)
d
5.50 (d, J¼8.6 Hz, 1H, NH), 5.36 (dd, J¼10.6,
9.3 Hz, 1H, H-3), 5.08 (dd, J¼10.0, 9.3 Hz, 1H, H-4), 4.83 (d, J¼8.3 Hz,
1H, H-1), 4.26 (dd, J¼12.3, 4.7 Hz, 1H, H-6a), 4.15 (dd, J¼12.3, 2.4 Hz,
1H, H-6b), 4.05 (ddd, J¼10.8, 4.7, 3.2 Hz, 1H, OCH2CH2N3), 3.80 (dt,
J¼10.7, 8.5 Hz, 1H, H-2), 3.74e3.66 (m, 2H, H-5, OCH2CH2N3), 3.51
(ddd, J¼13.5, 8.6, 3.3 Hz, 1H, OCH2CH2N3), 3.26 (ddd, J¼13.4, 4.7,
3.1 Hz, 1H, OCH2CH2N3), 2.09 (s, 3H), 2.03 (s, 3H), 2.03 (s, 3H) (each
OAc), 1.96 (s, 3H, NHAc); 13C NMR (126 MHz, CDCl3):
d 170.74,
170.65, 170.40, 169.38 (each C]O), 100.46 (C-1), 72.04 (C-4), 71.97
(C-5), 68.55 (C-3), 68.41 (OCH2CH2N3), 61.97 (C-6), 54.93 (C-2),
50.60 (OCH2CH2N3), 23.37 (NHAc), 20.74, 20.66, 20.62 (each OAc);
ES-HRMS calcd for C16H24N4O9Na 439.1441, found m/z 439.1429
(500 MHz, CDCl3):
d
5.56 (d, J¼9.3 Hz, 1H, NH), 5.16 (dd, J¼10.3,
9.3 Hz, 1H, H-3), 5.09 (t, J¼9.7 Hz, 1H, H-4), 4.65 (d, J¼10.3 Hz, 1H,
H-1), 4.22 (dd, J¼12.4, 5.1 Hz, 1H, H-6a), 4.15 (dd, J¼12.4, 2.4 Hz, 1H,
H-6b), 4.10 (td, J¼10.3, 9.2 Hz, 1H, H-2), 3.70 (ddd, J¼9.9, 5.0, 2.4 Hz,
1H, H-5), 3.56 (dt, J¼13.2, 6.7 Hz, 1H, SCH2CH2N3), 3.48 (dt, J¼12.6,
6.9 Hz, 1H, SCH2CH2N3), 2.99 (dt, J¼13.9, 6.9 Hz, 1H, SCH2CH2N3),
2.79 (dt, J¼13.8, 6.7 Hz, 1H, SCH2CH2N3), 2.09, 2.04, 2.03 (each s,
each 3H, each OAc), 1.96 (s, 3H, NHAc); 13C NMR (126 MHz, CDCl3):
[MþNa]þ; IR (cmꢁ1): 2106, 1744, 1656, 1566, 1436, 1368, 1225, 1170,
20
1125, 1033, 910; Rf: 0.41 (1:19 MeOH/CH2Cl2); [
CHCl3).
a]
ꢁ22.8 (c 0.36,
D
4.5. 1,3-Di[1-(ethyl 2-acetamido-2-deoxy-1-thio-
b-D-gluco-
d
171.08, 170.62, 170.12, 169.24 (each C]O), 84.69 (C-1), 76.09 (C-5),
pyranosyl)-1,2,3-triazol-4-ylmethyloxy]benzene 1
73.55 (C-3), 68.19 (C-4), 62.18 (C-6), 53.21 (C-2), 51.60 (SCH2CH2N3),
29.64 (SCH2CH2N3), 23.26 (NHAc), 20.71, 20.65, 20.58 (each OAc);
ES-HRMS calcd for C16H24N4O8NaS 455.1213, found m/z 455.1210
[MþNa]þ; IR (cmꢁ1): 2099, 1742, 1659, 1543, 1371, 1223, 1036, 916;
To a mixture of azide 13 (350 mg, 0.809 mmol) and meta-bis-
propargyloxybenzene 20 (63 mg, 0.338 mmol), in THF/H2O (1:1,
12 mL), were added sodium ascorbate (41 mg, 0.207 mmol) and
copper sulfate pentahydrate (51 mg, 0.204 mmol). The mixture was
Rf: 0.57 (3:47 MeOH/CH2Cl2); [
a
]
20 ꢁ60.5 (c 0.44, CHCl3).
D
ꢀ
Please cite this article in press as: Andre, S.; et al., Tetrahedron (2015), http://dx.doi.org/10.1016/j.tet.2015.07.020

ꢀ
S. Andre et al. / Tetrahedron xxx (2015) 1e14
9
stirred at room temperature for 18 h. Tetrahydrofuran was then
removed and CH2Cl2 was added and the mixture was then washed
with water. The aqueous layer was re-extracted further with
CH2Cl2. The combined organic layers were then washed with water,
dried over NaSO4 and the solvent removed. Chromatography of the
residue (95:5 CH2Cl2/MeOH) gave the acetylated intermediate
J¼10.0 Hz, 2H, H-3), 5.06 (t, J¼9.7 Hz, 2H, H-4), 4.67 (dt, J¼12.9,
6.2 Hz, 2H, SCH2CH2triazole), 4.56 (dt, J¼13.7, 6.6 Hz, 2H,
SCH2CH2triazole), 4.43 (d, J¼10.4 Hz, 2H, NH), 4.25e4.14 (m, 4H, H-
6a, H-6b), 4.06 (q, J¼9.9 Hz, 2H, H-2), 3.67 (ddd, J¼10.1, 4.8, 2.6 Hz,
2H, H-5), 3.30 (dt, J¼13.8, 6.7 Hz, 2H, SCH2CH2triazole), 3.06 (dt,
J¼14.6, 6.3 Hz, 2H, SCH2CH2triazole), 2.06, 2.03, 2.02 (each s, each
6H, each OAc), 1.93 (s, 6H, NHAc); 13C NMR (126 MHz, CDCl3)
(350 mg, w95%) as a white solid; 1H NMR (500 MHz, CDCl3)
d 7.76
(s, 2H, CCH, triazole), 7.20 (t, J¼8.3 Hz, 1H, aromatic H), 6.65e6.61
(m, 3H, aromatic H), 6.01 (d, J¼9.3 Hz, 2H, NH), 5.20 (s, 4H, ArOCH2),
5.17 (dd, J¼10.3, 9.4 Hz, 2H, H-3), 5.05 (dd, J¼10.1, 9.3 Hz, 2H, H-4),
4.67 (dt, J¼13.1, 6.4 Hz, 2H, SCH2CH2triazole), 4.61e4.55 (m, 2H,
SCH2CH2triazole), 4.53 (d, J¼10.4 Hz, 2H, H-1), 4.19e4.13 (m, 4H, H-
6a, H-6b), 4.05 (td, J¼10.3, 9.2 Hz, 2H, H-2), 3.67 (ddd, J¼10.1, 4.6,
3.0 Hz, 2H, H-5), 3.30 (dt, J¼14.6, 6.7 Hz, 2H, SCH2CH2triazole), 3.08
(dt, J¼14.6, 6.4 Hz, 2H, SCH2CH2triazole), 2.05, 2.03, 2.03 (each s,
each 6H, each OAc), 1.93 (s, 6H, NHAc); 13C NMR (126 MHz, CDCl3)
d 170.91, 170.56, 170.34, 169.30 (each C]O), 152.70 (aromatic C),
143.86 (CCH, triazole), 124.18 (CCH, triazole), 116.20 (aromatic CH),
84.54 (C-1), 76.08 (C5), 73.39 (C-3), 68.21 (C-4), 62.68 (CH2OAr),
62.06 (C-6), 53.02 (C-2), 50.45 (SCH2CH2triazole), 30.37
(SCH2CH2triazole), 23.17 (NHAc), 20.72, 20.64, 20.58 (each OAc);
ES-HRMS calcd for
C44H58N8O18S2Na 1073.3208, found m/z
1073.3193 [MþNa]þ; IR (ATR, cmꢁ1): 1743, 1661, 1536, 1508, 1372,
1221, 1043, 915, 731; Rf: 0.34 (2:23 MeOH/CH2Cl2). Deacetylation of
this intermediate (315 mg, 0.300 mmol) as described in the prep-
aration of 1 gave the title compound 2 (196 mg, 82%) as a white
d
170.88, 170.56, 170.40, 169.31 (each C]O), 159.30 (aromatic C),
143.60 (CCH, triazole), 130.40 (aromatic CH), 124.22 (CCH, triazole),
108.26 (aromatic CH), 102.45 (aromatic CH), 84.48 (C-1), 76.08 (C-
5), 73.34 (C-3), 68.25 (C-4), 62.04 (C-6), 62.04 (CH2OAr), 53.15 (C-2),
50.38 (SCH2CH2triazole), 30.44 (SCH2CH2triazole), 23.19 (NHAc),
20.72, 20.65, 20.59 (each OAc); ES-HRMS calcd for
solid; 1H NMR (500 MHz, DMSO-d6)
d 8.18 (s, 2H, CCH, triazole), 7.71
(d, J¼9.3 Hz, 2H, NH), 6.95 (s, 4H, aromatic H), 5.05 (d, J¼1.4 Hz, 4H,
ArOCH2), 5.04 (d, J¼3.9 Hz, 2H, OH-4), 4.99 (d, J¼5.5 Hz, 2H, OH-3),
4.63e4.54 (m, 6H, SCH2CH2triazole, OH-6), 4.40 (d, J¼10.3 Hz, 2H,
H-1), 3.71 (ddd, J¼11.9, 5.6, 2.0 Hz, 2H, H-6), 3.53 (q, J¼9.8 Hz, 2H,
H-2), 3.44 (dt, J¼11.9, 6.1 Hz, 2H, H-6b), 3.30e3.21 (m, 2H, H-3),
3.20e3.13 (m, 4H, H-5, SCH2CH2triazole), 3.09 (td, J¼9.1, 5.3 Hz, 2H,
H-4), 2.97 (dt, J¼13.9, 6.9 Hz, 2H, SCH2CH2triazole), 1.78 (s, 6H,
C
44H58N8O18S2Na 1073.3208, found m/z 1073.3201 [MþNa]þ; IR
(ATR, cmꢁ1): 1743, 1660, 1525, 1371, 1221, 1035, 733, 703; Rf: 0.44
(2:23 MeOH/CH2Cl2).
The acetylated intermediate (350 mg, 0.333 mmol) was stirred
in methanol (30 mL) and a catalytic amount of a freshly prepared
1 M NaOMe solution in MeOH (0.17 mL, 0.170 mmol) was added.
The resulting mixture was stirred for 2 h at room temperature, and
Amberlite IR-120Hþ was then added and the mixture stirred for
a further 10 min. The resin was removed by filtration and washed
with water and acetonitrile. The solvents were removed until
a minimal amount of water remained and the concentrate was
subjected to reverse-phase column chromatography. The solution
of the compound, in a minimal amount of water, which was heated
or made acidic if necessary to dissolve, was loaded onto the re-
verse-phase silica gel. Three volumes of water were first passed
through the column and the title compound (207 mg, 78%, white
solid) was then eluted with 1:1 MeCN/H2O; 1H NMR (500 MHz,
NHAc); 13C NMR (126 MHz, DMSO)
d 169.51 (C]O), 152.81 (aro-
matic C), 142.91 (CCH, triazole), 125.48 (CCH, triazole), 116.03 (ar-
omatic CH), 85.03 (C-1), 81.61 (C-5), 75.81 (C-3), 70.90 (C-4), 62.07
(CH2OAr), 61.68 (C-6), 54.70 (C-2), 50.11 (SCH2CH2triazole), 30.51
(SCH2CH2triazole),
23.46
(NHAc);
ES-HRMS
calcd
for
C
32H46N8O12S2Na 821.2574, found m/z 821.2584 [MþNa]þ; IR (ATR,
cmꢁ1): 3267, 1644, 1552, 1508, 1374, 1216, 1052, 1027, 1007, 822; Rf:
20
0.62 (1:1 MeCN/H2O) reverse-phase silica gel; [
DMSO).
a
]
ꢁ24.0 (c 0.27,
D
4.7. 1,3-Di[1-(ethyl 2-acetamido-2-deoxy-1-thio-
a-D-gluco-
pyranosyl)-1,2,3-triazol-4-ylmethyloxy]benzene 5
Reaction of azide 17 (335 mg, 0.775 mmol) and 20 as described
in preparation of 1 gave the acetylated intermediate (327 mg, 89%)
DMSO-d6)
d
8.20 (s, 2H, CCH, triazole), 7.72 (d, J¼9.3 Hz, 2H, NH),
7.18 (t, J¼8.2 Hz, 1H, aromatic H), 6.71 (t, J¼2.3 Hz, 1H, aromatic H),
6.62 (dd, J¼8.3, 2.3 Hz, 2H, aromatic H), 5.09 (s, 4H, ArOCH2), 5.04
(d, J¼5.3 Hz, 2H, OH-4), 4.99 (d, J¼5.4 Hz, 2H, OH-3), 4.67e4.52 (m,
6H, SCH2CH2triazole, OH-6), 4.40 (d, J¼10.3 Hz, 2H, H-1), 3.71 (ddd,
J¼11.8, 5.4, 2.0 Hz, 2H, H-6a), 3.53 (q, J¼9.8 Hz, 2H, H-2), 3.44 (dt,
J¼11.9, 6.1 Hz, 2H, H-6b), 3.29e3.23 (m, 2H, H-3), 3.20e3.13 (m, 4H,
H-5, SCH2CH2triazole), 3.09 (td, J¼9.1, 5.2 Hz, 2H, H-4), 2.97 (dt,
J¼13.9, 6.9 Hz, 2H, SCH2CH2triazole), 1.78 (s, 6H, NHAc); 13C NMR
as a white solid; 1H NMR (500 MHz, CDCl3)
d 7.68 (s, 2H, CCH, tri-
azole), 7.23e7.17 (m, 1H, aromatic H), 6.65e6.59 (m, 3H, aromatic
H), 5.88 (d, J¼8.0 Hz, 2H, NH), 5.58 (d, J¼5.4 Hz, 2H, H-1), 5.20 (s,
4H, CH2OAr), 5.11 (t, J¼9.6 Hz, 2H, H-4), 5.02 (dd, J¼11.1, 9.3 Hz, 2H,
H-3), 4.59 (t, J¼6.7 Hz, 4H, SCH2CH2triazole), 4.46 (ddd, J¼11.1, 8.0,
5.4 Hz, 2H, H-2), 4.30 (ddd, J¼9.9, 4.9, 2.2 Hz, 2H, H-5), 4.25 (dd,
J¼12.3, 4.8 Hz, 2H, H-6a), 4.12 (dd, J¼12.3, 2.2 Hz, 2H, H-6b), 3.18
(dt, J¼13.6, 6.7 Hz, 2H, SCH2CH2triazole), 3.09 (dt, J¼14.0, 6.8 Hz,
2H, SCH2CH2triazole), 2.06, 2.04, 2.04 (each s, each 6H, each OAc),
(126 MHz, DMSO)
d 169.52 (C]O), 159.75 (aromatic C), 142.62
(CCH, triazole), 130.45 (aromatic CH), 125.60 (CCH, triazole), 107.67
(aromatic CH), 102.01 (aromatic CH), 85.03 (C-1), 81.60 (C-5), 75.81
(C-3), 70.90 (C-4), 61.68 (C-6), 61.60 (CH2OAr), 54.70 (C-2), 50.14
(SCH2CH2triazole), 30.50 (SCH2CH2triazole), 23.46 (NHAc); ES-
HRMS calcd for C32H46N8O12S2Na 821.2574, found m/z 821.2573
1.97 (s, 6H, NHAc); 13C NMR (126 MHz, CDCl3)
d 171.72, 170.52,
170.20, 169.24 (each C]O), 159.37 (aromatic C), 144.26 (CCH, tri-
azole), 130.13 (aromatic CH), 123.05 (CCH, triazole), 107.78 (aro-
matic CH), 102.13 (aromatic CH), 84.63 (C-1), 70.96 (C-3), 68.75 (C-
5), 67.98 (C-4), 62.07 (CH2OAr), 62.01 (C-6), 52.75 (C-2), 49.52
(SCH2CH2triazole), 31.44 (SCH2CH2triazole), 23.18 (NHAc), 20.70,
20.68, 20.58 (each OAc); ES-HRMS calcd for C44H58N8O18S2Na
1073.3208, found m/z 1073.3237 [MþNa]þ; IR (ATR, cmꢁ1): 3286,
1742, 1540, 1369, 1227, 1150, 1086, 1039, 734; Rf: 0.47 (2:23 MeOH/
CH2Cl2).
[MþNa]þ; IR (ATR, cmꢁ1): 3269, 1645, 1551, 1373, 1264, 1153, 1048,
20
1027, 820. Rf: 0.61 (1:1 MeCN/H2O, reverse-phase silica gel); [
a]
D
ꢁ26.9 (c 0.26, DMSO).
4.6. 1,4-Di[1-(ethyl 2-acetamido-2-deoxy-1-thio-
b-D-gluco-
pyranosyl)-1,2,3-triazol-4-ylmethyloxy]benzene 2
Deacetylation of this intermediate (314 mg, 0.299 mmol) as
described in the preparation of 1 gave the title compound 3
Reaction of 13 (350 mg, 0.809 mmol) and para-bispropargy-
loxybenzene 19 (63 mg, 0.338 mmol) as described in preparation of
1 gave the acetylated intermediate (319 mg, 90%) as a white solid;
(226 mg, 95%) as a white solid; 1H NMR (500 MHz, DMSO-d6)
d 8.20
(s, 2H, CCH, triazole), 7.83 (d, J¼7.0 Hz, 2H, NH), 7.19 (t, J¼8.2 Hz, 1H,
aromatic H), 6.70 (t, J¼2.4 Hz, 1H, aromatic H), 6.62 (dd, J¼8.3,
2.3 Hz, 2H, aromatic H), 5.44 (d, J¼5.3 Hz, 2H, H-1), 5.11 (d, J¼5.6 Hz,
2H, OH-4), 5.09 (s, 4H, CH2OAr), 4.82 (d, J¼5.7 Hz, 2H, OH-3), 4.64 (t,
1H NMR (500 MHz, CDCl3)
d 7.72 (s, 2H, CCH, triazole), 6.91 (s, 4H,
aromatic H), 5.92 (d, J¼9.3 Hz, 2H, H-1), 5.16 (s, 4H, ArOCH2), 5.14 (t,
ꢀ
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10
J¼5.8 Hz, 2H, OH-6), 4.59 (dt, J¼13.7, 6.8 Hz, 2H, SCH2CH2triazole),
4.52 (dt, J¼13.9, 7.0 Hz, 2H, SCH2CH2triazole), 3.79 (ddd, J¼11.0, 7.0,
5.3 Hz, 2H, H-2), 3.75e3.67 (m, 4H, H-5, H-6a), 3.48 (dt, J¼11.9,
6.2 Hz, 2H, H-6b), 3.34 (ddd, J¼10.4, 8.4, 5.4 Hz, 2H, H-3), 3.11 (td,
J¼9.3, 5.6 Hz, 2H, H-4), 3.05 (dt, J¼13.9, 7.0 Hz, 2H, SCH2CH2tria-
zole), 2.96 (dt, J¼13.8, 6.8 Hz, 2H, SCH2CH2triazole), 1.81 (s, 6H,
intermediate (343 mg, 86%) as a white solid; 1H NMR (500 MHz,
CDCl3-d) 7.71 (s, 3H, CCH, triazole), 6.28 (s, 3H, aromatic H), 5.99
d
(d, J¼8.0 Hz, 3H, NH), 5.59 (d, J¼5.4 Hz, 3H, H-1), 5.17 (s, 6H,
CH2OAr), 5.11 (t, J¼9.6 Hz, 3H, H-4), 5.02 (dd, J¼11.1, 9.3 Hz, 3H, H-
3), 4.60 (t, J¼6.7 Hz, 6H, SCH2CH2triazole), 4.47 (ddd, J¼11.2, 8.0,
5.4 Hz, 3H, H-2), 4.31 (ddd, J¼10.0, 4.8, 2.2 Hz, 3H, H-5), 4.26 (dd,
J¼12.3, 4.9 Hz, 3H, H-6a), 4.12 (dd, J¼12.3, 2.2 Hz, 3H, H-6b), 3.18
(dt, J¼13.6, 6.7 Hz, 3H, SCH2CH2triazole), 3.10 (dt, J¼13.9, 6.8 Hz, 3H,
SCH2CH2triazole), 2.05, 2.04, 2.04 (each s, each 9H, each OAc), 1.97
NHAc); 13C NMR (126 MHz, DMSO)
d 170.03 (C]O), 159.74 (aro-
matic C), 142.87 (CCH, triazole), 130.47 (aromatic CH), 125.15 (CCH,
triazole), 107.68 (aromatic CH), 102.03 (aromatic CH), 84.47 (C-1),
74.19 (C-5), 71.42 (C-4), 71.08 (C-3), 61.61 (CH2OAr), 61.31 (C-6),
54.60 (C-2), 49.74 (SCH2CH2triazole), 30.54 (SCH2CH2triazole),
23.03 (NHAc); ES-HRMS calcd for C32H46N8O12S2Na 821.2574,
found m/z 821.2585 [MþNa]þ; IR (ATR, cmꢁ1): 3278, 1644, 1594,
(s, 9H, NHAc); 13C NMR (126 MHz, CDCl3)
d 171.65, 170.53, 170.25,
169.28 (each C]O), 160.02 (aromatic C), 143.99 (CCH, triazole),
123.21 (CCH, triazole), 95.38 (aromatic CH), 84.64 (C-1), 70.92 (C-3),
68.73 (C-5), 68.05 (C-4), 62.03 (CH2OAr), 62.03 (C-6), 52.71 (C-2),
49.53 (SCH2CH2triazole), 31.43 (SCH2CH2triazole), 23.17 (NHAc),
20.70, 20.69, 20.59 (each OAc); ES-HRMS calcd for
1547, 1283, 1150, 1041, 1028, 763; Rf: 0.57 (1:1 MeCN/H2O, reverse-
20
phase silica gel); [
a]
þ114.6 (c 0.42, DMSO).
D
C
63H84N12O27S3Na 1559.4629, found m/z 1559.4636 [MþNa]þ; IR
4.8. 1,4-Di[1-(ethyl 2-acetamido-2-deoxy-1-thio-
a
-D-gluco-
(ATR, cmꢁ1): 3276, 1744, 1635, 1541, 1509, 1376, 1230, 1152, 1118,
1044, 827, 803; Rf: 0.40 (2:23 MeOH/CH2Cl2). Deacetylation of this
intermediate (330 mg, 0.215 mmol) as described in preparation of 1
gave the title compound 8 (224 mg, 90%) as a white solid; 1H NMR
pyranosyl)-1,2,3-triazol-4-ylmethyloxy]benzene 6
Reaction of azide 17 (335 mg, 0.775 mmol) and 19 (65 mg,
0.349 mmol) as described in the preparation of 1 gave the acety-
lated intermediate (353 mg, 96%) as a white solid; 1H NMR
(500 MHz, DMSO-d6)
d
8.20 (s, 3H, CCH, triazole), 7.83 (d, J¼7.0 Hz,
3H, NH), 6.33 (s, 3H, aromatic H), 5.45 (d, J¼5.3 Hz, 3H, H-1), 5.11 (d,
J¼5.7 Hz, 3H, OH-4), 5.07 (s, 6H, CH2OAr), 4.81 (d, J¼5.3 Hz, 3H, OH-
3), 4.66e4.63 (m, 3H, OH-6), 4.59 (dt, J¼13.7, 6.9 Hz, 3H,
SCH2CH2triazole), 4.52 (dt, J¼14.0, 7.0 Hz, 3H, SCH2CH2triazole),
3.79 (ddd, J¼10.9, 7.0, 5.2 Hz, 3H, H-2), 3.71 (td, J¼12.4, 11.1, 5.2 Hz,
6H, H-5. H-6a), 3.48 (dt, J¼11.4, 5.3 Hz, 3H, H-6b), 3.38e3.33 (m, 3H,
H-3), 3.11 (dt, J¼9.6, 4.7 Hz, 3H, H-4), 3.05 (dt, J¼14.0, 7.2 Hz, 3H,
SCH2CH2triazole), 2.96 (dt, J¼13.9, 6.9 Hz, 3H, SCH2CH2triazole),
(500 MHz, CDCl3)
d 7.65 (s, 2H, CCH, triazole), 6.92 (s, 4H, aromatic
H), 5.83 (d, J¼8.0 Hz, 2H, NH), 5.57 (d, J¼5.4 Hz, 2H, H-1), 5.17 (s, 4H,
CH2OAr), 5.12 (t, J¼9.6 Hz, 2H, H-4), 5.02 (dd, J¼11.1, 9.3 Hz, 2H, H-
3), 4.59 (t, J¼6.7 Hz, 4H, SCH2CH2triazole), 4.46 (ddd, J¼11.1, 7.9,
5.5 Hz, 2H, H-2), 4.30 (ddd, J¼10.0, 4.9, 2.1 Hz, 2H, H-5), 4.26 (dd,
J¼12.3, 4.8 Hz, 2H, H-6a), 4.12 (dd, J¼12.3, 2.1 Hz, 2H, H-6b), 3.18
(dt, J¼13.6, 6.7 Hz, 2H, SCH2CH2triazole), 3.09 (dt, J¼13.9, 6.8 Hz,
2H, SCH2CH2triazole), 2.06, 2.04, 2.04 (each s, each 6H, each OAc),
1.81 (s, 9H, NHAc); 13C NMR (126 MHz, DMSO)
d 170.04 (C]O),
1.97 (s, 6H, NHAc); 13C NMR (126 MHz, CDCl3)
d
172.87, 171.77,
160.36 (Oaromatic C), 142.77 (CCH, triazole), 125.18 (CCH, triazole),
94.91 (aromatic CH), 84.47 (C-1), 74.18 (C-5), 71.42 (C-4), 71.08 (C-
3), 61.68 (ArOCH2), 61.31 (C-6), 54.60 (C-2), 49.75 (SCH2CH2tria-
zole), 30.53 (SCH2CH2triazole), 23.03 (NHAc); ES-HRMS calcd for
170.52, 170.18 (each C]O), 152.75 (aromatic C), 144.55 (CCH, tri-
azole), 122.96 (CCH, triazole), 115.88 (aromatic CH), 84.64 (C-1),
70.99 (C-3), 68.76 (C-5), 67.93 (C-4), 62.66 (CH2OAr), 62.01 (C-6),
52.77 (C-2), 49.50 (SCH2CH2triazole), 31.46 (SCH2CH2triazole),
23.18 (NHAc), 20.70, 20.69, 20.58 (each OAc); ES-HRMS calcd for
C
45H66N12O18S3Na 1181.3678, found m/z 1181.3660 [MþNa]þ; IR
(ATR, cmꢁ1): 3286, 1641, 1543, 1376, 1168, 1099, 1066, 1004, 943,
20
C
44H58N8O18S2Na 1073.3208, found m/z 1073.3203 [MþNa]þ; IR
810, 757; Rf: 0.71 (1:1 MeCN/H2O, reverse-phase silica gel); [
þ142.2 (c 0.45, DMSO).
a]
D
(ATR, cmꢁ1): 3278, 1744, 1635, 1547, 1376, 1228, 1085, 1043, 1027,
800; Rf: 0.47 (2:23 MeOH/CH2Cl2). Deacetylation of this in-
termediate (349 mg, 0.332 mmol) as described in the preparation of
1 gave the title compound (196 mg, 74%) as a white solid; 1H NMR
4.10. 1,1,2,2-Tetrakis[4-(1-(ethyl 2-acetamido-2-deoxy-1-thio-
-glucopyranosyl)-1,2,3-triazol-4-ylmethyloxy-phenyl)]eth-
ane 7
a-D
(500 MHz, DMSO-d6)
d
8.17 (s, 2H, CCH, triazole), 7.83 (d, J¼7.0 Hz,
2H, NH), 6.95 (s, 4H, aromatic H), 5.44 (d, J¼5.2 Hz, 2H, H-1), 5.12 (d,
J¼5.6 Hz, 2H, OH-4), 5.04 (s, 4H, CH2OAr), 4.82 (d, J¼5.7 Hz, 2H, OH-
3), 4.64 (t, J¼5.7 Hz, 2H, OH-6), 4.58 (dt, J¼13.7, 6.8 Hz, 2H,
SCH2CH2triazole), 4.51 (dt, J¼13.9, 7.0 Hz, 2H, SCH2CH2triazole),
3.79 (ddd, J¼11.0, 7.0, 5.2 Hz, 2H, H-2), 3.74e3.67 (m, 4H, H-5, H-
6a), 3.48 (dt, J¼12.0, 6.2 Hz, 2H, H-6b), 3.34 (ddd, J¼11.1, 8.7, 5.8 Hz,
2H, H-3), 3.10 (ddd, J¼9.9, 8.6, 5.6 Hz, 2H, H-4), 3.04 (dt, J¼13.9,
7.0 Hz, 2H, SCH2CH2triazole), 2.96 (dt, J¼13.8, 6.8 Hz, 2H,
SCH2CH2triazole), 1.81 (s, 6H, NHAc); 13C NMR (126 MHz, DMSO)
Reaction of azide 17 (333 mg, 0.770 mmol) and 22 (104 mg,
0.190 mmol) as described in the preparation of 1 gave, after chro-
matography (95:5 to 93:7 CH2Cl2/MeOH), the acetylated in-
termediate (357 mg, 83%) as a yellow solid; 1H NMR (500 MHz,
CDCl3-d)
d
7.66 (s, 4H, CCH, triazole), 6.93 (d, J¼8.7 Hz, 8H, aromatic
H), 6.72 (d, J¼8.8 Hz, 8H, aromatic H), 5.89 (d, J¼8.0 Hz, 4H, NH),
5.58 (d, J¼5.4 Hz, 4H, H-1), 5.14 (s, 8H, CH2OAr), 5.11 (t, J¼9.8 Hz, 4H,
H-4), 5.02 (dd, J¼11.1, 9.3 Hz, 4H, H-3), 4.60 (t, J¼6.8 Hz, 8H,
SCH2CH2triazole), 4.47 (ddd, J¼11.1, 8.0, 5.5 Hz, 4H, H-2), 4.31 (ddd,
J¼10.1, 4.8, 2.0 Hz, 4H, H-5), 4.26 (dd, J¼12.3, 4.8 Hz, 4H, H-6a), 4.13
(dd, J¼12.4, 2.2 Hz, 4H, H6b), 3.18 (dt, J¼13.7, 6.7 Hz, 4H,
SCH2CH2triazole), 3.10 (dt, J¼14.0, 6.9 Hz, 4H, SCH2CH2triazole),
2.06 (s, 12H), 2.04 (s, 12H), 2.04 (s, 12H) (each OAc), 1.96 (s, 12H,
d
170.02 (C]O), 152.80 (aromatic C), 143.15 (CCH, triazole), 125.03
(CCH, triazole), 116.04 (aromatic CH), 84.48 (C-1), 74.19 (C-5), 71.42
(C-4), 71.08 (C-3), 62.09 (CH2OAr), 61.32 (C-6), 54.61 (C-2), 49.72
(SCH2CH2triazole), 30.57 (SCH2CH2triazole), 23.03 (NHAc); ES-
HRMS calcd for C32H46N8O12S2Na1 821.2574, found m/z 821.2578
[MþNa]þ; IR (ATR, cmꢁ1): 3283, 1644, 1543, 1508, 1215, 1064, 1039,
NHAc); 13C NMR (126 MHz, CDCl3)
d 171.71, 170.54, 170.23, 169.25
20
1007, 823; Rf: 0.61 (1:1 MeCN/H2O, reverse-phase silica gel); [
a
]
(each C]O), 156.56 (aromatic C), 144.35 (CCH, triazole), 138.73
(Ar2eC]CeAr2), 137.20 (aromatic C), 132.56 (aromatic CH), 123.01
(CCH, triazole), 113.88 (aromatic CH), 84.60 (C-1), 70.97 (C-3), 68.76
(C-5), 68.00 (C4), 62.01 (CH2OAr), 62.01 (C-6), 52.71 (C-2), 49.52
(SCH2CH2triazole), 31.38 (SCH2CH2triazole), 23.17 (NHAc), 20.70,
20.70, 20.58 (each OAc); ES-HRMS calcd for C102H124N16O36S4Cl
2311.6935, found m/z 2311.6877 [MþCl]ꢁ; IR (ATR, cmꢁ1): 1741,
1669, 1508, 1367, 1225, 1039, 734; Rf: 0.39 (2:23 MeOH/CH2Cl2).
Deacetylation of this intermediate (355 mg, 0.311 mmol) as
D
þ146.4 (c 0.39, DMSO).
4.9. 1,3,5-Tri[1-(ethyl 2-acetamido-2-deoxy-1-thio-
a-D-gluco-
pyranosyl)-1,2,3-triazol-4-ylmethyloxy]benzene 8
Reaction of azide 17 (348 mg, 0.805 mmol) and tripropargyl
ether 21 (62 mg, 0.258 mmol) as described in preparation of 1, gave,
after chromatography (95:5 to 93:7 CH2Cl2/MeOH), the acetylated
ꢀ
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11
described in the preparation of 1 gave the title compound 7
(263 mg, 95%) as a yellow solid; 1H NMR (500 MHz, DMSO-d6)
8.18
4.12. 1,3-Di[1-(ethyl 2-acetamido-2-deoxy-
anosyl)-1,2,3-triazol-4-ylmethyloxy]benzene 3
b-D-glucopyr-
d
(s, 4H, CCH, triazole), 7.83 (d, J¼7.0 Hz, 4H, NH), 6.87 (d, J¼8.7 Hz,
8H, aromatic H), 6.80 (d, J¼8.8 Hz, 8H, aromatic H), 5.44 (d,
J¼5.3 Hz, 4H, H-1), 5.11 (d, J¼5.6 Hz, 4H, OH-4), 5.03 (s, 8H,
CH2OAr), 4.81 (d, J¼5.8 Hz, 4H, OH-3), 4.64 (t, J¼5.8 Hz, 4H, OH-6),
4.58 (dt, J¼13.7, 6.8 Hz, 4H, SCH2CH2triazole), 4.51 (dt, J¼14.0,
7.0 Hz, 4H, SCH2CH2triazole), 3.79 (ddd, J¼10.9, 7.0, 5.2 Hz, 4H, H-2),
3.75e3.67 (m, 8H, H5, H-6a), 3.48 (dt, J¼12.0, 6.2 Hz, 4H, H-6b),
3.37e3.31 (m, 4H, H-3), 3.11 (td, J¼9.1, 5.9 Hz, 4H, H-4), 3.04 (dt,
J¼13.9, 7.0 Hz, 4H, SCH2CH2triazole), 2.96 (dt, J¼13.8, 6.8 Hz, 4H,
SCH2CH2triazole), 1.80 (s, 12H, NHAc); 13C NMR (126 MHz, DMSO)
Reaction of azide 16 (320 mg, 0.769 mmol) and 20 (68 mg,
0.365 mmol) in THF/H2O (1:1, 12 mL) as described in preparation of
1 gave, after chromatography (95:5 CH2Cl2/MeOH), the acetylated
intermediate (311 mg, 84%) as a white solid; 1H NMR (500 MHz,
CDCl3)
d
7.76 (s, 2H, CCH, triazole), 7.21 (t, J¼8.2 Hz, 1H, aromatic H),
6.63 (dd, J¼8.2, 2.3 Hz, 2H, aromatic H), 6.58 (t, J¼2.4 Hz, 1H, aro-
matic H), 6.01 (d, J¼8.8 Hz, 2H, NH), 5.21 (dd, J¼10.6, 9.3 Hz, 2H, H-
3), 5.20e5.12 (m, 4H, ArOCH2), 5.03 (dd, J¼10.0, 9.3 Hz, 2H, H-4),
4.65 (d, J¼8.4 Hz, 2H, H-1), 4.64e4.59 (m, 2H, OCH2CH2triazole),
4.50 (ddd, J¼14.5, 9.0, 3.2 Hz, 2H, OCH2CH2triazole), 4.26e4.23 (m,
2H, OCH2CH2triazole), 4.21 (dd, J¼12.4, 4.8 Hz, 2H, H-6a), 4.12 (dd,
J¼12.3, 2.4 Hz, 2H, H-6b), 3.91 (ddd, J¼11.2, 9.1, 2.9 Hz, 2H,
OCH2CH2triazole), 3.87 (dt, J¼10.7, 8.6 Hz, 2H, H-2), 3.68 (ddd,
J¼10.0, 4.8, 2.4 Hz, 2H, H-5), 2.08, 2.01, 2.01 (each 6H, each s, each
d
170.03 (C]O), 156.89 (Oaromatic C), 142.86 (CCH, triazole), 138.51
(Ar2eC]CeAr2), 136.95 (aromatic C), 132.46 (aromatic CH), 125.15
(CCH, triazole), 114.33 (aromatic CH), 84.49 (C-1), 74.19 (C-5), 71.42
(C-4), 71.08 (C-3), 61.46 (CH2OAr), 61.31 (C-6), 54.60 (C-2), 49.76
(SCH2CH2triazole), 30.56 (SCH2CH2triazole), 23.03 (NHAc); ES-
HRMS calcd for
C
78H100N16O24S4Na1 1795.5877, found m/z
OAc), 1.84 (s, 6H, NHAc); 13C NMR (126 MHz, CDCl3)
d 170.82,170.61,
1795.5903 [MþNa]þ; IR (ATR, cmꢁ1): 3279, 1644, 1506, 1235, 1175,
170.61, 169.33 (each C]O), 159.28 (aromatic C), 143.57 (CCH, tri-
azole), 130.24 (aromatic CH), 124.54 (CCH, triazole), 107.88 (aro-
matic CH), 102.20 (aromatic CH), 100.59 (C-1), 72.09 (C-3), 71.92 (C-
5), 68.43 (C-4), 67.30 (OCH2CH2triazole), 61.92 (CH2OAr), 61.76 (C-
6), 54.34 (C-2), 50.14 (OCH2CH2triazole), 23.16 (NHAc), 20.74, 20.64,
20.60 (each OAc); ES-HRMS calcd for C44H58N8O20Na1 1041.3665,
found m/z 1041.3691 [MþNa]þ; IR (ATR, cmꢁ1): 1740, 1659, 1369,
1256, 1153, 1038, 733; Rf: 0.43 (2:23 MeOH/CH2Cl2). Deacetylation
of this intermediate (303 mg, 0.297 mmol) as described in the
preparation of 1 gave the title compound 3 (146 mg, 64%) as a white
1051, 1004, 830, 761; Rf: 0.62 (1:1 MeCN/H2O, reverse-phase silica
gel); [
a
]
20 þ132.6 (c 0.33, DMSO).
D
4.11. 1,4-Di[1-(ethyl 2-acetamido-2-deoxy-b-D-glucopyr-
anosyl)-1,2,3-triazol-4-ylmethyloxy]benzene 4
Reaction of azide 16 (320 mg, 0.769 mmol) and 19 (68 mg,
0.365 mmol) as described in the preparation of 1 gave, after
chromatography (95:5 CH2Cl2/MeOH), the acetylated intermediate
(315 mg, 85%) as a white solid; 1H NMR (500 MHz, CDCl3)
d
7.74 (s,
solid; 1H NMR (500 MHz, DMSO-d6)
d 8.07 (s, 2H, CCH, triazole),
2H, CCH, triazole), 6.92 (s, 4H, aromatic H), 5.68 (d, J¼8.9 Hz, 2H,
NH), 5.16 (dd, J¼10.7, 9.3 Hz, 2H, H-3), 5.13 (d, J¼2.5 Hz, 4H,
ArOCH2), 5.06 (t, J¼9.6 Hz, 2H, H-4), 4.62 (ddd, J¼14.5, 4.5, 2.9 Hz,
2H, OCH2CH2triazole), 4.55 (d, J¼8.4 Hz, 2H, H-1), 4.50 (ddd,
J¼14.5, 9.0, 3.2 Hz, 2H, OCH2CH2triazole), 4.27e4.23 (m, 2H,
OCH2CH2triazole), 4.23 (dd, J¼12.3, 4.7 Hz, 2H, H-6a), 4.13 (dd,
J¼12.3, 2.4 Hz, 2H, H-6b), 3.94 (dt, J¼10.6, 8.6 Hz, 2H, H-2),
3.92e3.87 (m, 2H, OCH2CH2triazole), 3.67 (ddd, J¼10.0, 4.7, 2.4 Hz,
2H, H-5), 2.08, 2.02, 2.01 (each s, each 6H, each OAc), 1.84 (s, 6H,
7.62 (d, J¼9.0 Hz, 2H, NH), 7.20 (t, J¼8.2 Hz, 1H, aromatic H), 6.71 (t,
J¼2.4 Hz, 1H, aromatic H), 6.63 (dd, J¼8.2, 2.3 Hz, 2H, aromatic H),
5.08 (s, 4H, ArOCH2), 4.56 (ddd, J¼14.5, 5.8, 3.8 Hz, 2H, OCH2CH2
-
triazole), 4.50 (ddd, J¼14.4, 7.4, 3.8 Hz, 2H, OCH2CH2triazole), 4.31
(d, J¼8.5 Hz, 2H, H-1), 4.06 (ddd, J¼11.2, 5.8, 3.8 Hz, 2H,
OCH2CH2triazole), 3.80 (ddd, J¼11.1, 7.4, 3.8 Hz, 2H, OCH2CH2
-
triazole), 3.68 (dd, J¼11.9, 2.0 Hz, 2H, H-6a), 3.46e3.39 (m, 4H, H-2,
H-6b), 3.24 (dd, J¼10.3, 8.4 Hz, 2H, H-3), 3.10 (ddd, J¼9.8, 5.9,
2.0 Hz, 2H, H-5), 3.08e3.01 (m, 2H, H-4), 1.74 (s, 6H, NHAc); 13C
NHAc); 13C NMR (126 MHz, CDCl3)
d
170.93, 170.62, 170.42, 169.30
NMR (126 MHz, DMSO) d 169.68 (C]O), 159.74 (aromatic C), 142.79
(each C]O), 152.72 (aromatic C), 143.91 (CCH, triazole), 124.43
(CCH, triazole), 115.87 (aromatic CH), 100.73 (C-1), 72.17 (C-3),
71.98 (C-5), 68.28 (C-4), 67.32 (OCH2CH2triazole), 62.40 (CH2OAr),
61.87 (C-6), 54.21 (C-2), 50.10 (OCH2CH2triazole), 23.18 (NHAc),
20.74, 20.63, 20.59 (each OAc); ES-HRMS calcd for C44H58N8O20Na
1041.3665, found m/z 1041.3674 [MþNa]þ; IR (ATR, cmꢁ1): 1742,
1662, 1592, 1508, 1490, 1374, 1231, 1219, 1039, 1003, 734; Rf: 0.43
(2:23 MeOH/CH2Cl2). Deacetylation of this intermediate (310 mg,
0.304 mmol) as described in preparation of 1 gave the title com-
pound 4 (207 mg, 89%) as a white solid; 1H NMR (500 MHz,
(CCH, triazole), 130.47 (aromatic CH), 125.51 (CCH, triazole), 107.62
(aromatic CH), 102.00 (aromatic CH), 101.14 (C-1), 77.53 (C-5), 74.56
(C-3), 71.00 (C-4), 66.96 (OCH2CH2triazole), 61.59 (CH2OAr), 61.49
(C-6), 55.55 (C-2), 49.98 (OCH2CH2triazole), 23.45 (NHAc); ES-
HRMS calcd for C32H46N8O14Na 789.3031, found m/z 789.3037
[MþNa]þ; IR (ATR, cmꢁ1): 3269, 1644, 1605, 1547, 1294, 1185, 1161,
1054, 1028, 825, 765; Rf: 0.61 (1:1 MeCN/H2O, reverse-phase silica
20
gel); [
a
]
ꢁ30.0 (c 0.34, DMSO).
D
4.13. Reaction of azide 17 with tripropargylamine 2322
DMSO-d6)
d
8.05 (s, 2H, CCH, triazole), 7.62 (d, J¼9.0 Hz, 2H, NH),
6.97 (s, 4H, aromatic H), 5.03 (s, 4H, ArOCH2), 4.55 (ddd, J¼14.5,
5.8, 3.8 Hz, 2H, OCH2CH2triazole), 4.49 (ddd, J¼14.5, 7.4, 3.8 Hz,
2H, OCH2CH2triazole), 4.31 (d, J¼8.5 Hz, 2H, H-1), 4.06 (ddd,
J¼11.2, 5.8, 3.8 Hz, 2H, OCH2CH2triazole), 3.79 (ddd, J¼11.1, 7.4,
3.7 Hz, 2H, OCH2CH2triazole), 3.68 (dd, J¼11.9, 2.0 Hz, 2H, H-6a),
3.46e3.40 (m, 4H, H-2, H-6b), 3.25 (dd, J¼10.2, 8.3 Hz, 2H, H-3),
3.10 (ddd, J¼10.0, 5.9, 2.0 Hz, 2H, H-5), 3.05 (dd, J¼9.7, 8.3 Hz, 2H,
To azide 17 (251 mg, 0.580 mmol) in THF/H2O (1:1, 10 mL) were
added sodium ascorbate (22 mg, 0.111 mmol) and copper sulfate
pentahydrate (27 mg, 0.108 mmol) and tripropargylamine (38 ml,
0.25 mmol). The mixture was stirred at room temperature for 18 h.
Tetrahydrofuran was then removed under diminished pressure
followed by the dilution of the solution with CH2Cl2. This mixture
was then washed with water. The aqueous layer was re-extracted
with a further portion of CH2Cl2. The combined organic layers
were then washed with water, dried over Na2SO4 and the solvent
removed. Chromatography of the residue (95:5 CH2Cl2/MeOH) gave
the monomeric product 24 (49 mg, 36%) as a white solid, the di-
meric intermediate (131 mg, 51%) as a white solid, and the trimeric
intermediate (46vmg, 13%) as a white solid. Analytical data for 24:
H-4), 1.74 (s, 6H, NHAc); 13C NMR (126 MHz, DMSO)
d 169.67 (C]
O), 152.80 (aromatic C), 143.07 (CCH, triazole), 125.40 (CCH, tri-
azole), 115.97 (aromatic CH), 101.15 (C-1), 77.53 (C-5), 74.56 (C-3),
71.00 (C-4), 66.97 (OCH2CH2triazole), 62.01 (CH2OAr), 61.48 (C-6),
55.55 (C-2), 49.96 (OCH2CH2triazole), 23.45 (NHAc); ES-HRMS
calcd for
C32H46N8O14Na 789.3031, found m/z 789.3014
[MþNa]þ; IR (ATR, cmꢁ1): 3283, 644, 1549, 1512, 1375, 1236, 1226,
1H NMR (500 MHz, CDCl3)
d 7.57 (s, 1H, CCH, triazole), 5.85 (d,
1101, 1055, 1030, 822, 806, 708; Rf: 0.70 (1:1 MeCN/H2O, reverse-
phase silica gel); [
J¼8.1 Hz, 1H, NH), 5.55 (d, J¼5.4 Hz, 1H, H-1), 5.11 (t, J¼9.6 Hz, 1H,
20
a]
ꢁ32.1 (c 0.41, DMSO).
H-4), 5.01 (dd, J¼11.2, 9.3 Hz, 1H, H-3), 4.57 (td, J¼6.8, 1.5 Hz, 2H,
D
ꢀ
Please cite this article in press as: Andre, S.; et al., Tetrahedron (2015), http://dx.doi.org/10.1016/j.tet.2015.07.020

ꢀ
S. Andre et al. / Tetrahedron xxx (2015) 1e14
12
SCH2CH2triazole), 4.46 (ddd, J¼11.1, 8.1, 5.4 Hz, 1H, H-2), 4.30 (ddd,
J¼9.9, 4.7, 2.1 Hz, 1H, H-5), 4.26 (dd, J¼12.2, 4.7 Hz, 1H, H-6a), 4.12
(dd, J¼12.3, 2.1 Hz, 1H, H-6b), 3.86 (s, 2H, NCH2triazole), 3.46 (d,
J¼2.4 Hz, 4H, NCH2CCH), 3.16 (dt, J¼13.6, 6.7 Hz, 1H, SCH2CH2tria-
zole), 3.07 (dt, J¼14.0, 6.9 Hz, 1H, SCH2CH2triazole), 2.27 (t,
J¼2.4 Hz, 2H, NCH2CCH), 2.07 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H) (each
NMR (126 MHz, DMSO) d 170.04 (C]O), 143.53 (CCH, triazole),
124.58 (CCH, triazole), 84.36 (C-1), 74.13 (C-5), 71.38 (C-4), 71.06 (C-
3), 61.28 (C-6), 54.57 (C-2), 49.64 (SCH2CH2triazole), 47.78
(NCH2triazole), 41.38 (NCH2CCH), 40.49 (NCH2CCH), 30.50
(SCH2CH2triazole),
23.01
(NHAc);
ES-HRMS
calcd
for
C
29H46N9O10S2 744.2809, found m/z 744.2811 [MþH]þ; IR (ATR,
OAc), 1.97 (s, 3H, NHAc); 13C NMR (126 MHz, CDCl3)
d
171.74,170.54,
cmꢁ1): 3278, 1645, 1544, 1431, 1373, 1299, 1097, 1051, 1030, 1005,
170.23, 169.22 (each C]O), 144.76 (CCH, triazole), 123.18 (CCH,
triazole), 84.62 (C-1), 78.47 (NCH2CCH), 73.49 (NCH2CCH), 70.99 (C-
3), 68.74 (C-5), 67.92 (C-4), 61.98 (C-6), 52.69 (C-2), 49.44
(SCH2CH2triazole), 47.99 (NCH2triazole), 41.97 (NCH2CCH), 31.48
(SCH2CH2triazole), 23.19 (NHAc), 20.70, 20.69, 20.57 (each OAc);
ES-HRMS calcd for C25H34N5O8S 564.2128, found m/z 564.2116
[MþH]þ; IR (ATR, cmꢁ1): 3280, 1742, 1665, 1367, 1227, 1090, 1040,
910, 729; Rf:0.35 (1:19 MeOH/CH2Cl2). Analytical data for dimeric
848, 761; [a]
20 165.9 (c 0.14, DMSO).
D
4.15. N,N,N-Tris[1-(1-(ethyl 2-acetamido-2-deoxy-1-thio-a-D-
glucoopyranosyl)-1,2,3-triazol-4-ylmethyl)]amine 10
Deacetylation of 26 (100 mg, 0.070 mmol) as described in the
preparation of 1 gave the title compound 10 (44 mg, 59%) as a white
solid; 1H NMR (500 MHz, DMSO-d6)
d 8.02 (s, 3H, CCH, triazole),
intermediate 25: 1H NMR (500 MHz, CDCl3)
d
7.66 (s, 2H, CCH,
7.83 (d, J¼7.0 Hz, 3H, NH), 5.44 (d, J¼5.2 Hz, 3H, H-1), 5.11 (d,
J¼5.6 Hz, 3H, OH-4), 4.81 (d, J¼5.7 Hz, 3H, OH-3), 4.65 (t, J¼5.8 Hz,
3H, OH-6), 4.58 (dt, J¼13.8, 6.9 Hz, 3H, SCH2CH2triazole), 4.50 (dt,
J¼14.0, 7.1 Hz, 3H, SCH2CH2triazole), 3.79 (ddd, J¼11.8, 7.0, 5.3 Hz,
3H, H-2), 3.75e3.66 (m, 6H, H-5, H-6a), 3.61 (s, 6H, NCH2triazole),
3.49 (dt, J¼11.9, 6.2 Hz, 3H, H-6b), 3.34 (ddd, J¼10.8, 8.7, 5.8 Hz, 3H,
H-3), 3.11 (ddd, J¼9.9, 8.4, 5.6 Hz, 3H, H-4), 3.04 (dt, J¼14.0, 7.1 Hz,
3H, SCH2CH2triazole), 2.96 (dt, J¼13.8, 7.0 Hz, 3H, SCH2CH2triazole),
triazole), 6.13 (d, J¼8.1 Hz, 2H, NH), 5.55 (d, J¼5.4 Hz, 2H, H-1), 5.10
(t, J¼9.6 Hz, 2H, H-4), 5.00 (dd, J¼11.2, 9.3 Hz, 2H, H-3), 4.64e4.51
(m, 4H, SCH2CH2triazole), 4.47 (ddd, J¼11.2, 8.1, 5.4 Hz, 2H, H-2),
4.30 (ddd, J¼9.9, 4.7, 2.2 Hz, 2H, H-5), 4.26 (dd, J¼12.2, 4.6 Hz, 2H,
H-6a), 4.12 (dd, J¼12.2, 2.1 Hz, 2H, H-6b), 3.85 (d, J¼2.1 Hz, 4H,
NCH2triazole), 3.35 (dd, J¼6.7, 2.4 Hz, 2H, NCH2CCH), 3.16 (dt,
J¼14.2, 6.4 Hz, 2H, SCH2CH2triazole), 3.07 (dt, J¼14.0, 6.9 Hz, 2H,
SCH2CH2triazole), 2.31 (t, J¼2.2 Hz, 1H, NCH2CCH), 2.07 (s, 6H), 2.03
(s, 6H), 2.02 (s, 6H) (each OAc), 1.97 (s, 6H, NHAc); 13C NMR
1.78 (s, 9H, NHAc); 13C NMR (126 MHz, DMSO)
d 170.04 (C]O),
143.67 (CCH, triazole), 124.58 (CCH, triazole), 84.41 (C-1), 74.15 (C-
5), 71.39 (C-4), 71.08 (C-3), 61.29 (C-6), 54.59 (C-2), 49.68
(SCH2CH2triazole), 47.39 (NCH2triazole), 30.55 (SCH2CH2triazole),
23.00 (NHAc); ES-HRMS calcd for C39H64N13O17S3 1094.3705, found
m/z 1094.3727 [MþFAꢁH]ꢁ; IR (ATR, cmꢁ1): 3277, 1645, 1544, 1432,
(126 MHz, CDCl3)
d 171.59, 170.57, 170.34, 169.26 (each C]O),
144.45 (CCH, triazole), 123.67 (CCH, triazole), 84.51 (C-1), 78.25
(NCH2CCH), 73.96 (NCH2CCH), 70.90 (C-3), 68.70 (C-5), 68.09 (C-4),
61.96 (C-6), 52.57 (C-2), 49.37 (SCH2CH2triazole), 47.76 (NCH2tria-
zole), 42.28 (NCH2CCH), 31.40 (SCH2CH2triazole), 23.13 (NHAc),
20.71, 20.71, 20.58 (each OAc); ES-HRMS calcd for C41H57N9O16S2Na
1018.3262, found m/z 1018.3289 [MþNa]þ; IR (ATR, cmꢁ1): 1742,
1665, 1535, 1367, 1227, 1090, 1040, 910, 729; Rf: 0.25 (1:19 MeOH/
CH2Cl2). Analytical data for trimeric intermediate 26: 1H NMR
1373, 1300, 1221, 1097, 1051, 1028, 1005, 848, 762; [
DMSO).
a]
20 191.5 (c 0.14,
D
4.16. 1,4-Di[1-(ethyl 2-acetamido-2-deoxy-1-thio-b-D-gal-
actopyranosyl)-1,2,3-triazol-4-ylmethyloxy]benzene 11
(500 MHz, CDCl3)
d
7.85 (s, 3H, CCH, triazole), 6.29 (d, J¼8.2 Hz, 3H,
NH), 5.54 (d, J¼5.4 Hz, 3H, H-1), 5.09 (t, J¼9.6 Hz, 3H, H-4), 5.01 (dd,
J¼11.1, 9.2 Hz, 3H, H-3), 4.60 (ddq, J¼20.8, 13.9, 6.5 Hz, 6H,
SCH2CH2triazole), 4.48 (ddd, J¼11.1, 8.2, 5.4 Hz, 3H, H-2), 4.30 (ddd,
J¼9.9, 4.6, 2.2 Hz, 3H, H-5), 4.26 (dd, J¼12.3, 4.6 Hz, 3H, H-6a), 4.12
(dd, J¼12.4, 2.2 Hz, 3H, H-6b), 3.74 (q, J¼14.2 Hz, 6H, NCH2triazole),
3.18 (dt, J¼14.2, 6.3 Hz, 3H, SCH2CH2triazole), 3.09 (dt, J¼13.9,
6.8 Hz, 3H, SCH2CH2triazole), 2.07 (s, 9H), 2.02 (s, 9H), 2.00 (s, 9H)
Reaction of azide 18 (215 mg, 0.497 mmol) and 19 (44 mg,
0.236 mmol) as described in the preparation of 1 gave, after chro-
matography (95:5 CH2Cl2/MeOH), the acetylated intermediate
(232 mg, 94%) as a white solid; 1H NMR (500 MHz, CDCl3)
d 7.72 (s,
2H, CCH, triazole), 6.91 (s, 4H, AreH), 5.81 (d, J¼8.7 Hz, 2H, NH),
5.37 (dd, J¼3.4, 1.2 Hz, 2H, H-4), 5.17 (s, 4H, ArOCH2), 5.04 (dd,
J¼10.2, 3.2 Hz, 2H, H-3), 4.70 (dt, J¼13.0, 6.2 Hz, 2H, SCH2CH2tria-
zole), 4.57 (dt, J¼13.8, 6.7 Hz, 2H, SCH2CH2triazole), 4.30e4.24 (m,
4H, H-1, H-2), 4.11 (dd, J¼6.5, 2.5 Hz, 4H, H-6a, H-6b), 3.87 (td,
J¼6.2, 1.1 Hz, 2H, H-5), 3.33 (dt, J¼14.6, 6.4 Hz, 2H, SCH2CH2tria-
zole), 3.07 (dt, J¼14.5, 6.3 Hz, 2H, SCH2CH2triazole), 2.16 (s, 6H),
2.03 (s, 6H), 1.99 (s, 6H) (each OAc), 1.94 (s, 6H, NHAc); 13C NMR
(each OAc), 1.97 (s, 9H, NHAc); 13C NMR (126 MHz, CDCl3)
d 171.48,
170.60, 170.42, 169.28 (each C]O), 143.65 (CCH, triazole), 124.44
(CCH, triazole), 84.55 (C-1), 70.84 (C-3), 68.67 (C-5), 68.20 (C-4),
61.97 (C-6), 52.47 (C-2), 49.47 (SCH2CH2triazole), 47.12 (NCH2tria-
zole), 31.40 (SCH2CH2triazole), 23.11 (NHAc), 20.72, 20.71 20.57
(each OAc); ES-HRMS calcd for C57H82N13O24S3 1428.4758, found
m/z 1428.4789 [MþH]þ; IR (ATR, cmꢁ1): 1742, 1665, 1535, 1367,
1227, 1090, 1042, 912, 730; Rf: 0.18 (1:19 MeOH/CH2Cl2).
(126 MHz, CDCl3)
d 170.52, 170.45, 170.40, 170.15 (each C]O),
152.71 (AreC), 143.83 (CCH, triazole), 124.14 (CCH, triazole), 116.16
(AreCH), 84.98 (C-1), 74.77 (C-5), 71.11 (C-3), 66.82 (C-4), 62.70
(CH2OAr), 61.74 (C-6), 50.54 (SCH2CH2triazole), 49.10 (C-2), 30.28
(SCH2CH2triazole), 23.26 (NHAc), 20.72, 20.66, 20.65 (each OAc);
4.14. N,N-Bis[1-(1-(ethyl 2-acetamido-2-deoxy-1-thio-a-D-
glucopyranosyl)-1,2,3-triazol-4-ylmethyl)]-N-prop-2-yn-1-
amine 9
ES-HRMS calcd for C44H58N8O18S2Na1 1073.3208, found m/z
1073.3197 [MþNa]þ; IR (ATR, cmꢁ1): 1746, 1663, 1508, 1371, 1234,
1054, 1032, 1016, 749; Rf: 0.38 (2:23 MeOH/CH2Cl2). Deacetylation
of this intermediate (25 mg, 0.024 mmol) as described in the
preparation of 1 gave the title compound 11 (16 mg, 84%) as a white
Deacetylation of 25 (117 mg, 0.117 mmol) as described in the
preparation of 1 gave the title compound (52 mg, 60%) as an off-
white solid; 1H NMR (500 MHz, DMSO-d6)
d
7.98 (s, 2H, CCH, tri-
solid; 1H NMR (500 MHz, DMSO-d6)
d 8.17 (s, 2H, CCH, triazole),
azole), 7.82 (d, J¼7.0 Hz, 2H, NH), 5.43 (d, J¼5.3 Hz, 2H, H-1), 5.11 (br
s, 2H, OH), 4.80 (br s, 2H, OH), 4.63 (br s, 2H, OH), 4.56 (dt, J¼13.8,
6.9 Hz, 2H, SCH2CH2triazole), 4.48 (dt, J¼14.0, 7.1 Hz, 2H,
SCH2CH2triazole), 3.79 (ddd, J¼11.7, 7.0, 5.2 Hz, 2H, H-2), 3.74e3.66
(m, 8H, H-5, H-6a, NCH2triazole), 3.48 (dd, J¼12.0, 6.3 Hz, 2H, H-
6b), 3.31 (m, 4H, H-3, NCH2CCH), 3.11 (t, J¼9.3 Hz, 2H, H-4), 3.03 (dt,
J¼13.9, 7.0 Hz, 2H, SCH2CH2triazole), 2.94 (dt, J¼13.8, 6.9 Hz, 2H,
SCH2CH2triazole), 2.48 (m, 1H, NCH2CCH), 1.80 (s, 6H, NHAc); 13C
7.64 (d, J¼9.5 Hz, 2H, NH), 6.96 (s, 4H, AreH), 5.05 (s, 4H, ArOCH2),
4.71 (d, J¼6.2 Hz, 2H, OH-3), 4.63 (d, J¼5.5 Hz, 1H, OH-6), 4.61 (d,
J¼4.5 Hz, 1H, OH-4), 4.61e4.56 (m, 4H, SCH2CH2triazole), 4.35 (d,
J¼10.3 Hz, 2H, H-1), 3.89 (q, J¼10.0 Hz, 2H, H-2), 3.69 (t, J¼3.6 Hz,
2H, H-4), 3.52 (t, J¼5.4 Hz, 4H, H-6a, H-6b), 3.47e3.36 (m, 4H, H-3,
H-5), 3.20 (dt, J¼13.7, 6.7 Hz, 2H, SCH2CH2triazole), 2.94 (dt, J¼14.1,
7.1 Hz, 2H, SCH2CH2triazole),1.78 (s, 6H, NHAc); 13C NMR (126 MHz,
DMSO)
d 169.74 (C]O), 152.80 (AreC), 142.94 (CCH, triazole),
ꢀ
Please cite this article in press as: Andre, S.; et al., Tetrahedron (2015), http://dx.doi.org/10.1016/j.tet.2015.07.020

ꢀ
S. Andre et al. / Tetrahedron xxx (2015) 1e14
13
2009; pp 31e52; (c) Oscarson, S. In The Sugar Code. Fundamentals of Glyco-
sciences; Gabius, H.-J., Ed.; Wiley-VCH: Weinheim, Germany, 2009; pp 53e70;
(d) Kiessling, L. L.; Splain, R. A. Annu. Rev. Biochem. 2010, 79, 619e653; (e)
125.37 (CCH, triazole), 116.03 (AreCH), 85.16 (C-1), 79.91 (C-5),
72.77 (C-3), 68.16 (C-4), 62.07 (CH2OAr), 61.25 (C-6), 50.80 (C-2),
50.13 (SCH2CH2triazole), 30.24 (SCH2CH2triazole), 23.51 (NHAc);
ES-HRMS calcd for C32H46N8O12S2Na 821.2574, found m/z 821.2551
[MþNa]þ; IR (ATR, cmꢁ1): 3271, 1635, 1549, 1507, 1372, 1312, 1225,
ꢀ
Murphy, P. V.; Andre, S.; Gabius, H.-J. Molecules 2013, 18, 4026e4053; (f) Solís,
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D.; Bovin, N. V.; Davis, A. P.; Jimenez-Barbero, J.; Romero, A.; Roy, R.; Smetana, K.
, Jr.; Gabius, H.-J. Biochim. Biophys. Acta 2015, 1850, 186e235; (g) Gabius, H.-J.
Trends Biochem. Sci. 2015, 40, 341.
1115, 1041, 1016, 978, 864, 820, 705; Rf: 0.68 (1:1 MeCN/H2O, re-
2. (a) Gready, J. E.; Zelensky, A. N. In The Sugar Code. Fundamentals of Glyco-
sciences; Gabius, H.-J., Ed.; Wiley-VCH: Weinheim, Germany, 2009;
pp 329e345; (b) Kaltner, H.; Gabius, H.-J. Histol. Histopathol. 2012, 27, 397e416;
20
verse-phase silica gel); [
a
]
8.3 (c 0.17, DMSO).
D
ꢀ
(c) Dawson, H.; Andre, S.; Karamitopoulou, E.; Zlobec, I.; Gabius, H.-J. Anticancer
4.17. Lectin purification and biotinylation
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Res. 2013, 33, 3055e3059; (d) Katzenmaier, E.-M.; Andre, S.; Kopitz, J.; Gabius,
H.-J. Anticancer Res. 2014, 34, 5429e5438; (e) Gabius, H.-J.; Kaltner, H.; Kopitz,
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WGA was purified by affinity chromatography, after gel elec-
trophoretic controls for purity biotinylated under activity-
preserving conditions and checked for bioactivity by haemag-
glutination.23 Labelled GSA-II was purchased from Vector
Laboratories.
3. (a) Lee, R. T.; Lee, Y. C. In Neoglycoconjugates. Preparation and Applications; Lee,
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Y. C., Lee, R. T., Eds.; Academic: San Diego, CA, 1994; pp 23e50; (b) Andre, S.;
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BioChem 2008, 9, 1649e1661; (c) Andre, S.; Jarikote, D. V.; Yan, D.; Vincenz, L.;
Wang, G.-N.; Kaltner, H.; Murphy, P. V.; Gabius, H.-J. Bioorg. Med. Chem. Lett.
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2012, 22, 313e318; (d) Andre, S.; Wang, G.-N.; Gabius, H.-J.; Murphy, P. V.
Carbohydr. Res. 2014, 389, 25e38.
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Res. 2006, 26, 4293e4298; (b) Satomaa, T.; Heiskanen, A.; Leonardsson, I.;
€
Angstrom, J.; Olonen, A.; Blomqvist, M.; Salovuori, N.; Haglund, C.; Teneberg, S.;
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Natunen, J.; Carpen, O.; Saarinen, J. Cancer Res. 2009, 69, 5811e5819; (c) North,
As lectin ligands, the glycoproteins fetuin, ASF or the agalacto
S. J.; von Gunten, S.; Antonopoulos, A.; Trollope, A.; MacGlashan, D. W., Jr.; Jang-
Lee, J.; Dell, A.; Metcalfe, D. D.; Kirshenbaum, A. S.; Bochner, B. S.; Haslam, S. M.
Glycobiology 2012, 22, 12e22.
version of ASF obtained by treatment with
as the neoglycoproteins based on bovine serum albumin presenting
22e26 -GlcNAc (or -GalNAc as control) moieties as p-iso-
thiocyanatophenyl derivative were adsorbed to the surface of
plastic microtiter plate wells, using a solution of 50 l phosphate-
b-galactosidase as well
5. (a) East, L.; Isacke, C. M. Biochim. Biophys. Acta 2002, 1572, 364e386; (b) Lu, J.;
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b
b
m
ꢀ
V.; Smith, D. F.; Lacroix, M.; Dumestre-Perard, C.; Lunardi, T.; Martin, L.; Ces-
buffered saline overnight at 4 ꢀC. Remaining sites for protein
binding were saturated with a solution of carbohydrate-free albu-
min (1% (w/v)) at 37 ꢀC for 1 h. The binding assay was carried out in
successive steps, starting with an incubation for 1 h at 37 ꢀC using
the biotinylated lectin without/with test compounds, continued
with washing, and the application of the indicator conjugate
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W.; Drickamer, K.; Weis, W. I.; Taylor, M. E. J. Biol. Chem. 2013, 288,
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streptavidin-peroxidase (0.5
spectrophotometric quantitation at 490 nm was developed enzy-
m
g/mL; Sigma). Finally, the signal for
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