Defining the potential of aglycone modifications for affinity/selectivity enhancement against medically relevant lectins: Synthesis, activity screening, and HSQC-Based NMR Analysis

Article abstract of DOI:10.1002/cbic.201402474

The emerging significance of lectins for pathophysiological processes provides incentive for the design of potent inhibitors. To this end, systematic assessment of contributions to affinity and selectivity by distinct types of synthetic tailoring of glyco

Full text of DOI:10.1002/cbic.201402474

CHEMBIOCHEM
FULL PAPERS
DOI: 10.1002/cbic.201402474
Defining the Potential of Aglycone Modifications for
Affinity/Selectivity Enhancement against Medically
Relevant Lectins: Synthesis, Activity Screening, and HSQC-
Based NMR Analysis
[
a]
[a]
[b]
[c]
[a]
Subhash R. Rauthu, Tze Chieh Shiao, Sabine Andrꢀ, Michelle C. Miller, ꢁlodie Madej,
[
c]
[b]
[a]
Kevin H. Mayo, Hans-Joachim Gabius, and Renꢀ Roy*
The emerging significance of lectins for pathophysiological
processes provides incentive for the design of potent inhibi-
tors. To this end, systematic assessment of contributions to af-
finity and selectivity by distinct types of synthetic tailoring of
glycosides is a salient step, here taken for the aglyconic modifi-
cations of two disaccharide core structures. Firstly we report
the synthesis of seven N-linked-lactosides and of eight O-
linked N-acetyllactosamines, each substituted with a 1,2,3-tri-
azole unit, prepared by copper-catalyzed azide–alkyne cycload-
dition (CuAAC). The totally regioselective b-d-(1!4) galactosy-
lation of a 6-O-TBDPSi-protected N-acetylglucosamine acceptor
provided efficient access to the N-acetyllactosamine precursor.
The resulting compounds were then systematically tested for
lectin reactivity in two binding assays of increasing biorelev-
ance (inhibition of lectin binding to a surface-presented glyco-
protein and to cell surfaces). As well as a plant toxin, we also
screened the relative inhibitory potential with adhesion/
growth-regulatory galectins (total of eight proteins). This type
of modification yielded up to 2.5-fold enhancement for proto-
type proteins, with further increases for galectins-3 and -4.
15
Moreover, the availability of N-labeled proteins and full as-
1
15
signments enabled H, N HSQC-based measurements for hu-
man galectins-1, -3, and -7 against p-nitrophenyl lactopyrano-
side, a frequently tested standard inhibitor containing an aro-
matic aglycone. The measurements confirmed the highest af-
finity against galectin-3 and detected chemical shift differences
in its hydrophobic core upon ligand binding, besides common
alterations around the canonical contact site for the lactoside
residue. What can be accomplished in terms of affinity/selectiv-
ity by this type of core extension having been determined, the
applied combined strategy should be instrumental for pro-
ceeding with defining structure–activity correlations at other
bioinspired sites in glycans and beyond the tested lectin types.
Introduction
The discovery of new ways in which structural information of
biomolecules is translated into cellular functions is the starting
point for the design of new classes of biopharmaceuticals to
turn these insights into practical applications. Glycans of cellu-
lar glycoconjugates constitute versatile platforms for encoding
of a plethora of signals, as embodied in the term “sugar
position in the flow of glycan-based information qualifies lec-
tins as pharmaceutical targets, with the aim to develop com-
pounds that interfere potently and selectively with glycan–
[3–6]
lectin recognition.
Steps in this direction can be successfully
taken, if the emerging rules of physiologic affinity regulation,
from the level of the minimum-contact site (mono- or disac-
[
1]
[2]
code”.
One route from glycan structures to bioactivity makes use of
charides) to supramolecular formation of glycan clusters, are
applied. Indeed, lessons learned from how the intriguingly
high natural levels of selectivity and specificity are achieved
hold promise for ligand (lead) optimization. Efforts in these
respects include investigation of the chemical structure of the
[
2]
specific carbohydrate-binding receptors (lectins). This central
[a] Dr. S. R. Rauthu, T. C. Shiao, ꢀ. Madej, Prof. R. Roy
PharmaQAM, Department of Chemistry, Universitꢁ du Quꢁbec ꢂ Montrꢁal
P.O. Box 8888, Succ. Centre-ville, Montrꢁal, QC, H3C 3P8 (Canada)
reactive region and spatial features (geometry, topology) of its
[7,8]
presentation, for example, by dendrimeric scaffolds.
Neces-
[
b] Priv.-Doz.Dr. S. Andrꢁ, Prof. H.-J. Gabius
sarily, each parameter should be examined separately in a sys-
tematic process to assemble a toolbox for rational design of
the most potent inhibitors.
Institute of Physiological Chemistry
Faculty of Veterinary Medicine, Ludwig-Maximilians-University
Veterinꢃrstrasse 13, 80539 Munich (Germany)
[
c] Dr. M. C. Miller, Prof. K. H. Mayo
Department of Biochemistry, Molecular Biology and Biophysics
of Minnesota Health Sciences Center
In this study we focus on testing synthetic extensions to a
disaccharide unit (lactose and N-acetyllactosamine, (Lac/
LacNAc) for two classes of medically relevant b-d-galactopyra-
noside-binding lectins: a plant toxin akin to ricin (Viscum
6
-155 Jackson Hall, 321 Church Street, Minneapolis, MN 55455 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201402474: H and C NMR spectra of all
new compounds.
[9]
1
13
album L. agglutinin, VAA) and adhesion/growth-regulatory
[10]
galectins.
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&
1
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
The plant toxin preferentially binds to the non-reducing-end
b-d-galactoside moieties of glycoconjugates, with its extension
by a p-nitrophenyl aglycone or a further sugar unit resulting in
potency. The obtained results defined the range for affinity en-
hancements and relative reactivity. They guided us to examine
structural aspects of the binding of p-nitrophenyl b-d-lactopyr-
anoside (PNPL, 45, Scheme 2, below), an established model
inhibitor containing an aromatic aglycone for human galectins,
[
11–14]
up to sixfold increases in inhibitory potency.
However, the
potential for affinity enhancements by elongation beyond the
[
15,16]
15
disaccharide moiety has so far appeared rather limited
in
In
in solution. The availability of N-labeled proteins—that is,
[3b]
[43–45]
1
15
comparison with modifications at the sugar backbone.
Gal-1, Gal-3 and Gal-7
—facilitated H, N HSQC analysis.
contrast, galectins can apparently accommodate structural
motifs beyond a disaccharide core, first inferred with p-amino/
nitrophenyl (or benzyl) derivatives of lactose/N-acetyllactos-
These measurements afforded a full view of ligand-induced
chemical shift differences, the first time that this type of NMR
analysis has been comparatively applied in studies on inhibitor
design for several members of a lectin family.
[
17–19]
amine (Lac/LacNAc).
Fittingly, LacNAc repeats in glycan
chains belong to the panel of natural counter-receptors, as
documented for mammalian galectins-1 and -3 (Gal-1 and Gal-
[
18,20–25]
3
).
In comparative terms, a marked susceptibility to pre- Results and Discussion
sentation of internal LacNAc units was noted for Gal-3 and Gal-
Synthetic part
[26–31]
9
, and also for Gal-4.
The binding to terminal and internal
LacNAc units was crystallographically verified for the N-termi-
Synthesis of triazole LacNAc derivatives: Our two series of com-
pounds encompassed eight O-LacNAc derivatives containing
1’-substituted triazole moieties attached through the 4’-posi-
tion (Scheme 1, compounds 15–22) and seven Lac derivatives
containing 4’-substituted triazole moieties attached through
the 1’-position (Scheme 2, below, compounds 30–32 and 41–
44).
[
32]
nal domain of Gal-9. Evidently, chemical tailoring of an agly-
cone beyond Lac/LacNAc follows this bioinspiration. Building
on the previously noted favorable effect of a triazole agly-
[
16,33–35]
cone,
two series of eight O-triazolated LacNAc deriva-
tives and seven N-lactose derivatives containing such agly-
cones substituted with aliphatic, cyclic, or aromatic groups
were synthesized. In order to map similarities and differences
of compound reactivity for galectins as a platform to plan se-
lectivity improvements, we first took advantage of the availa-
bility of the complete set of these proteins from one organism:
that is, chicken galectins (CGs), with the three homodimeric
family members (CG-1A, CG-1B, CG-2), as well as one chimera
The first synthetic route started from propargyl b-d-LacNAc
disaccharide 6, prepared through a highly regioselective glyco-
sylation (Scheme 1). Our strategy was based on the early ob-
[46]
servation that,
when
a
suitable N-acetylglucosamine
(GlcNAc) acceptor was protected with a bulky silyl ether
(TBDPSi) at O6, b-d-galactosylation of the remaining GlcNAc
O3 and O4 diol occurs preferentially at O4. Hence, known
prop-2-ynyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-b-d-gluco-
[
36–42]
type (CG-3), and one tandem-repeat type (CG-8).
Next,
human Gal-1 (prototype) and Gal-3 (chimera-type), as well as
the tandem-repeat-type Gal-4, were analyzed comparatively.
This was done first in a solid-phase assay with the glycoprotein
asialofetuin (ASF) as matrix for lectin binding and then by in-
hibition of lectin binding to cells, to assess relative inhibitory
[47,48]
pyranoside (2) was used as starting material.
It was readily
prepared in 74% yield from the peracetylated glucosamine
1 by treatment with propargyl alcohol and BF ·OEt catalyst
3
2
with a short reaction time in dichloromethane under reflux. It
Scheme 1. The synthetic route to propargyl LacNAc derivative 6 and to 1’-substituted 1H-1,2,3-triazoles as O-glycosides. a) HCꢀCCH
to reflux, 5 h (75%); b) NaOMe, MeOH, RT, 2 h (quant.); c) TBDPSCl, pyridine, RT, 12 h (98%); d) BF ·Et O, CH Cl
, ꢁ458C, 1 h (86%); e) R-N
CuSO , vit C, RT, 12 h (91–95%); f) TBAF/THF, 12 h; g) MeONa/MeOH, RT, 2 h (91–97%, 2 steps).
2
OH, BF
3
·Et
2
O, CH
2 2
Cl , 08C
3
2
2
2
3
, THF/H
2
O (1:1),
4
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&
2
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
[
49]
is worth mentioning that, as opposed to previous claims,
Synthesis of triazolyl lactoside derivatives: This synthetic route
[52]
our yield was essentially the same as that reported with use of
started from the known lactosyl azide 23 (Scheme 2). Firstly,
[
49,50]
the milder Yb(OTf) as catalyst.
After standard Zemplꢀn de-
23 was treated under the above CuAAC conditions (CuSO , as-
3
4
O-acetylation of 1 (NaOMe, MeOH, RT, 2 h, quant.), triol 3 was
protected at O6 with tert-butyldiphenylsilyl chloride (TBDPSCl,
C H N, RT, 12 h) to afford 4 in 98% yield. Regioselective glyco-
corbate, THF/H O 1:1, RT, 12 h, Method B) with commercially
2
available alkynyl alcohols 24–26 to afford triazoles 27–29 in
33, 99, and 94% yields, respectively. The yield of compound 27
was significantly improved, to 90%, when the cycloaddition
5
5
sylation of diol 4 with the galactosyl trichloroacetimidate
[
51]
donor 5
exclusively provided the LacNAc derivative 6, in
was carried out under microwave conditions (CuSO , ascorbate,
4
8
6% yield. Unambiguous linkage characterization was firmly
DMF, mw, 708C, 30 min, Method A). Zemplꢀn de-O-acetylation
as above led to the set of (lactosyl)-1H-1,2,3-triazoles 30–32 in
quantitative yields.
1
13
established by H and C NMR 2D homonuclear COSY and
HMQC spectroscopy and by assessing long-range coupling
between Gal-H1 and GlcNAc-C4 in the HMBC spectrum. Fig-
ures S1 and S2 in the Supporting Information illustrate full
high-field NMR spectra of compound 6 for the signal attribu-
tions, together with the proton and carbon connectivity (COSY,
HMQC, and HMBC). In addition, using the bulkier TBDPS pro-
Naphthyl analogue 41 was also readily prepared from azide
23, which was condensed with naphthyl propargyl ether 33
under the microwave CuAAC conditions described above to
provide peracetylated triazole derivative 37 (99%), which upon
acetate removal afforded 41 quantitatively. Similarly, peracety-
lated series 38–40 was obtained by the same procedure with
use of propargyl aryl ethers 34–36 in 96, 85, and 95% yields,
respectively. Zemplꢀn transesterification (NaOMe, MeOH) re-
sulted in triazoles 42–44 quantitatively.
The two test panels thus having been completed, the 15
synthetic compounds, unsubstituted Lac/LacNAc, and the de-
protected azide 23 were tested in inhibition assays to deter-
mine the impact of the structural additions on affinity/selectivi-
ty to lectin panels.
[
46]
tecting group at O6 afforded a better yield (86%) than the
7% obtained with a slightly less bulky TBDMS O6-protected
6
[
49]
silyl ether. In our opinion, this strategy represents the most
effective one published for the chemical synthesis of LacNAc
derivatives.
Turning 6 into the family of triazoles 7–14 was accomplished
in 91–95% yields by means of copper-catalyzed alkyne–azide
cycloaddition (CuAAC) under standard conditions (RꢁN ,
3
CuSO , sodium ascorbate, THF/H O 1:1, RT, 12 h). Removal of
4
2
the TBDPS protecting group (TBAF) followed by Zemplꢀn de-
O-acetylation as above afforded LacNAc triazoles 15–22
(Scheme 1).
Scheme 2. The synthetic route from lactosyl azide 23 to 4’-substituted 1H-1,2,3-triazoles as N-glycosides, together with the structure of p-nitrophenyl b-d-lac-
topyranoside (45) used in the HSQC NMR experiments. a) CuSO , vit C, DMF, microwave, 708C, 30 min; or b) THF/H O (1:1), CuSO , vit C, RT, 12 h; c) MeONa/
4 2 4
MeOH (quant.).
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&
3
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
Inhibition of lectin binding to glycoprotein and cell surfaces
In the first experimental setting, the glycoprotein asialofetuin,
a known ligand for (ga)lectins containing up to nine LacNAc
[
53,54]
termini on three N-glycan chains,
was adsorbed to the
plastic surface of microtiter plate wells. The branch ends of the
three mostly triantennary N-glycans per protein then served as
docking sites for the labeled lectins, and running the binding
assay in the presence of our panel of sugar derivatives was ex-
pected to assess their capacity to interfere with the glycopro-
tein–lectin interaction. At least, the compounds of the two sets
should reach the inhibitory capacity of unsubstituted lactose:
that is, 0.6 mm for VAA and CG-1A, 4–5 mm for CG-1B, CG-2,
and Gal-1, and 1.5–2 mm for the Gal-3 proteins, CG-8, and Gal-
4. In the search for the best-case scenario for each lectin, the
Gal-3 proteins (chicken and human) and Gal-4 proved most
susceptible to certain compounds, whereas the inhibitory ca-
pacities of the derivatives did not surpass the 2.5-fold thresh-
old for the plant toxin in any of the tested prototype galectins
or CG-8 (except for 44, with a threefold enhancement). In
detail, 18 showed improvements of about three- to fourfold
for CG-3 and Gal-3 at 0.4–0.5 mm, with 16, 17, and 22 being
a little less potent, and 43 and 44 perfoming similarly, in these
cases and especially for Gal-4 (at 0.25–0.3 mm). Thus, the sub-
stitutions did not impair binding capacity and could in several
cases enhance the relative activity to block lectin binding to
the surface-presented N-glycans. It is to be noted that these
cases concerned distinct proteins and so identify instances of
selectivity rather than affinity between galectins.
Figure 1. Cytofluorimetric determination of binding of CG-1A/Gal-1 to cell
surfaces and the effect of the presence of carbohydrate inhibitors on bind-
ing parameters. Control values representing marker-independent (back-
ground) staining and the 100% level are represented by the gray area and
the black line, respectively. Quantitative data on percentage of positive
cells/mean fluorescence intensity are added to each panel, and lines connect
these data to the corresponding scan profile. Numbers are printed from top
(background—gray) to bottom (100% level—black) in the order given in
ꢁ
1
each listing. A), B) Binding of biotinylated CG-1A (20 mgmL ) to CHO cells
in the presence of A) 5, 1, 0.5, and 0.1 mm lactose, and B) 0.5 mm of com-
To take the evaluations to the level of cells, we next exam-
ined the relative inhibitory potencies of our compounds on
lectin binding to cell-surface glycoconjugates. In fact, galectins
target distinct glycolipids/glycoproteins for triggering outside-
in signaling: for example, the a b -integrin on pancreas carci-
ꢁ
1
pounds 18, 20, and 30. C), D) Binding of FTC-labeled Gal-1 (20 mgmL ) to
the human pancreatic carcinoma (Capan-1 reconstituted for the expression
INK4a
of the tumor suppressor p16
) cells in the presence of C) 2 mm com-
pound 18 and 2 mm lactose, and D) 2 mm of compounds 20 and 30.
5
1
[
55]
noma cells for inducing anoikis. Interfering with binding on
this level would then have cell biological (therapeutic) conse-
quences.
aliquots of the same cell suspension to ensure identical cell pa-
rameters. Reflecting the consistently about twofold increase in
inhibitory activity on complex formation with glycoprotein in
the solid-phase assay, the signals in the cytofluorimetric assays
were similarly reduced (Figure 1B). Cell binding of the human
orthologue Gal-1 was slightly more sensitive to the presence
of the derivatives (Figure 1C, D).
Prior to these assays, toxicity had to be excluded. Increasing
the concentration up to 5 mm did not lead to any indication of
a negative impact on growth of human colon adenocarcinoma
(SW480) cells, and medium with 10 mm of compound 32 as
test case sustained cell growth at about 80% of the extent in
parallel controls, with and without lactose. Obviously, over this
concentration range, as used in the lectin assays, no cytotoxici-
ty was encountered, thus further pointing toward aglycone
modifications for improving the therapeutic potential.
The much stronger inhibitory capacity of compound 18 on
CG-3 binding is documented in Figure 2A. Compound 44
(tested at 0.25 mm vs. 1 mm Lac) reached maximum inhibition
for Gal-4 (Figure 2B).
The cell assays measured the extent of inhibition on lectin
binding that each derivative can provide. Data are expressed
as percentages of positive cells and mean fluorescence intensi-
ties. In each setting (cell line/lectin), a lectin concentration in
the linear range of signal increase was identified, and then the
assumed inhibitability of binding by sugar used as hapten was
ascertained, as convincingly shown for the prototype CG-1A in
Figure 1A. Starting from the 100% value, cell positivity step-
wisely decreased with increasing sugar concentration (Fig-
ure 1A). A control with noncognate mannose excluded osmo-
larity effects. Routinely, comparative analysis was performed on
Overall, these assays extended the data basis on inhibition
of lectin activity, here on their binding to cell surface glycans,
acquiring an increased level of biomedical relevance relative to
the solid-phase-based experimental series. Interestingly, rela-
tive values of the two data sets were rather similar. With
regard to inter-galectin comparison, binding of the prototype
proteins (and the plant toxin) was less affected by the deriva-
tives than that of the chimera/tandem-repeat-type proteins.
The difference was quantitative. In a previous calorimetric anal-
[15]
ysis, such a difference in affinity between Gal-1 and Gal-3
ꢁ
1
(0.4 kcalmol in numbers measured for p-nitrophenyl lacto-
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&
4
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
Figure 2. Cytofluorimetric determination of binding of CG-3 and Gal-4 to cell
surfaces and the effect of presence of carbohydrates on binding parameters
(
for further details, please see legend to Figure 1). A) Binding of biotinylated
ꢁ
1
CG-3 (5 mgmL ) to CHO cells in the presence of compound 18 and 0.5 mm
lactose. B) Binding of Alexa Fluor 647-labeled Gal-4 (2 mgmL ) to the
ꢁ
1
human pancreatic carcinoma (Capan-1 reconstituted for the expression of
INK4a
the tumor suppressor p16
4 and 1 mm lactose.
) cells in the presence of 0.25 mm compound
4
pyranoside (45)) was mostly attributed to the entropic contri-
ꢁ
1
butions (4.8 vs. 5.3 kcalmol ). The availability of these data for
a frequently used (standard) inhibitor and the similarity of its
properties to those of the compounds tested prompted us,
15
given the in-house availability of N-labeled human galectins,
to perform a comparative binding study, in which we focused
on chemical shift differences between ligand-free and ligand-
loaded prototype (Gal-1 and Gal-7) and chimera-type (Gal-3)
proteins.
Figure 3. Differences in chemical shifts induced by ligand PNPL (45) and re-
D
sulting titration curves for K determination. Expansions of distinct sections
1
5
1
15
of N, H HSQC spectra for N-labeled A) Gal-1, C) Gal-3, and E) Gal-7 are
shown. Expansion overlays are provided for HSQC data acquired during the
titration. Black cross-peaks are for the HSQC spectrum at the beginning of
the titration in the absence of ligand 45, and red cross-peaks are for HSQC
data at the end of the titration. N- and H-weighted chemical shift changes
(Dd) are plotted for the top 15–22 shifted resonances for B) Gal-1, D) Gal-3,
and F) Gal-7 as a function of ligand concentration. Binding curves were
fitted to calculate apparent equilibrium dissociation constants.
Monitoring lectin binding by chemical-shift mapping
In the first stage of this experimental part of our study, we per-
formed titrations with ligand 45 to determine the dissociation
constants under these conditions, especially with use of the
15
1
1
5
N-labeled proteins. As illustrated by expansions of distinct
regions from each HSQC spectrum, chemical-shift differences
Dd) of specifically assigned cross-peaks during the courses of
(
titrations are shown (Figure 3A, C, E), along with plots of Dd
values of the 15 to 22 most shifted resonances (Figure 3B, D,
F). Individual titration curves were fit with a Gaussian/Boltzman
function for two-state binding, yielding averaged dissociation
constants for the two prototype galectins of 385ꢂ68 mm for
Gal-1 and 460ꢂ80 mm for Gal-7, together with, as expected in
ping is illustrated in Dd values (absolute value from chemical
shifts at the beginning of the titration minus those at the end
of the titration) for each amino acid in the sequence (Figure 4).
Although the largest Dd values are generally observed for a
similar set of backbone NH residues in each instance, primarily
those within b-strands 4, 5, and 6 (shown as loops in Gal-1 and
Gal-7), the profile for Gal-3 includes other resonances that are
inexplicably shifted. In quantitative terms, whereas linear re-
gression analysis of Dd correlation plots for pairs indicates a
[
15]
terms of affinity ranking based on calorimetry,
values of
2
50ꢂ60 mm for Gal-3. Fits to titration curves are demonstrated
for Gal-1 in Figure S84 in the Supporting Information. In all
2
2
instances, R values ranged from 0.97 to 0.99, indicating excel-
relatively high degree of correlation for Gal-1 and Gal-7 (R =
2
lent fits to the data. The same was observed for fits to PNPL
binding curves with Gal-7 and Gal-3.
0.62), pairing data for Gal-3 to Gal-1/-7 resulted in R values of
0.32 for the Gal-1/-3 pair and 0.29 for the Gal-7/-3 pair, and
thus a weak correlation.
The differences in affinity having been ascertained, the ob-
1
15
tained H, N HSQC-based information was then examined in
terms of whether it provided indications for an impact of
ligand binding beyond the immediate contact site, and this
differentially among the three galectins. The corresponding
data revealed that the lactoside derivative 45 interacts with all
three galectins at their canonical carbohydrate-binding sites
around the central Trp residue. Detailed chemical-shift map-
To provide a visualization of differences, Figure 4 highlights
residues most perturbed by binding of the ligand in the
models of crystal structures of Gal-1 (B, PDB ID: 1GZW), Gal-3
(D, PDB ID: 1A3K), and Gal-7 (F, PDB ID: 4GAL). A red color indi-
cates Dd values at least two standard deviations (SD) above
the average, orange between 2 SD and 1 SD, pink between
1 SD and average, and gray below average. For topological ref-
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&
5
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
Conclusion
The presented data help appraise the potential of an extension
of Lac/LacNAc beyond the anomeric center to enhance lectin
affinity/selectivity. Among galectins, corresponding factors of
up to 2.5 for the prototype proteins, about fourfold increases
for Gal-3, and up to sixfold for the human tandem-repeat-type
galectin were reached. Our combined experimental approach,
merging synthesis with two types of activity testing (glycopro-
tein/cell), thus draws attention to 1) cross-reactivity in inter-ga-
lectin comparison, and 2) the requirement to bring several in-
dividual approaches together for eventually reaching an opti-
mal selectivity. The bioinspired 3’-substitution at the Gal unit
has already proven instrumental in widening the gap between
[3b,15]
prototype and chimera-/tandem-repeat-type galectins;
presentation on distinct scaffolds such as 1,3,5-triiodobenzene
[
57a–d]
[57e]
or pentaerythrytol,
cone-4-type calixarene
or glycoden-
[58]
drimersomes furthers this separation. Lessons learned from
identification of natural galectin binders such as the poly-b1,3-
linked galactosyl epitope of Leishmania lipophosphoglycan or
[
28,59,60]
the xenoantigen
can then be the source for inspirations
for optimal tailoring of the sugar headgroup or a mimetic
thereof.
In this context, the added NMR spectroscopy analysis with
15
N-labeled galectins shows that note should be taken of the
significance of structural alterations in the lectin beyond the
Figure 4. Profiles of differences in chemical shifts induced by ligand 45 and
direct contact sites. In the monitoring of binding of Lac and
1
5
the relative positions of affected residues in the galectin structures. N- and
15
1
two 3’-substituted LacNAc derivatives to truncated Gal-3,
N
H-weighted chemical shift changes (Dd) between the experiments in the
2
and H spin-relaxation experiments have revealed that aspects
of conformational entropy (backbone fluctuations and dynamic
responses in the hydrophobic core) can come strongly into
absence and in the presence of 1 mm ligand are plotted versus the amino
acid sequence. Data are shown for A) Gal-1, C) Gal-3, and E) Gal-7. The larg-
1
5
1
est N, H-weighted chemical shift changes are highlighted at the corre-
sponding positions in the crystal structures of B) Gal-1, D) Gal-3, and F) Gal-
[61,62]
play, depending on the type of ligand.
These insights un-
7. Residues with signals that have been shifted significantly by binding of
derscore the merits of the fine-structural analysis in solution,
because the contribution of conformational entropy was re-
ported to be comparable in magnitude to that of the enthal-
ligand are colored in red (over 2 SD), orange (2–1 SD), pink (1 SD to aver-
age), and gray (below average). The contact position for Lac/LacNAc is given
by adding this disaccharide (in blue).
[62]
py.
In aggregate, the systematic quantitative assessment of the
impact of structural alterations at distinct sites of the ligand on
inhibitory capacity, done for a lectin family, provides a toolbox.
Of course, if a therapeutic perspective is considered, cross-re-
activity to other types of Lac/LacNAc-binding lectins—C-type
lectins, for example—will also have to be determined. The
stepwise analysis for each structural section (sugar headgroup,
substitution pattern, reducing-end extension, and oligovalen-
cy) is the prerequisite for fully achieving affinity and selectivity
goals. Moreover, the inherent delineation of how proteins of a
family react to ligand accommodation in solution will deepen
our understanding of the entropic contributions to affinity.
erence, a model of lactose has been introduced to denote its
contact site. Both types of illustration in Figure 4 converge to
highlight common features of accommodating the lactoside
core. However, binding the derivative has an impact on
a number of residues away from the canonical lactose-binding
[56]
site, which hosts the ligand as its low-energy conformer.
Some of these residues are located at the backside face of the
b-sandwich. Notably, residues significantly affected in b-
strands 3 and 10 of Gal-3 are primarily hydrophobic and orient-
ed towards the interior of the b-sandwich: for example, I145,
L147, F149 in b-strand 3, as well as L234 and I236 in b-
strand 10. Overall, it appears that binding of 45 in particular in-
duced changes in the hydrophobic core of the b-sandwich. In
line with the rather constant enthalpy change measured for
Experimental Section
[
15]
Gal-1 and Gal-3, our data did not reveal any strong effect of
the ring current of the substituent on the position of resonan-
ces as evidence for interactions. It is thus likely that the p-nitro-
phenyl substituent will primarily be exposed to the solvent.
Chemistry
General: The reactions were carried out in organic media under ni-
trogen with use of freshly distilled solvents (dichloromethane was
freshly distilled over P O ; DMF was distilled on ninhydrin and then
2
5
stored over 4 ꢃ molecular sieves). All reagents were used as sup-
plied without prior purification unless otherwise stated and were
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&
6
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
obtained from Acros Organics and Sigma–Aldrich. Microwave reac-
tions were conducted with a Biotage Initiator Microwave Synthesiz-
er at normal absorption level. After workup, the organic layers
were dried over anhydrous Na SO . Progress of reactions was
give the title compound 4 as a white solid (1.882 g, 3.78 mmol,
23
98%). R =0.19 (CH Cl /MeOH 15:1); m.p. 78–808C; [a] =ꢁ64 (c=
f
2
2
D
1
3
1.0, CHCl ); H NMR (300 MHz, CDCl ): d=7.69 (dd,
1.8 Hz, 4H; Harom), 7.53–7.30 (m, 6H; H ), 6.21 (d, J =5.9 Hz,
1H; NH), 5.34 (d, J =3.4 Hz, 1H; OH-3), 4.66 (d, J =8.2 Hz, 1H;
H,H 1,2
H-1), 4.45–4.22 (m, 2H; OCH ), 3.97 (dd, J
J
=7.6,
H,H
3
3
3
2
4
arom
H,H
3
3
monitored by analytical thin-layer chromatography with silica gel
0 F254 precoated plates (Merck). The visualization of molecules
3
3
6
=11.1, J =3.1 Hz,
6a,6b 5,6a
2
3
3
was performed by different methods depending on the class of
compounds and functional groups present in the molecule: 1) by
irradiation under UV light (l=254 nm), 2) by soaking in specific
mixed acid (sulfuric acid/methanol/water 5:45:45, v/v/v), 3) by use
of an oxidizing solution of molybdate [prepared from ammonium
molybdate (25 g) and ceric sulfate (10 g) dissolved in water
1H; H-6a), 3.88 (dd, J6 =11.0, J =5.3 Hz, 1H; H-6b), 3.76–3.44
a,6b 5,6b
4
(m, 5H; H-3, H-2, H-4, H-5, OH-4), 2.48 (t, J =2.4 Hz, 1H; CꢀCH),
H,H
1
3
2.04 (s, 3H; NCOCH ), 1.04 ppm (s, 9H; SiCMe ); C NMR (75 MHz,
3
3
CDCl ): d=172.6 (CONH), 135.6, 135.5 (Cq-arom), 133.3, 133.2, 129.6,
3
127.6, 127.6 (Carom), 98.1 (C-1), 78.8 (CꢀCH), 76.1 (CꢀCH), 75.4 (C-
5), 74.9 (C-3), 71.4 (C-4), 63.8 (OCH ), 56.5 (C-6), 55.3 (C-2), 26.7
2
+
(
900 mL) and concentrated sulfuric acid (100 mL)], followed by
(SiCMe ), 23.4 (NHCOCH ), 19.2 ppm (SiCMe ); HRMS (ESI ): m/z
calcd for C H NO Si: 498.2306 [M+H] ; found: 498.2300.
27 36 6
3
3
3
+
heating to 3008C, and 4) by soaking in KMnO solution [prepared
4
by mixing equal quantities of KMnO solution (1%) and Na CO so-
4
2
3
Prop-2-ynyl 2-acetamido-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-d-gal-
actopyranosyl)-6-O-tert-butyldiphenylsilyl-b-d-glucopyranoside (6):
Powdered molecular sieves (4 ꢃ, 0.5 g) were added under nitrogen
lution (2%)]. Purification by flash column chromatography was per-
formed with silica gel (Silicycle, 60 ꢃ, 40–63 mm) and the indicated
eluent. The solvents employed for chromatography were of ACS or
HPLC quality and were not distilled before use. They were evapo-
rated under reduced pressure (rotary evaporator under vacuum
generated by a glass filter pump system). Water was nanopure
grade, purified through a Barnstead NANOPure II Filter with Barn-
stead MegOhm-CM Sybron meter. Lyophilization was performed
with a Freeze Mobile 24 instrument (Virtis). Optical rotations were
measured with a JASCO P-1010 polarimeter. Melting points were
to a solution of acceptor 4 (950 mg, 1.90 mmol, 1.0 equiv) and
[51]
2
,3,4,6-tetra-O-acetyl-a-d-galactopyranosyl trichloroacetimidate
(5, 1.41 g, 2.86 mmol, 1.5 equiv) in dry dichloromethane (10 mL).
The mixture was stirred for 1 h at room temperature, and then
cooled to ꢁ458C. A solution of BF ·Et O (50 mL, 0.41 mmol) was
3
2
then added dropwise. After 1 h the mixture was neutralized with
triethylamine, diluted with dichloromethane (10 mL), filtered
through celite, and washed with dichloromethane. The combined
1
measured with a Fisher–Johns apparatus and are uncorrected. H
1
3
filtrate and washings were dried over MgSO , filtered, and concen-
and C NMR spectra were recorded at 300 or 600 MHz and 75 or
50 MHz, respectively, with Varian Gemini 2000 (300 MHz) and
4
trated. The syrupy product was purified by silica gel chromatogra-
1
phy to give 6 as a white solid (1.35 g, 1.63 mmol, 86%); R =0.31
Varian Inova (600 MHz) spectrometers. All NMR spectra were mea-
f
(
CH Cl /MeOH 98:2); m.p. 143–1448C (CH Cl /petrolum ether);
sured at 258C in the indicated deuterated solvents. Two-dimen-
2
3
2
2
2
2
1
1
1
[a] =ꢁ6 (c=1.0, CHCl ); H NMR (600 MHz, CDCl ): d=7.80–7.66
sional homonuclear correlation H, H COSY experiments were used
to confirm NMR peak assignments. Proton and carbon chemical
shifts are reported downfield from internal references of residual
solvents. Analysis and assignments were made with the aid of
COSY and HETCOR experiments. Signal annotations are described
as “prime” for the Gal residues, whereas letters are used for the
aglycones. Accurate mass measurements (HRMS) were carried out
with a HPLC Agilent Technologies 1200 Series+Agilent Technolo-
gies 6210 MS TOF instrument in positive electrospray mode. Low-
resolution mass spectra were recorded with the same apparatus or
with a LCQ Advantage ion trap instrument from Thermo Fisher Sci-
entific in positive electrospray mode (Mass Spectrometry Laborato-
ry, University of Montreal, QC, Canada or at l’Universitꢀ du Quꢀbec
ꢄ Montrꢀal, Quꢀbec, Canada). Either protonated molecular ions
D
3
3
3
(
m, 4H; H ), 7.51–7.33 (m, 6H; H ), 5.70 (d, J
=8.2 Hz, 1H;
arom
arom
NH,H2
3
3
3
AcNH), 5.37 (dd, J = J =2.6 Hz, 1H; H-4’), 5.20 (dd, J =8.1,
3,4
4,5
1,2
3
3
3
J2,3 =10.4 Hz, 1H; H-2’), 4.98 (dd, J =3.5, J =10.5 Hz, 1H; H-
3,4
2,3
3
3
3
4
’), 4.87 (d, J =8.3 Hz, 1H; H-1), 4.73 (d, J =8.1 Hz, 1H; H-1’),
1
,
2
1
,
2
.46–4.24 (m, 2H; OCH ), 4.15 (m, J=6.6 Hz, 2H; H-6’), 3.99 (m, 1H;
2
H-5), 3.92 (m, 2H; H-5’, H-6a), 3.89–3.77 (m, 3H; H-4, H-6b, OH-3),
4
3
.61 (m, 1H; H-2), 3.41 (m, 1H; H-3), 2.45 (t, J =2.3 Hz, 1H; Cꢀ
H,H2
CH), 2.16, 2.07, 2.04, 2.00 (4s, 12H; COCH ), 1.73 (s, 3H; NCOCH ),
C NMR (75 MHz, CDCl ): d=170.6,
3
70.4, 170.1, 169.9, 169.1 (CO), 135.9, 135.4, 133.4, 132.4, 129.9,
3
3
13
1
1
1
.08 ppm (s, 9H; SiCMe3);
29.8, 127.8, 127.6 (Carom), 100.9 (C-1’), 97.6 (C-1), 79.8 (C-4), 78.7
(
6
CꢀCH), 75.0 (C-3), 74.5 (CꢀCH), 71.6 (C-5), 71.2 (C-5’), 70.7 (C-3’),
8.8 (C-2’), 66.8 (C-4’), 61.6 (C-6), 61.1 (C-6’), 56.3 (C-2), 55.2 (OCH ),
2
n+
+
26.8 (SiCMe ), 23.6 (NHCOCH ), 20.6, 20.5, 20.5, 20.3 (COCH ),
3 3 3
[
M+nH] or sodium adducts [M+Na] were used for empirical
+
1
8
9.3 ppm (SiCMe ); HRMS (ESI ): m/z calcd for C H NO SiNa:
formula confirmation.
3
+
41 53
15
50.3077 [M+Na] ; found: 850.3070.
Synthesis of the LacNAc triazoles
4-[2-Acetamido-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyra-
Prop-2-ynyl 2-acetamido-2-deoxy-6-O-tert-butyldiphenylsilyl-b-d-glu-
copyranoside (4): Detailed syntheses and spectroscopic data for
known intermediates 1–3 can be found in the Supporting Informa-
tion. The procedure described below follows conditions estab-
nosyl)-6-O-tert-butyldiphenylsilyl-b-d-glucopyranosyloxymethyl]-1-
methyl-1H-1,2,3-triazole (7): Methyl azide was distilled at 908C. It
was freshly prepared from MeI (3 mL) and NaN3 (3.6 g) in DMF/
water (4 mL, 1:1, v/v) with stirring at 508C overnight. The distillate
was immediately added to a round-bottomed flask containing a so-
lution of compound 6 (83 mg, 0.10 mmol, 1.0 equiv), CuSO ·5H O
[46]
lished in the literature for an analogous anomeric azide or for
[47]
a 6-O-tert-butyldimethylsilyl analogue. tert-Butylchlorodiphenylsi-
4
2
lane (1.06 mL, 4.05 mmol, 1.05 equiv) was added to a solution of
(5.0 mg, 0.02 mmol, 0.2 equiv), and sodium ascorbate (8.0 mg,
[48]
compound 3
(1.00 g, 3.86 mmol, 1.0 equiv) in dry pyridine
0.04 mmol, 0.4 equiv) in THF/H O (4 mL, 1:1, v/v) and stirred at
2
(
25 mL). The solution was stirred at room temperature for 6 h. The
room temperature overnight. The crude reaction mixture was dilut-
reaction mixture was poured into ice-water and extracted with di-
chloromethane (3ꢅ20 mL). The combined extracts were washed
ed with EtOAc and washed with H O (with a little amount of
2
hexane, several times). The organic layer was treated with an aque-
ous solution of EDTA (5%, 2ꢅ), dried over Na SO , filtered, and con-
successively with hydrochloric acid (5%), saturated NaHCO solu-
3
2
4
tion, and water. The organic layer was dried under Na SO , filtered,
centrated under reduced pressure. The residue was purified by
flash chromatography on silica gel [CH Cl (100%) to CH Cl /MeOH
96:4]. The LacNAc triazole derivative 7 was isolated as a white
2
4
and concentrated. The crude product was purified by flash chro-
matography on silica gel [CH Cl (100%) to CH Cl /MeOH 15:1] to
2
2
2
2
2
2
2
2
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&
7
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
solid (84 mg, 0.095 mmol, 95%). R =0.28 (CH Cl /MeOH 92:8);
(300 MHz, CDCl ): d=7.88–7.65 (m, 4H; Harom), 7.49 (s, 1H; Htriazole),
f
2
2
3
2
3
1
3
[
a] =ꢁ2.2 (c=1.0, CHCl ); H NMR (300 MHz, CDCl ): d=7.73 (m,
7.46–7.29 (m, 6H; H ), 6.10 (d, J
=7.7 Hz, 1H; AcNH), 5.36
NH,H2
D
3
3
arom
3
3
3
3
4
H; H ), 7.49 (s, 1H; H
), 7.47–7.32 (m, 6H; H ), 6.02 (d,
(dd, J = J =3.4 Hz, 1H; H-4’), 5.19 (dd, J =8.1, J =10.4 Hz,
3,4 4,5 1,2 2,3
3 3
arom
triazole
arom
3
3
3
JNH,H2 =8.0 Hz, 1H; AcNH), 5.36 (dd, J = J =3.4 Hz, 1H; H-4’),
1H; H-2’), 4.97 (dd, J =3.0, J =10.3 Hz, 2H; H-3’, H-1), 4.83–
3,4 2,3
4
3,4
4,5
3
3
3
5
.19 (dd, J1,2 =8.0, J2,3 =10.4 Hz, 1H; H-2’), 4.97 (dd, J3,4 =3.4,
4.71 (m, 4H; H-6a’, H-6b’, H-1’, N-CH(CH ) ), 4.13 (d, J =6.7 Hz,
3 2 H,H
3
J2,3 =10.4 Hz, 2H; H-3’, H-1), 4.73 (m, J=5.6, 7.8 Hz, 3H; H-6a’, H-
2H; OCH ), 4.09–3.79 (m, 6H; H-3, H-4, H-6a, H-6b H-5, H-5’), 3.70
2 ,
3
4
6
b’, H-1’), 4.14 (d, JH,H =6.5 Hz, 2H; OCH ), 4.05 (s, 3H; NCH ),
(dd, J=8.1, 14.8 Hz, 1H; H-2), 3.44 (d, J3,OH =8.4 Hz, 1H; OH-3),
2
3
4
1
1
.00–3.88 (m, 4H; H-3, H-4, H-6a, H-6b), 3.71 (dd, J=8.3, J=
2.16, 2.06, 1.99, 1.98, (4s, 12H; COCH ), 1.73 (s, 3H; NCOCH ) 1.56
3
3
3
7.5 Hz, 1H; H-2), 3.43 (d, J3 =5.8 Hz, 1H; OH-3), 2.15, 2.06, 1.99,
(dd, J=6.7, 1.4 Hz, 2H; N-CH(CH ) ), 1.08 ppm (s, 9H; SiCMe );
3 2 3
,OH
13
.97, (4s, 12H; COCH ), 1.73 (s, 3H; NCOCH ), 1.08 ppm (s, 9H;
C NMR (75 MHz, CDCl ): d=170.8, 170.3, 170.1, 169.8, 169.1 (CO),
3
3
3
1
3
SiCMe3); C NMR (75 MHz, CDCl ): d=170.8, 170.3, 170.0, 169.8,
144.3 (triazole-C ), 135.8, 135.4, 133.4, 132.4, 129.8, 127.8, 127.6
3
4
1
1
69.1 (CO), 144.9 (triazole-C ) 135.8, 135.4, 133.4, 132.4, 129.9,
(Carom), 119.9 (triazole-C ), 100.8 (C-1’), 99.6 (C-1), 79.6 (C-4), 74.7 (C-
4
5
27.8, 127.6 (Carom), 123.4 (triazole-C ) 100.8 (C-1’), 99.6 (C-1), 79.5
5), 72.2 (C-3), 71.1 (C-5’), 70.8 (C-3’), 68.8 (C-2’), 66.8 (C-4’), 62.1 (C-
6), 61.8 (C-6’), 61.1 (OCH ), 56.4 (C-2), 53.0 (N-CH(CH ) ), 26.8
5
(
C-4), 74.6 (C-5), 72.1 (C-3), 71.1 (C-5’), 70.7 (C-3’), 68.8 (C-2’), 66.8
2
3 2
(
C-4’), 61.9 (C-6), 61.8 (C-6’), 61.1 (OCH ), 56.3 (C-2), 36.6 (N-CH ),
(SiCMe ), 23.5 (NHCOCH ), 23.0, 22.9 (N-CH(CH ) ), 20.6, 20.5, 20.5,
2
3
3
3
3 2
+
2
1
8
6.8 (SiCMe ), 23.4 (NHCOCH ), 20.6, 20.5, 20.5, 20.3 (COCH ),
20.3 (COCH ), 19.3 ppm (SiCMe ); HRMS (ESI ): m/z calcd for
3
3
3
3
3
+
+
9.3 ppm (SiCMe ); HRMS (ESI ): m/z calcd for C H N O Si:
85.3584 [M+H] ; found: 885.3574.
C H N O Si: 913.3897 [M+H] ; found: 913.3888.
44 61 4 15
3
42 57
4
15
+
4-[2-Acetamido-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyra-
4
-[2-Acetamido-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyra-
nosyl)-6-O-tert-butyldiphenylsilyl-b-d-glucopyranosyloxymethyl]-1-
propyl-1H-1,2,3-triazole (10): A mixture of 6 (83 mg, 0.10 mmol), n-
propyl azide (13 mg, 0.15 mmol, 1.5 equiv), CuSO ·5H O (5 mg,
nosyl)-6-O-tert-butyldiphenylsilyl-b-d-glucopyranosyloxymethyl]-1-
ethyl-1H-1,2,3-triazole (8): Compound 8 was synthetized under the
same conditions as described above for 7. Fresh ethyl azide was
prepared from a mixture of iodoethane (3.6 mL) and sodium azide
4
2
0.02 mmol, 0.2 eqiuv.), and sodium ascorbate (8.0 mg, 0.04 mmol,
0.4 equiv) in THF/H O (4 mL, 1:1, v/v) was stirred at room tempera-
2
(
3.6 g) in DMF/H O (18 mL/6 mL). After distillation, a slight excess
ture for 12 h. The desired compound 10 was purified by flash
2
of ethyl azide was added to a solution of compound 6 (83 mg,
.10 mmol, 1.0 equiv) and CuSO ·5H O (5.0 mg, 0.02 mmol, 0.2
eqiuv.) and sodium ascorbate (8.0 mg, 0.04 mmol, 0.4 equiv) in
column chromatography (CH Cl /MeOH 92:8) and isolated as
a white solid (87 mg, 0.095 mmol, 95%). R =0.42 (CH Cl /MeOH
f 2 2
2
2
0
4
2
2
D
3
1
92:8); [a] =ꢁ7.2 (c=1.0, CHCl ); H NMR (300 MHz, CDCl ) d=
3
3
THF/H O (6 mL, 1:1, v/v). The mixture was stirred at room tempera-
7.79–7.69 (m, 4H; H ), 7.50 (s, 1H; H ), 7.48–7.32 (m, 6H;
triazole
Harom), 6.06 (d, JNH,H2 =7.7 Hz, 1H; AcNH), 5.37 (dd, J3,4 = J =
4,5
2
arom
3
3
3
ture for 12 h. The triazole derivative 8 was isolated as a white solid
2
D
3
3
3
(
85 mg, 0.095 mmol, 95%). R =0.27 (CH Cl /MeOH 97:3); [a] =
3.4 Hz, 1H; H-4’), 5.20 (dd, J =8.1, J =10.4 Hz, 1H; H-2’), 4.97
1,2 2,3
3 3 ,
f
2
2
1
ꢁ
1.2 (c=1.0, CHCl ); H NMR (300 MHz, CDCl ): d=7.72 (dd, J=9.2,
.1 Hz, 4H; Harom), 7.52 (s, 1H; Htriazole), 7.48–7.30 (m, 6H; Harom), 6.15
=7.8 Hz, 1H; AcNH), 5.36 (dd, J = J =3.4 Hz, 1H; H-
’), 5.18 (dd, J =8.1, J =10.3 Hz, 1H; H-2’), 4.96 (m, J =3.0,
J2,3 =10.3 Hz, 2H; H-3’ H-1), 4.77–4.72 (m, 3H; H-6a’, H-6b’, H-1’),
.36 (q, J=7.4 Hz, 2H; N-CH CH ), 4.14 (d, J =6.5 Hz, 2H; OCH2),
(m, J =3.0, J =10.3 Hz, 2H; H-3’ H-1), 4.79–4.71 (m, 3H; H-6a’,
3,4 2,3
3 2
3
3
4
H-6b’, H-1’), 4.29 (t, J =7.2 Hz, 2H; N-CH CH CH ), 4.15 (d, J
=
H,H
2
2
3
H,H
3
3
3
(
d, J
6.7 Hz, 2H; OCH ), 3.99–3.82 (m, 6H; H-3, H-4, H-6a, H-6b H-5, H-
NH,H2
3,4
4,5
2
,
3
3
3
3
4
5’), 3.75–3.73 (m, 1H; H-2), 3.45 (d, J =8.4 Hz, 1H; OH-3), 2.16,
1
,2
2,3
3,4
3,OH
3
,
2.06, 1.99, 1.98, (4s, 12H; COCH ), 1.92–1.89 (m, 2H; N-CH CH CH ),
3
2
2
3
4
4
1.73 (s, 3H; NHCOCH ), 1.08 (s, 9H; SiCMe ), 0.96 ppm (t, J=7.4 Hz,
3 3
2
3
H,H
13
3
1
.92–3.84 (m, 4H; H-3, H-4, H-6a, H-6b), 3.72 (dd, J=8.1, 14.8 Hz,
3H; N-CH CH CH ); C NMR (75 MHz, CDCl ): d=170.8, 170.4,
2 2 3 3
3
H; H-2), 3.42 (d, J =8.4 Hz, 1H; OH-3), 2.14, 2.05, 1.98, 1.97
170.1, 169.9, 169.1 (CO), 144.9 (triazole-C ), 135.9, 135.4, 133.5,
3
,OH
4
(
4s, 12H; COCH ), 1.72, (s, 3H; NCOCH ), 1.52 (t, J=7.4 Hz, 3H; N-
132.5, 129.9, 127.8, 127.6 (Carom), 122.4 (triazole-C ) 100.8 (C-1’), 99.7
(C-1), 79.7 (C-4), 74.7 (C-5), 72.2 (C-3), 71.2 (C-5’), 70.8 (C-3’), 68.8
3
3
5
13
CH CH ), 1.07 ppm (s, 9H; SiCMe ); C NMR (75 MHz, CDCl ): d=
2
3
3
3
1
1
1
7
5
2
70.8, 170.3, 170.0, 169.8, 169.1 (CO), 144.9 (triazole-C ) 135.8,
(C-2’), 66.8 (C-4’), 61.9 (C-6), 61.8 (C-6’), 61.1 (OCH ), 56.4 (C-2), 52.1
4
2
35.4, 133.4, 132.4, 129.9, 127.8, 127.6 (Carom), 121.8 (triazole-C5),
00.7 (C-1’), 99.6 (C-1), 79.5 (C-4), 74.6 (C-5), 72.1 (C-3), 71.1 (C-5’),
0.7 (C-3’), 68.7 (C-2’), 66.8 (C-4’), 61.9 (C-6), 61.7 (C-6’), 61.1 (OCH2),
6.3 (C-2), 45.2 (N-CH CH ), 26.7 (SiCMe ), 23.4 (NHCOCH ), 20.53,
(N-CH -CH -CH ), 26.8 (SiCMe ), 23.4 (NHCOCH ), 23.5 (N-CH -CH -
2
2
3
3
3
2
2
CH ), 20.6, 20.6, 20.5, 20.3 (COCH ), 19.4 (SiCMe ), 11.0 ppm (N-
3
3
3
+
CH CH ); HRMS (ESI ): m/z calcd for C H N O Si: 913.3897
2
3
+
44 61
4
15
[M+H] ; found: 913.3889.
2
3
3
3
0.49, 20.45, 20.28 (COCH ), 19.3 (SiCMe ), 15.4 ppm (N-CH CH );
3
3
2
3
+
+
2-{4-[2-Acetamido-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyr-
anosyl)-6-O-tert-butyldiphenylsilyl-b-d-glucopyranosyloxymethyl]-1H-
HRMS (ESI ): m/z calcd for C H N O Si: 899.3741 [M+H] ; found:
43
59
4
15
8
99.3734.
1,2,3-triazol-1-yl}ethanol (11): A mixture of 6 (83 mg, 0.10 mmol), 2-
4-[2-Acetamido-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyra-
azidoethanol (13 mg, 0.15 mmol, 1.5 equiv), CuSO ·5H O (5 mg,
4
2
nosyl)-6-O-tert-butyldiphenylsilyl-b-d-glucopyranosyloxymethyl]-1-iso-
propyl-1H-1,2,3-triazole (9): A solution of compound 6 (83 mg,
0.02 mmol, 0.2 equiv), and sodium ascorbate (8.0 mg, 0.04 mmol,
0.4 equiv) in THF/H O (4 mL, 1:1, v/v) was stirred at room tempera-
2
0
.10 mmol), isopropyl azide (13 mg, 0.15 mmol), CuSO ·5H O
ture. After 12 h reaction time according to the general procedure
described above, compound 11 was purified by flash column chro-
matography [CH Cl (100%) to CH Cl /MeOH 94:6] and isolated as
4
2
(
5 mg, 0.02 mmol, 0.2 equiv), and sodium ascorbate (8.0 mg,
.04 mmol, 0.4 equiv) in THF/H O (4 mL, 1:1, v/v) was stirred at
0
2
2
2
2
2
room temperature for 12 h. The crude product was diluted with
a white solid (83 mg, 0.091 mmol, 91%). R =0.24 (CH Cl /MeOH
f 2 2
23
1
EtOAc and washed with H O (with a little amount of hexane, sever-
94:6); [a] =ꢁ24.1 (c=1.0, CHCl ); H NMR (300 MHz, CDCl ) d=
2
D
3
3
al times). The organic layer was treated with an aqueous solution
of EDTA (5%, 2ꢅ), dried over Na SO , filtered, and concentrated
7.78–7.72 (m, 4H; H ), 7.60 (s, 1H; H
), 7.51–7.33 (m, 6H;
triazole
arom
3
3
3
Harom), 6.32 (d, JNH,H2 =7.2 Hz, 1H; AcNH), 5.36 (dd, J3,4 = J =
4,5
2
4
3
3
under reduced pressure. The residue was purified by flash chroma-
tography on silica gel [CH Cl (100%) to CH Cl /MeOH 95:5]. The
3.2 Hz, 1H; H-4’), 5.18 (dd, J1 =8.0, J =10.4 Hz, 1H; H-2’), 5.01–
,2 2,3
4.90 (m, 3H; H-3’, H-1, N-CH -CH -OH), 4.78–4.65 (m, 3H; N-CH -
2
2
2
2
2
2
2
triazole 9 was isolated as a white solid (87 mg, 0.095 mmol, 95%).
CH -OH, H-1’, H-6a’), 4.58–4.33 (m, 3H; N-CH -CH -OH, H-6b’), 4.13
2 2 2
2
23
1
R =0.23 (CH Cl /MeOH 95:5); [a] =ꢁ20.6 (c=1.0, CHCl ); H NMR
(d, J =6.6 Hz, 2H; OCH ), 3.83–3.71 (m, 6H; H-3, H-4, H-6a, H-6b,
H,H 2
f
2
2
D
3
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&
8
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
3
H-5, H-5’), 3.44 (d, J3,OH =7.9 Hz, 1H; OH-3), 3.13 (dd, J=8.1,
26.7 (SiCMe ), 25.0, 24.9 (N-C
), 23.3 (NHCOCH ), 20.5 (N-
3
cyclopentyl
3
1
3
4.8 Hz, 1H; H-2), 2.15, 2.04, 1.98, 1.85 (4s, 12H; COCH ), 1.73 (s,
C
), 20.6, 20.6, 20.5, 20.3 (COCH ), 19.4 ppm (SiCMe ); HRMS
3
cyclopentyl
+
3
3
13
+
H; NCOCH ), 1.08 ppm (s, 9H; SiCMe ); C NMR (75 MHz, CDCl3):
(ESI ): m/z calcd for C H N O Si: 939.4059 [M+H] ; found:
3
3
46 63
4
15
d=171.4, 170.2, 170.1, 169.9, 169.1 (CO), 143.2 (triazole-C ), 135.9,
939.4054.
4
1
1
7
35.4, 133.4, 132.4, 129.9, 127.8, 127.6 (Carom), 124.6 (triazole-C5)
00.8 (C-1’), 96.1 (C-1), 80.2 (C-4), 74.5 (C-5), 71.1 (C-3), 70.7 (C-5’),
0.3 (C-3’), 68.7 (C-2’), 66.8 (C-4’), 66.7 (C-6), 61.7 (C-6’), 61.1 (N-
4
-[2-Acetamido-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyra-
nosyl)-6-O-tert-butyldiphenylsilyl-b-d-glucopyranosyloxymethyl]-1-cy-
clohexyl-1H-1,2,3-triazole (14): A mixture of 6 (83 mg, 0.10 mmol),
cyclohexyl azide (13 mg, 0.15 mmol, 1.5 equiv), CuSO ·5H O (5 mg,
0.02 mmol, 0.2 eqiuv.), and sodium ascorbate (8.0 mg, 0.04 mmol,
0.4 equiv) in THF/H O (4 mL, 1:1, v/v) was stirred at room tempera-
CH CH -OH) 60.7 (OCH ), 58.0 (OCH ), 56.4 (C-2), 53.40 (N-CH -CH -
OH), 26.6 (SiCMe ), 23.4 (NHCOCH ), 23.28 (N-CH -CH -CH ), 20.56,
2
2
2
2
2
2
2
4
2
3
3
2
2
3
+
0.51, 20.48, 20.3 (COCH ), 19.31 ppm (SiCMe ); HRMS (ESI ): m/z
3 3
+
2
calcd for C H N O Si: 915.3689 [M+H] ; found: 915.3678.
4
3
59
4
16
ture as above for 12 h. Compound 14 was purified by flash column
4
-[2-Acetamido-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyra-
chromatography (CH
Cl /MeOH 98:2) and isolated as a white solid
2 2
2
3
nosyl)-6-O-tert-butyldiphenylsilyl-b-d-glucopyranosyloxymethyl]-1-
benzyl-1H-1,2,3-triazole (12): A mixture of 6 (83 mg, 0.10 mmol),
benzyl azide (20 mg, 0.15 mmol, 1.5 equiv), CuSO ·5H O (5 mg,
0
0
(90 mg, 0.094 mmol, 94%). R
f
=0.15 (CH
); H NMR (300 MHz, CDCl
4H; Harom), 7.50 (s, 1H; Htriazole), 7.47–7.23 (m, 6H; Harom), 6.24 (d,
2
Cl
2
/MeOH 98:2); [a]
D
=
1
ꢁ8.2 (c=1.0, CHCl
): d=7.75–7.71 (m,
3
3
4
2
3
3
3
.02 mmol, 0.2 eqiuv.), and sodium ascorbate (8.0 mg, 0.04 mmol,
JNH,H2 =11.6 Hz, 1H; AcNH), 5.35 (dd, J3,4 = J4,5 =3.2 Hz, 1H; H-4’),
3 3
.4 equiv) in THF/H O (4 mL, 1:1, v/v) was stirred at room tempera-
5.18 (dd, J1,2 =8.0, J2,3 =10.4 Hz, 1H; H-2’), 4.98–4.93 (m, 3H; H-3’,
2
ture for 12 h. Compound 12 was purified by flash column chroma-
H-6a’, H-6b’), 4.81–4.69 (m, 3H; H-1, H-6a, H-6b), 4.44–4.31 (m, 1H;
4
tography (CH Cl /MeOH 98:2) and isolated as a white solid (91 mg,
N-CHcyclohexyl), 4.13 (d, JH,H =6.6 Hz, 2H; CH
2
), 4.04–3.83 (m, 6H; H-3,
2
2
2
D
3
3
0
1
.095 mmol, 95%). R =0.35 (CH Cl /MeOH 98:2); [a] =ꢁ2.3 (c=
H-4, H-6a, H-6b, H-5, H-5’), 3.71–3.69 (m, 1H; H-2), 3.42 (d, J3,OH
7.9 Hz, 1H; OH-3), 2.29–2.20 (m, 2H; CH2cyclohexyl), 2.15, 2.05, 1.98,
1.97 (4s, 12H; COCH ), 1.71 (s, 3H; NCOCH ), 1.88–1.85 (m, 2H;
=
f
2
2
1
.0, CHCl ); H NMR (300 MHz, CDCl ): d=7.82–7.62 (m, 4H; H ),
3
3
arom
7
.47–7.31 (m, 10H; Htriazole, Harom), 7.29–7.21 (m, 2H; H ), 6.07 (d,
3
3
arom
3
3
3
JNH,H2 =7.2 Hz, 1H; AcNH), 5.48 (s, 2H; NCH ), 5.36 (dd, J = J =
4,5
CH2cyclohexyl), 1.07 (s, 9H; SiCMe ), 1.0–0.95 (m, 2H; CH2cyclohexyl), some
3
2
3,4
3
3
3
4
.2 Hz, 1H; H-4’), 5.19 (dd, J1 =8.0, J =10.4 Hz, 1H; H-2’), 5.00–
,2 2,3
multiplets belonging to CH2cyclohexyl masked under singlets at 2.4–
1
3
.69 (m, 2H; H-3’, H-6a’), 4.72 (m, 2H; H-6b’, H-1), 4.14 (m, 3H;
1.5 ppm; C NMR (75 MHz, CDCl
169.0 (CO), 143.99 (triazole-C ), 135.8, 135.3, 133.3, 132.4, 129.8,
127.7, 127.5 (Carom), 120.3 (triazole-C ), 100.7 (C-1’), 99.6 (C-1), 79.5
(C-4), 74.6 (C-5), 72.2 (C-3), 71.0 (C-5’), 70.7 (C-3’), 68.7 (C-2’), 66.8
(C-4’), 62.0 (C-6), 61.7 (C-6’), 61.0 (OCH
2), 33.4, 33.3 (N-Ccyclohexyl), 26.7 (SiCMe
(NHCOCH ), 20.5 (N-Ccyclohexyl), 20.6, 20.6, 20.5, 20.3 (COCH
19.4 ppm (SiCMe
3
): d=170.8, 170.3, 170.0, 169.8,
OCH , H-1’), 4.01–3.77 (m, 6H; H-3, H-4, H-6a, H-6b, H-5, H-5’), 3.41
4
2
3
(
d, J =8.4 Hz, 1H; OH-3), 2.15, 2.04, 1.98, 1.85 (4s, 12H; COCH ),
5
3
,OH
3
13
1
1
1
1
.73 ppm (s, 3H; NCOCH3); C NMR (75 MHz, CDCl ): d=170.7,
3
70.3, 170.0, 169.8, 169.0 (CO), 144.9 (triazole-C ) 135.8, 135.4,
2
), 60.0 (N-Ccyclohexyl), 56.4 (C-
), 25.0, 24.9 (N-Ccyclohexyl) 23.3
3
4
34.4, 133.4, 132.4, 129.8, 129.0, 128.7, 128.0, 127.7, 127.6 (Carom),
22.4 (triazole-C ) 100.7 (C-1’), 99.5 (C-1), 79.5 (C-4), 74.5 (C-5), 72.1
3
),
3
5
+
(
(
(
C-3), 71.1 (C-5’), 70.7 (C-3’), 68.7 (C-2’), 66.8 (C-4’), 61.8 (C-6), 61.7
); HRMS (ESI ): m/z calcd for C47H N O15Si:
3 65 4
+
C-6’), 61.0 (OCH ), 56.2 (C-2), 54.07 (N-CH -Ph), 26.7 (SiCMe ), 23.3
953.4216 [M+H] ; found: 953.4216.
2
2
3
NHCOCH ), 20.5, 20.5, 20.4, 20.3 (COCH ), 19.2 ppm (SiCMe );
3
+
3
3
+
4-[2-Acetamido-2-deoxy-4-O-(b-d-galactopyranosyl)-b-d-glucopyrano-
syloxymethyl]-1-methyl-1H-1,2,3-triazole (15): A solution of TBAF/THF
HRMS (ESI ): m/z calcd for C H N O Si: 961.3897 [M+H] ; found:
48
61
4
15
9
61.3895.
(
(
1m, 56 mL, 2 equiv) was added to a solution of compound 7
25 mg, 28 mmol) in dry THF (1.5 mL), and the reaction mixture was
4-[2-Acetamido-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyra-
stirred under nitrogen at room temperature for 12 h. The reaction
mixture was then concentrated under reduced pressure. The solu-
tion of this crude product in sodium methoxide in methanol (1m,
nosyl)-6-O-tert-butyldiphenylsilyl-b-d-glucopyranosyloxymethyl]-1-cy-
clopentyl-1H-1,2,3-triazole (13): A mixture of 6 (83 mg, 0.10 mmol),
cyclopentyl azide (17 mg, 0.15 mmol, 1.5 equiv), CuSO ·5H O
4
2
1
mL, pH 10) was stirred at room temperature until complete con-
(
0
5 mg, 0.02 mmol, 0.2 eqiuv.), and sodium ascorbate (8.0 mg,
.04 mmol, 0.4 equiv) in THF/H O (4 mL, 1:1, v/v) was stirred at
sumption of starting material. After 1.5 h, the solution was neutral-
2
+
ized by the addition of ion-exchange resin (Amberlite IR 120, H ),
room temperature for 12 h as above. Compound 13 was purified
filtered, and washed with MeOH, and the solvent was removed
under reduced pressure. The reaction mixture was then washed
with mixed acid/base ion-exchange resin (Amberlite) in water to
remove traces of TBAF, and lyophilized to afford 15 as a white
foam-like compound (13 mg, 27 mmol, 97%). R =0.11 (MeCN/H O
by flash column chromatography (CH Cl /MeOH 98:2) and isolated
2
2
as a white solid (87 mg, 0.093 mmol, 93%). R =0.30 (CH Cl /MeOH
f
2
2
2
D
3
1
9
7
8:2); [a] =ꢁ15.2 (c=1.0, CHCl ); H NMR (300 MHz, CDCl ): d=
3
3
.75–7.70 (m, 4H; H ), 7.50 (s, 1H; H
) 7.48–7.29 (m, 6H;
triazole
arom
3
3
3
Harom), 6.24 (d, JNH,H2 =11.6 Hz, 1H; AcNH) 5.35 (dd, J3,4 = J
.2 Hz, 1H; H-4’), 5.18 (dd, J1 =8.0, J =10.4 Hz, 1H; H-2’), 5.03–
.92 (m, 3H; H-3’, H-6a’, H-6b’), 4.81–4.69 (m, 3H; H-1, H-6a’, H-
b’), 4.39 (ddd, J=2.8, 7.2, 11.8 Hz, 1H; N-CHcyclopentyl), 4.13 (d,
JH,H =6.6 Hz, 2H; OCH ), 3.92 (ddd, J=25.1, 21.3, 11.3 Hz, 6H; H-3,
=
f
2
4,5
23
1
3
3
85:15); [a] = ++ 8.6 (c=1.0, H O); H NMR (300 MHz, D O): d=7.94
D
2 2
3
4
,2
2,3
(
s, 1H), 4.91 (d, J=12.9 Hz, 1H), 4.80 (d, J=8.5 Hz, 1H), 4.60 (d, J=
.1 Hz, 1H), 4.46 (d, J=7.8 Hz, 1H), 4.11 (m, J=5.0 Hz, 3H), 4.01–
3.97 (m, 1H), 3.92 (d, J=3.5 Hz, 1H), 3.84 (dd, J=12.3, 5.2 Hz, 1H),
.80–3.62 (m, 8H), 3.59 (ddd, J=9.3, 5.2, 2.2 Hz, 1H), 3.57–3.50 (m,
8
6
4
2
3
H-4, H-6a, H-6b, H-5, H-5’), 3.70 (dd, J=9.2 Hz, 1H; H-2), 3.42 (d,
13
3
1H), 1.91 ppm (s, 3H); C NMR (75 MHz, D O): d=175.0, 144.1,
2
J3,OH =7.9 Hz, 1H; OH-3), 2.40–2.19 (m, 2H; CH2cyclopentyl), 2.14, 2.04,
1
6
26.8, 103.5, 100.6, 79.0, 76.0, 75.5, 73.1, 72.9, 72.7, 71.6, 69.2, 63.1,
1
.97, 1.95 (4s, 12H; COCH ), 1.87 (d, J=6.5 Hz, 2H; CH2cyclopentyl),
3
+
2.6, 61.7, 60.7, 55.7, 37.2, 22.7 ppm; HRMS (ESI ): m/z calcd for
1
.81–1.66 (m, 2H; CH2cyclopentyl), 1.71 (s, 3H; NCOCH ), 1.07 (s, 9H;
3
+
13
C H N O : 479.1989 [M+H] ; found: 479.1984.
SiCMe ), 1.01–0.78 ppm (m, 2H; CH2cyclopentyl); C NMR (75 MHz,
18 30
4
11
3
CDCl ): d=170.8, 170.3, 170.0, 169.8, 169.0 (CO), 144.00 (triazole-
C4), 135.8, 135.3, 133.3, 132.4, 129.8, 127.7, 127.5 (Carom), 120.3 (tria-
3
4
-[2-Acetamido-2-deoxy-4-O-(b-d-galactopyranosyl)-b-d-glucopyrano-
syloxymethyl]-1-ethyl-1H-1,2,3-triazole (16): Compound 16 was iso-
lated from 8 (38 mg, 42 mmol) under the conditions described
above as a white foam (19 mg, 38 mmol, 91%). R =0.15 (Me/H O
zole-C ), 100.7 (C-1’), 99.6 (C-1), 79.5 (C-4), 74.6 (C-5), 72.2 (C-3),
5
7
6
1.0 (C-5), 70.7 (C-3’), 68.7 (C-2’), 66.8 (C-4’), 62.0 (C-6), 61.7 (C-6’),
f
2
1.0 (OCH ), 60.03 (N-Ccyclopentyl), 56.4 (C-2), 33.4, 33.3 (N-Ccyclopentyl),
23
1
2
8
5:15); [a] =ꢁ22.3 (c=1.0, H O); H NMR (300 MHz, D O): d=8.01
D
2
2
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&
9
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
(
7
3
s, 1H), 4.91 (d, J=12.9 Hz, 1H), 4.83 (d, J=8.5 Hz, 1H), 4.60 (d, J=
.8 Hz, 1H), 4.49–4.42 (m, 3H), 3.98 (dd, J=12.2, 2.0 Hz, 1H), 3.94–
.90 (m, 2H), 3.83 (dd, J=12.3, 5.1 Hz, 1H), 3.79–3.64 (m, 8H), 3.59
4-[2-Acetamido-2-deoxy-4-O-(b-d-galactopyranosyl)-b-d-glucopyrano-
syloxymethyl]-1-cyclopentyl-1H-1,2,3-triazole (21): Compound 13
(61 mg, 65 mmol) was stirred under the same conditions as de-
scribed above for 15 to give 21 as a white foam (31 mg, 59 mmol,
(
dd, J=12.4, 10.3 Hz, 1H), 3.53 (dd, J=10.0, 7.8 Hz, 1H), 1.90 (s,
13
23
3
1
6
H), 1.51 ppm (t, J=7.4 Hz, 3H); C NMR (75 MHz, D O): d=175.0,
91%). R =0.10 (CH Cl /MeOH 4:1); [a] =ꢁ8.7 (c=1.0, H O);
2
f
2
2
D
2
1
44.1, 125.3, 103.5, 100.7, 79.0, 76.0, 75.5, 73.2, 72.9, 71.6, 69.2,
H NMR (300 MHz, D O): d=8.06 (s, 1H), 5.08–5.00 (m, 1H), 4.92 (d,
2
+
2.6, 61.7, 60.7, 55.7, 46.3, 22.7, 15.3 ppm; HRMS (ESI ): m/z calcd
J=13.0 Hz, 1H), 4.82 (d, J=9.4 Hz, 1H), 4.65–4.59 (m, 1H), 4.49 (d,
J=7.8 Hz, 1H), 4.00 (dd, J=12.2, 1.8 Hz, 1H), 3.96–3.92 (m, 1H),
+
for C H N O : 493.2146 [M+H] ; found: 493.2146.
1
9
32
4
11
3
3
1
.86 (dd, J=12.3, 5.1 Hz, 1H), 3.82–3.66 (m, 9H), 3.63–3.60 (m, 1H),
4
-[2-Acetamido-2-deoxy-4-O-(b-d-galactopyranosyl)-b-d-glucopyrano-
.56 (dd, J=9.8, 8.0 Hz, 1H), 2.33–2.27 (m, 2H), 2.02–2.97 (m, 2H),
syloxymethyl]-1-isopropyl-1H-1,2,3-triazole (17): Compound 17 was
isolated from 9 (67 mg, 73 mmol) under the same conditions as
described above as a white foam (35 mg, 69 mmol, 94%). R =0.16
13
.92 (s, 3H), 1.88–1.83 (m, 2H), 1.81–1.75 ppm (m, 2H); C NMR
(75 MHz, D O): d=124.4, 103.5, 100.8, 79.0, 76.0, 75.4, 73.2, 72.8,
2
f
71.6, 69.2, 63.0, 62.6, 61.6, 60.7, 55.7, 33.5, 24.2, 22.7 ppm; HRMS
(ESI ): m/z calcd for C H N O : 532.2381 [M+H] ; found:
22 36 4 11
532.2450.
2
D
3
1
(
MeN/H O 85:15); [a] =ꢁ10.7 (c=1.0, H O); H NMR (300 MHz,
+
+
2
2
D O): d=8.08 (s, 1H), 4.92 (d, J=12.9 Hz, 1H), 4.88–4.85 (m, 1H),
2
4
1
8
1
1
6
.62 (d, J=6.8 Hz, 1H), 4.49 (d, J=7.8 Hz, 1H), 4.00 (d, J=10.8 Hz,
H), 3.96–3.91 (m, 1H), 3.86 (dd, J=12.2, 5.1 Hz, 1H), 3.81–3.66 (m,
H), 3.65–3.59 (m, 1H), 3.57 (dd, J=17.6, 9.5 Hz, 1H), 1.93 (s, 3H),
4-[2-Acetamido-2-deoxy-4-O-(b-d-galactopyranosyl)-b-d-glucopyrano-
syloxymethyl]-1-cyclohexyl-1H-1,2,3-triazole (22): Under the condi-
tions described above for compound 15, compound 22 was isolat-
ed from compound 14 (68 mg, 71 mmol) as a white foam (36 mg,
13
.58 ppm (d, J=6.8 Hz, 6H); C NMR (75 MHz, D O): d=175.0,
2
23.6, 103.5, 100.7, 79.0, 76.0, 75.4, 73.1, 72.8, 71.6, 69.2, 62.6, 61.6,
+
23
0.6, 55.6, 54.4, 31.0, 22.7, 22.6 ppm; HRMS (ESI ): m/z calcd for
66 mmol, 93%). R =0.11 (CH Cl /MeOH 4:1); [a] =ꢁ9.7 (c=1.0,
f
2
2
D
+
1
C H N O : 507.2302 [M+H] ; found: 507.2295.
H O); H NMR (300 MHz, D O): d=8.07 (s, 1H), 4.93 (d, J=13.0 Hz,
2 2
2
0
34
4
11
1
3
H), 4.83 (d, J=11.9 Hz, 1H), 4.64–4.60 (m, 1H), 4.57–4.52 (m,
.7 Hz, 1H), 4.50 (d, J=7.8 Hz, 1H), 4.01 (d, J=10.7 Hz, 1H), 3.95
4
-[2-Acetamido-2-deoxy-4-O-(b-d-galactopyranosyl)-b-d-glucopyrano-
syloxymethyl]-1-propyl-1H-1,2,3-triazole (18): Compound 18 was iso-
lated from 10 (80 mg, 87 mmol) under the same conditions as
described above as a white foam (40 mg, 79 mmol, 91%). R =0.16
(d, J=3.2 Hz, 1H), 3.87 (dd, J=12.3, 5.1 Hz, 1H), 3.83–3.66 (m, 9H),
3
.64–3.61 (m, 1H), 3.59–3.54 (m, 1H), 2.18 (d, J=11.0 Hz, 2H), 1.93
f
(s, 3H), 1.82–1.74 (m, 2H), 1.50 (q, J=12.9 Hz, 2H), 1.38–1.27 ppm
(m, 2H); C NMR (75 MHz, D O): d=174.9, 143.7, 123.8, 103.6,
100.8, 79.1, 76.0, 76.0, 75.5, 75.4, 73.2, 72.9, 71.6, 69.2, 62.7, 61.7,
2
D
3
1
(
Me/H O 85:15); [a] =ꢁ16.9 (c=1.0, H O); H NMR (300 MHz,
13
2
2
2
D O): d=8.03 (s, 1H), 4.93 (d, J=12.9 Hz, 1H), 4.83 (d, J=12.4 Hz,
2
1
3
5
7
H), 4.66–4.60 (m, 1H), 4.49 (d, J=7.8 Hz, 1H), 4.41 (t, J=6.9 Hz,
H), 4.03–3.98 (m, 1H), 3.94 (d, J=3.4 Hz, 1H), 3.86 (dd, J=12.3,
.1 Hz, 1H), 3.81–3.67 (m, 8H), 3.65–3.59 (m, 1H), 3.56 (dd, J=9.9,
.9 Hz, 1H), 1.96–1.89 (m, 5H), 0.88 ppm (t, J=7.4 Hz, 4H);
+
6
1.3, 60.7, 55.7, 33.6, 25.2, 22.7 ppm; HRMS (ESI ): m/z calcd for
+
C H N O : 547.2615 [M+H] ; found: 547.2613.
23
38
4
11
Synthesis of the (lactosyl)-1H-1,2,3-triazole series 30–32
1-(2,2’,3,3’,4,6,6’-Hepta-O-acetyl-b-d-lactopyranosyl)-1H-1,2,3-triazol-
1
3
C NMR (75 MHz, D O): d=175.0, 144.0, 125.8, 103.5, 100.7, 79.0,
2
[
7
2
5.9, 75.4, 73.1, 72.8, 71.6, 69.2, 62.6, 61.6, 60.6, 55.6, 52.6, 23.6,
+
4-yl]methanol (27)—microwave method A: Propargyl alcohol
(176 mL, 3.0 mmol), sodium ascorbate (7 mg, 0.038 mmol), and
CuSO ·5H O (3 mg, 0.011 mmol) were added to a solution of
azide 23 (250 mg, 0.38 mmol) in dry DMF (1.3 mL) in a microwave
sealed tube. The mixture was stirred under microwave irradiation
for 30 min at 708C. The reaction mixture was then diluted with
EtOAc and washed with H O (with a little amount of hexane). The
organic layer was treated with an aqueous solution of EDTA (5%,
2ꢅ), dried over Na SO , filtered, and concentrated under pressure
2.6, 10.7 ppm; HRMS (ESI ): m/z calcd for C H N O : 507.2302
20
34
4
11
+
[M+H] ; found: 507.2294.
4
2
52]
[
2-{4-[2-Acetamido-2-deoxy-4-O-(b-d-galactopyranosyl)-b-d-glucopyra-
nosyloxymethyl]-1H-1,2,3-triazol-1-yl}ethanol (19): Compound 11
22 mg, 24 mmol) was stirred under the same conditions as used
for 15 above to give 19 as a white foam (12 mg, 22 mmol, 92%).
(
2
2
D
3
1
R =0.10 (ACN/H O 85:15); [a] =ꢁ5.5 (c=1.0, H O); H NMR
f
2
2
(
300 MHz, D O): d=8.05 (s, 1H), 4.95 (d, J=13.0 Hz, 1H), 4.87 (d,
2
2
4
J=8.5 Hz, 1H), 4.64 (d, J=8.0 Hz, 2H), 4.61–4.57 (m, 1H), 4.49 (d,
J=7.8 Hz, 1H), 4.05–4.00 (m, 3H), 3.95 (d, J=3.4 Hz, 1H), 3.90–3.83
vacuum. The residue was purified by flash chromatography on
silica gel [CH Cl2 (100%) to CH Cl /MeOH 98:2] to give 27 as
2
2
2
(
m, 1H), 3.80–3.67 (m, 8H), 3.63 (dd, J=7.3, 5.3 Hz, 1H), 3.56 (dd,
a white solid (89.3 mg, 0.12 mmol, 33%).
13
J=9.8, 8.0 Hz, 1H), 1.93 ppm (s, 3H); C NMR (75 MHz, D O): d=
2
General method B: Azide 23 (200 mg, 0.3 mmol), propargyl alcohol
(54 mL, 0.91 mmol), CuSO ·5H O (23 mg, 0.09 mmol), and sodium
1
6
74.9, 144.1, 126.2, 103.5, 100.5, 79.0, 76.0, 75.4, 73.1, 72.8, 71.6,
+
9.1, 62.5, 61.6, 60.7, 55.6, 53.1, 22.6 ppm; HRMS (ESI ): m/z calcd
4
2
+
ascorbate (18 mg, 0.09 mmol) were dissolved in a mixture of tetra-
hydrofuran and water (1:1, 4 mL). After 12 h at room temperature,
sodium ascorbate (10 mg) and CuSO4 (10 mg) were added. The
mixture was stirred for 48 h at room temperature. The reaction
mixture was then treated as described above and then purified by
flash column chromatography (CH Cl /MeOH 98:2) to give 27 as
for C H N O : 509.2095 [M+H] ; found: 509.2088.
1
9
32
4
12
4-[2-Acetamido-2-deoxy-4-O-(b-d-galactopyranosyl)-b-d-glucopyrano-
syloxymethyl]-1-benzyl-1H-1,2,3-triazole (20): Under the conditions
described above for compound 15, compound 20 was isolated
from compound 12 (50 mg, 52.02 mmol) as a white foam (27 mg,
2
2
2
D
3
4
1
3
1
7
2
1
7
8.69 mmol, 94%). R =0.19 (CH Cl /MeOH 4:1); [a] =+19.4 (c=
a white solid (193.7 mg, 0.27 mmol, 90%). R =0.26, CH Cl /MeOH
f 2 2
f
2
2
1
23
1
.0, H O); H NMR (300 MHz, D O): d=8.01 (s, 1H), 7.50–7.41 (m,
95:5; [a]D ꢁ11.7 (c=1.0, CHCl ); H NMR (300 MHz, CDCl ): d=7.72
2
2
3
3
3
H), 7.38 (d, J=6.7 Hz, 2H), 5.65–5.57 (s, 2H), 4.88 (d, J=13.1 Hz,
H), 4.79 (d, J=13.2 Hz, 1H), 4.59 (d, J=7.8 Hz, 1H), 4.49 (d, J=
.8 Hz, 1H), 3.97–3.90 (m, 2H), 3.87–3.63 (m, 8H), 3.60–3.52 (m,
(s, 1H; H-a), 5.83 (d, J =8.4 Hz, 1H; H-1), 5.40–5.33 (m, 3H; H-2,
1,2
H-3, H-4’), 5.16–5.03 (m, 1H; H-2’), 4.96 (dd, J2’,3’ =10.4, J3’,4’
3
3
=
2.5 Hz, 1H; H-3’), 4.75 (s, 1H; H-c), 4.54–4.44 (m, 2H; H-1’, H-6a),
13
H), 1.81 ppm (s, 3H); C NMR (75 MHz, D O): d=175.0, 144.5,
4.21–4.01 (m, 3H; H-6 , H-6a’, H-6b’), 3.93–3.88 (m, 3H; H-5, H-4, H-
2
b
1
3
35.4, 129.8, 129.5, 128.8, 125.8, 103.5, 100.8, 78.9, 76.0, 75.4, 73.1,
2.8, 71.6, 69.2, 62.6, 61.6, 60.6, 55.6, 54.6, 22.6 ppm HRMS (ESI ):
5’), 2.67 (s, 1H; OH), 2.20–1.69 ppm (7s, 21H; 7ꢅ COCH3); C NMR
+
(75 MHz, CDCl ): d=170.2, 170.1, 170.0, 169.9, 169.4, 169.1, 169.0,
3
+
m/z calcd for C H N O : 555.2302 [M+H] ; found: 555.2300.
148.2, 120.1, 100.9, 85.3, 75.7, 75.4, 72.4, 70.7, 70.6, 70.3, 68.8, 66.4,
2
4
34
4
11
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&10
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
1
3
6
1.6, 60.6, 56.2, 20.6, 20.6, 20.5, 20.5, 20.4, 20.2, 20.1 ppm; HRMS
C NMR (75 MHz, D O): d=145.1, 122.5, 102.5, 86.8, 77.2, 76.9,
2
+
+
(
ESI ): m/z calcd for C H N O : 718.2301 [M+H] ; found:
75.0, 74.1, 72.1, 71.6, 70.5, 68.1, 60.6, 60.0, 59.3, 27.3 ppm; HRMS
(ESI ): m/z calcd for C H N O : 438.1718 [M+H] ; found:
16 28 3 11
29
40
3
18
+
+
7
18.2299.
4
38.1719.
2
-[1-(2,2’,3,3’,4,6,6’-Hepta-O-acetyl-b-d-lactopyranosyl)-1H-1,2,3-tria-
4-(1-b-d-Lactopyranosyl-1H-1,2,3-triazol-1-yl)butan-1-ol (32): After
zol-4-yl]ethanol (28): But-3-yn-1-ol (91 mL, 1.2 mmol), sodium ascor-
bate (6 mg, 0.03 mmol), and CuSO ·5H O (3 mg, 0.009 mmol) were
Zemplꢀn transesterification as described above, compound 29
4
2
(
0
100 mg, 0.13 mmol) gave 32 as a white powder (60.5 mg,
added to a solution of azide 23 (200 mg, 0.3 mmol) in dry DMF
1.0 mL). Under the microwave conditions described above and
standard workup, the crude triazole was purified by flash column
2
3
.13 mmol, quantitative). R =0.27, CH Cl /MeOH 6/4; [a] ꢁ2.1
(
f
2
2
D
1
(
c=1.0, MeOH); H NMR (300 MHz, D O, 228C): d=8.02 (s, 1H; H-
2
3 3
a), 5.74 (d, J =9.2 Hz, 1H; H-1), 4.51 (d, J =7.7 Hz, 1H; H-1’),
chromatography (CH Cl /MeOH 98:2) to give 28 as a white solid
1,2
1’,2’
2
2
23
4
6
2
1
6
.15–3.45 (m, 14H; H-2, H-2’, H-3, H-3’, H-4, H-4’, H-5, H-5’, H-6a, H-
(
ꢁ
216.2 mg, 0.30 mmol, 99%). R =0.23, CH Cl /MeOH 95:5; [a]
f 2 2
D
11.7 (c=1.0, CHCl ); H NMR (300 MHz, CDCl ): d=7.59 (s, 1H; H-
3
1
b, H-6a’, H-6b’, H-c), 2.75 (t, J =7.3 Hz, 1H; H-f), 1.73–1.65 (m,
4,5
3
3
13
3
H; H-d), 1.60–1.54 ppm (m, 2H; H-e); C NMR (75 MHz, D O): d=
a), 5.80 (d, J =9.0 Hz, 1H; H-1), 5.48–5.20 (m, 3H; H-2, H-3, H-4’),
2
1
,2
3
3
3
48.1, 121.7, 102.5, 86.7, 77.2, 76.9, 75.0, 74.2, 72.1, 71.5, 70.5, 68.1,
5
.12 (dd, J =10.4, J =7.9 Hz, 1H; H-2’), 4.96 (dd, J =10.4,
1’,2’ 2’,3’ 2’,3’
+
3
0.9, 60.6, 59.3, 30.2, 24.4, 23.8 ppm; HRMS (ESI ): m/z calcd for
J3’,4’ =3.2 Hz, 1H; H-3’), 4.57–4.41 (m, 2H; H-1’, H-6 ), 4.13–4.08 (m,
a
+
C H N O : 466.2031 [M+H] ; found: 466.2030.
3
H; H-6b, H-6a’, H-6b’), 3.95–3.86 (m, 5H; H-5, H-4, H-5’, H-c), 2.93
18 32
3
11
3
(
t, J =5.8 Hz, 2H; H-d), 2.41 (s, 1H; OH), 2.20–1.79 ppm (7s, 21H;
c,d
From propargyl aryl ethers 33–36 to N-glycosides 41–44
1
3
7
1
7
2
7
COCH3); C NMR (75 MHz, CDCl ): d=170.3, 170.2, 170.0, 170.0,
3
69.4, 169.3, 169.0, 146.0, 120.2, 101.0, 85.4, 75.8, 75.6, 72.3, 70.8,
0.7, 70.7, 68.9, 66.5, 61.6, 61.3, 60.7, 28.7, 20.7, 20.6, 20.6, 20.6,
0.5, 20.4, 20.2 ppm; HRMS (ESI ): m/z calcd for C H N O :
Propargyl aryl ethers 33–36: Compounds 33–36 were prepared
under literature conditions in 88 to 96% yields by alkylation of the
corresponding naphthol or phenol with propargyl bromide. Briefly,
+
30
42
3
18
+
[63]
3
[64]
32.2458 [M+H] ; found: 732.2453.
one equivalent of aryl alcohol and dry Cs CO
or dry K CO
3
in
2
2
[65]
dry DMF or acetone were treated with 1.3 equivalent of proparg-
yl bromide at room temperature to reflux until completion of re-
action as monitored by TLC. After standard workup by extraction
with EtOAc, the solvent was evaporated, and the residue was puri-
fied by flash chromatography on silica gel (hexane!hexane/CH Cl
4
-[1-(2,2’,3,3’,4,6,6’-Hepta-O-acetyl-b-d-lactopyranosyl)-1H-1,2,3-tria-
zol-4-yl]butan-1-ol (29): Under the same conditions as described
above for compound 28, azide 23 (200 mg, 0.3 mmol), hex-5-yn-1-
ol (66 mL, 0.6 mmol), sodium ascorbate (6 mg, 0.03 mmol), and
CuSO ·5H O (3 mg, 0.009 mmol) were stirred in dry DMF (1.0 mL).
2
2
4
2
8
5:15).
Triazole 29 was isolated after purification by flash column chroma-
tography (CH Cl /MeOH 97:3) as a white solid (215 mg, 0.28 mmol,
1-(2,2’,3,3’,4,6,6’-Hepta-O-acetyl-b-d-lactopyranosyl)-4-[(naphthalen-2-
yloxy)methyl]-1H-1,2,3-triazole (37): The lactosyl azide 23 (200 mg,
0.3 mmol), naphthyl propargyl ether 33 (109 mg, 0.6 mmol),
2
2
23
9
4%). R =0.25 (CH Cl /MeOH 95:5); [a] ꢁ11.6 (c=1.0, CHCl );
f
2
2
D
3
1
3
H NMR (300 MHz, CDCl ): d=7.47 (s, 1H; H-a), 5.79 (d,
J =
1,2
3
3
9
1
1
4
.3 Hz, 1H; H-1), 5.49–5.22 (m, 3H; H-2, H-3, H-4’), 5.12 (dd, J
=
’,2’
sodium ascorbate (6 mg, 0.03 mmol), and CuSO ·5 O (3 mg,
4 2
1
3
3
3
0.4, J2’,3’ =7.8 Hz, 1H; H-2’), 4.96 (dd, J2’,3’ =10.4, J3’,4’ =3.4 Hz,
0.009 mmol) in dry DMF (1.0 mL) were stirred under microwave
conditions as described above. After standard workup, the crude
triazole was purified by flash column chromatography (AcOEt/
3
H; H-3’), 4.52 (d, J =7.9 Hz, 1H; H-1’), 4.48–4.45 (m, 1H; H-6a),
1’,2’
.17–4.04 (m, 3H; H-6b, H-6a’, H-6b’), 3.97–3.82 (m, 3H; H-5, H-4,
3
3
H-5’), 3.63 (t, J =6.3 Hz, 2H; H-c), 2.74 (t, J =7.3 Hz, 2H; H-f),
hexane 6:4) to give compound 37 as a white solid (250 mg,
c,d
e,f
1
2
1
1
7
2
.24–1.83 (9s, 23H; 8COCH , OH), 1.80–1.68 (m, 2H; H-d), 1.62–
0.30 mmol, 99%). R
CDCl
J
f
=0.22 (AcOEt/hex 6:4); H NMR (300 MHz,
3
13
.49 ppm (m, 2H; H-e); C NMR (75 MHz, CDCl ): d=170.3, 170.2,
3
): d=7.82 (s, 1H; H-a), 7.79–7.69 (m, 3H; H-g,j,m), 7.44 (t,
k,i =7.5 Hz, 1H; H-k), 7.34 (t, Jl,m =7.4 Hz, 1H; H-l), 7.25–7.15 (m,
3
3
3
70.0, 169.99, 169.4, 169.3, 169.0, 148.6, 119.0, 101.0, 85.3, 75.8,
5.6, 72.4, 70.8, 70.7, 70.5, 68.9, 66.5, 62.2, 61.7, 60.7, 31.8, 25.1,
2H; H-e,f), 5.90–5.74 (m, 1H; H-1’), 5.49–5.28 (m, 5H; H-c, H-2, H-3,
+
3
3
3
5.1, 20.7, 20.6, 20.6, 20.6, 20.6, 20.4, 20.2 ppm; HRMS (ESI ): m/z
H-4’), 5.13 (dd, J2’,3’ =10.4, J2’,1’ =7.8 Hz, 1H; H-2’), 4.97 (dd, J2’,3’ =
3
+
calcd for C H N O : 760.2771 [M+H] ; found: 760.2748.
10.4, J3’,4’ =3.4 Hz, 1H; H-3’), 4.53–4.46 (m, 2H; H-1’, H-6a’), 4.19–
3
2
46
3
18
4
2
1
1
.04 (m, 3H; H-6b, H-6a’, H-6b’), 4.00–3.84 (m, 3H; H-5, H-4, H-5’),
13
(
2
1-b-d-Lactopyranosyl-1H-1,2,3-triazol-4-yl)methanol (30): Compound
7 (102 mg, 0.14 mmol) was treated under Zemplꢀn conditions as
described above. Compound 30 was isolated after lyophilization as
white powder (60.5 mg, 0.14 mmol, quantitative). Rf =0.20,
.27–1.68 ppm (m, 23H; 4ꢅCOCH3); C NMR (75 MHz, CDCl ): d=
3
70.3, 170.1, 170.0, 170.0, 169.4, 169.0, 169.0, 155.9, 144.8, 134.3,
29.5, 129.1, 127.6, 126.9, 126.4, 123.9, 121.1, 118.7, 107.2, 101.1,
a
85.5, 75.9, 75.6, 72.5, 70.8, 70.8, 70.4, 69.0, 66.5, 61.8, 61.7, 60.8,
20.7, 20.7, 20.6, 20.6, 20.6, 20.4, 20.0 ppm; HRMS (ESI ): m/z calcd
for C H N O Na: 866.2590 [M+Na] ; found: 866.2578.
23
1
+
CH Cl /MeOH 6/4; [a] 4.1 (c=1.0, MeOH); H NMR (300 MHz,
D O): d=8.23 (s, 1H; H-a), 5.80 (d, J =9.2 Hz, 1H; H-1), 4.76 (s,
2
H-2, H-2’, H-3, H-3’, H-4, H-4’, H-5, H-5’, H-6a, H-6b, H-6a’, H-6b’, H-
c); C NMR (75 MHz, D O): d=146.7, 122.8, 102.5, 86.8, 77.3, 76.8,
7
2
2
D
3
+
2
1,2
39
45
3
18
3
H; H-c), 4.52 (d, J =7.6 Hz, 1H; H-1’), 4.17–3.47 ppm (m, 14H;
1’,2’
1-(2,2’,3,3’,4,6,6’-Hepta-O-acetyl-b-d-lactopyranosyl)-4-(phenoxymeth-
1
3
yl)-1H-1,2,3-triazole (38): The triazole 38 was synthesized under the
same conditions as described above, starting with lactosyl azide 23
(200 mg, 0.3 mmol), phenyl propargyl ether (34, 80 mg, 0.6 mmol),
sodium ascorbate (6 mg, 0.03 mmol), and CuSO ·5H O ((3 mg,
2
+
5.0, 74.1, 72.1, 71.5, 70.5, 68.1, 60.6, 59.3, 54.1 ppm; HRMS (ESI ):
+
m/z calcd for C H N O : 424.1562 [M+H] ; found: 424.1563.
1
5
26
3
11
4
2
2-(1-b-d-Lactopyranosyl)-1H-1,2,3-triazol-4-yl)ethanol (31): Com-
0.009 mmol) in DMF. After purification over silica gel (AcOEt/
pound 28 (102 mg, 0.14 mmol) was deacetylated under the same
conditions as described above to give compound 31 after lyophili-
hexane 6:4), the desired compound 38 was isolated as a white
1
solid (230 mg, 0.29 mmol, 96%). R =0.29, AcOEt/hex 6:4; H NMR
f
zation as a white powder (61.2 mg, 0.14 mmol, quantitative). R =
(300 MHz, CDCl ): d=7.74 (s, 1H; H-a), 7.30–7.15 (m, 2H; H-g,h),
f
3
23
1
0
.18, CH Cl /MeOH 6/4; [a] 7.0 (c=1.0, MeOH); H NMR (300 MHz,
6.97–6.88 (m, 3H; H-e,f,i), 5.83–5.77 (m, 1H; H-1), 5.40–5.29 (m, 3H;
2
2
D
3
3
3
D O): d=8.09 (s, 1H; H-a), 5.77 (d, J =9.1 Hz, 1H; H-1), 4.52 (d,
H-2, H-3, H-4’), 5.16 (s, 2H; H-c), 5.08 (dd, J =7.8, J =10.4 Hz,
1’,2’ 2’,3’
3 3
2
1,2
3
J1’,2’ =7.7 Hz, 1H; H-1’), 4.15–3.46 (m, 14H; H-2, H-2’, H-3, H-3’, H-4,
1H; H-2’), 4.93 (dd, J =10.4, J =3.4 Hz, 1H; H-3’), 4.49–4.41
2’,3’ 3’,4’
H-4’, H-5, H-5’, H-6a, H-6b, H-6a’, H-6b’, H-c), 2.98 ppm (s, 2H; H-d),
(m, 2H; H-1’, H-6 ’), 4.15–4.00 (m, 3H; H-6 , H-6 ’, H-6 ’), 3.95–3.81
a b a b
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&
11
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
(
m, 3H; H-5, H-4, H-5’), 2.11–1.78 ppm (m, 23H; 4ꢅCOCH );
1-(b-d-Lactopyranosyl)-4-(phenoxymethyl)-1H-1,2,3-triazole (42): Un-
der the Zemplꢀn conditions described above, triazole 38 (103 mg,
0.13 mmol) gave 42 as a white powder (63 mg, 0.13 mmol, quanti-
3
1
3
C NMR (75 MHz, CDCl ): d=170.3, 170.1, 170.0, 170.0, 169.3,
3
1
7
2
69.1, 169.0, 158.0, 144.9, 129.5, 121.3, 121.1, 114.7, 101.0, 85.4,
5.8, 75.6, 72.5, 70.8, 70.8, 70.4, 69.0, 66.5, 61.7, 61.7, 60.8, 20.7,
1
tative). R =0.18, MeCN/H O 95:5; H NMR (300 MHz, D O): d=8.15
f
2
2
3
+
3
0.7, 20.6, 20.6, 20.6, 20.4, 20.1 ppm; HRMS (ESI ): m/z calcd for
(s, 1H; H-a), 7.24 (t, J =7.8 Hz, 2H; H-g,h), 6.93 (d, J =7.7 Hz,
3H; H-e,f,i), 5.64 (d, J =9.1 Hz, 1H; H-1), 5.12 (s, 2H; H-c), 4.37 (d,
J1’,2’ =7.6 Hz, 1H; H-1’), 3.98–3.39 ppm (m, 12H; H-2, H-2’, H-3, H-
’, H-4, H-4’, H-5, H-5’, H-6a, H-6b, H-6a’, H-6b’); C NMR (75 MHz,
g,(e,i)
f,h
+
3
C H N O Na: 816.2434 [M+Na] ; found: 816.2419.
3
5
43
3
18
1,2
3
1
-(2,2’,3,3’,4,6,6’-Hepta-O-acetyl-b-d-lactopyranosyl)-4-[(4-methoxy-
13
3
phenoxy)methyl]-1H-1,2,3-triazole (39): Under the microwave condi-
tions described above, lactosyl azide 23 (200 mg, 0.3 mmol) and 4-
methoxyphenyl propargyl ether (35, 100 mg, 0.6 mmol) gave the
desired triazole 39 as a white solid (209.3 mg, 0.25 mmol, 85%).
D O): d=157.0, 143.3, 129.6, 123.9, 121.6, 114.7, 102.6, 87.0, 77.4,
2
7
7.0, 75.1, 74.3, 72.2, 71.7, 70.6, 68.2, 60.7, 60.6, 59.4 ppm; HRMS
+
+
(ESI ): m/z calcd for C H N O : 500.1875 [M+H] ; found:
21 30 3 11
5
00.1872.
1
R =0.20, AcOEt/hex 6:4; H NMR (300 MHz, CDCl ): d=7.77 (s, 1H;
f
3
1
-b-d-Lactopyranosyl-4-[(4-methoxyphenoxy)methyl]-1H-1,2,3-triazole
H-a), 7.95–7.87 (m, 2H; H-g,h), 6.85–6.78 (m, 2H; H-e,f), 5.89–5.79
m, 1H; H-1), 5.43–5.34 (m, 3H; H-2, H-3, H-4’), 5.19–5.09 (m, 3H;
(43): Triazole 39 (103.5 mg, 0.12 mmol) was deacetylated under the
(
3
3
general Zemplꢀn conditions described above to give 43 as a white
powder after lyophilization (65.2 mg, 0.13 mmol, quantitative). R =
0
6.85 (d, J =7.5 Hz, 2H; H-g,h), 6.79 (d, J =9.4 Hz, 2H; H-e,f),
g,e f,h
5
7
H-2’, H-c), 4.97 (dd, J2 =10.4, J =3.4 Hz, 1H; H-3’), 4.54–4.46
’,3’
3’,4’
(
(
m, 2H; H-1’, H-6a’), 4.18–4.05 (m, 3H; H-6 , H-6a’, H-6b’), 3.96–3.89
f
b
1
.18, MeCN/H O 95:5; H NMR (300 MHz, D O): d=8.13 (s, 1H; H-a),
m, 3H; H-5, H-4, H-5’), 2.21–1.81 ppm (m, 23H; 4ꢅCOCH );
2
2
3
3
3
1
3
C NMR (75 MHz, CDCl ): d=170.3, 170.1, 170.0, 170.0, 169.4,
3
3
3
.64 (d, J1,2 =9.0 Hz, 1H; H-1), 5.04 (s, 2H; H-c), 4.38 (d, J1’,2’
=
1
7
2
69.1, 169.0, 154.2, 152.1, 145.1, 121.0, 115.8, 114.6, 101.0, 85.4,
5.8, 75.6, 72.6, 70.8, 70.8, 70.4, 69.0, 66.5, 62.6, 61.7, 60.8, 55.6,
.6 Hz, 1H; H-1’), 4.03–3.38 ppm (m, 15H; H-2, H-2’, H-3, H-3’, H-4,
13
+
H-4’, H-5, H-5’, H-6a, H-6b, H-6a’, H-6b’, H-j); C NMR (75 MHz,
D O): d=153.4, 151.4, 143.6, 124.2, 116.7, 114.9, 102.8, 87.2, 77.6,
0.7, 20.7, 20.6, 20.6, 20.6, 20.4, 20.1 ppm; HRMS (ESI ): m/z calcd
+
for C H N O Na: 846.2539 [M+Na] ; found: 846.2534.
2
3
6
45
3
19
7
7.2, 75.3, 74.4, 72.4, 71.9, 70.8, 68.4, 61.8, 60.9, 59.6, 55.6 ppm;
+
+
4
-{[1-(2,2’,3,3’,4,6,6’-Hepta-O-acetyl-b-d-lactopyranosyl)-1H-1,2,3-tria-
zol-4-yl]methoxy}benzonitrile (40): The azide 23 (200 mg, 0.3 mmol),
-(prop-2-ynyloxy)benzonitrile (36, 189 mg, 1.2 mmol), sodium as-
corbate (6 mg, 0.03 mmol), and CuSO ·5H O (3 mg, 0.009 mmol) in
DMF (1.0 mL) were stirred under the conditions described above.
After 30 min under microwave irradiation, the reaction mixture was
HRMS (ESI ): m/z calcd for C H N O : 530.1980 [M+H] ; found:
22 32 3 12
530.1973.
4
4-[(1-b-d-Lactopyranosyl-1H-1,2,3-triazol-4-yl)methoxy]benzonitrile
4
2
(44): Transesterification of triazole 40 (101.3 mg, 0.124 mmol)
under Zemplꢀn conditions gave 44 in quantitative yield as a white
solid (70.3 mg, 0.124 mmol). R =0.27, CH Cl /MeOH 6:4; H NMR
1
f
2
2
3
diluted with EtOAc and washed with H O (with a little amount of
2
(
6
300 MHz, D O): d=8.20 (s, 1H; H-a), 7.53 (d, J =8.8 Hz, 2H; H-e),
.97 (d, J =8.9 Hz, 2H; H-f), 5.66 (d, J =9.2 Hz, 1H; H-1), 5.16
e,f 1,2
2
e,f
hexane). The organic layer was treated with an aqueous solution of
3
3
EDTA (5%, 2ꢅ), dried over Na SO , filtered, and concentrated under
2
4
3
(s, 2H; H-c), 4.37 (d, J1’,2’ =7.6 Hz, 1H; H-1’), 3.99–3.34 ppm (m,
reduced pressure. The crude triazole was purified by flash chroma-
1
2H; H-2, H-2’, H-3, H-3’, H-4, H-4’, H-5, H-5’, H-6a, H-6b, H-6a’, H-
tography on silica gel (hexane/AcOEt 4:6) to give compound 40 as
13
1
6b’); C NMR (75 MHz, D
O): d=160.7, 142.6, 134.0, 124.0, 119.4,
2
a white solid (233 mg, 0.29 mmol, 95%); H NMR (300 MHz, CDCl ):
3
3
3
115.1, 102.9, 102.4, 86.8, 77.2, 76.8, 74.9, 74.0, 72.0, 71.5, 70.4, 68.1,
d=7.80 (s, 1H; H-a), 7.59 (d, J =8.0 Hz, 2H; H-e), 7.04 (d, J =
.1 Hz, 2H; H-f), 5.84 (d, J1 =8.2 Hz, 1H; H-1), 5.47–5.32 (m, 3H;
,2
e,f
e,f
+
3
60.6, 59.2 ppm; HRMS (ESI ): m/z calcd for C22H N O11Na: 547.1647
28 4
8
+
[
M+Na] ; found: 547.1643.
H-2, H-3, H-4’), 5.24 (s, 2H; H-c), 5.15–5.06 (m, 1H; H-2’), 4.97 (dd,
3
3
J2 =10.5, J =2.6 Hz, 1H; H-3’), 4.53–4.47 (m, 2H; H-1’, H-6a’),
’,3’
3’,4’
Biochemistry
4
.20–4.03 (m, 3H; H-6b, H-6a’, H-6b’), 3.96–3.88 (m, 3H; H-5, H-4,
13
Lectins: Extracts of dried mistletoe leaves and pellets of bacteria
after recombinant galectin production under conditions optimized
for yield and protein integrity were obtained in phosphate buffer
H-5’), 2.24–1.76 ppm (m, 23H; 4ꢅCOCH3); C NMR (75 MHz,
CDCl ): d=170.3, 170.1, 170.0, 170.0, 169.3, 169.1, 169.0, 161.2,
3
1
7
2
43.6, 134.0, 121.3, 118.9, 115.5, 104.6, 101.0, 85.5, 76.0, 75.5, 72.3,
0.8, 70.8, 70.4, 68.9, 66.5, 61.8, 61.6, 60.7, 34.0, 22.3, 20.7, 20.6,
(pH 7.2, 20 mm) containing NaCl (150 mm). Affinity chromatogra-
+
phy on lactosylated Sepharose 4B, prepared by divinyl sulfone acti-
vation to allow high yields, was performed as key separation step,
with preparative gel filtration ensuring the quaternary structure, as
0.6, 20.6, 20.4, 20.1, 20.1 ppm; HRMS (ESI ): m/z calcd for
+
C H N O : 819.2567 [M+H] ; found: 819.2562.
3
6
43
4
18
[
39,66]
1-(b-d-Lactopyranosyl)-4-[(naphthalen-2-yloxy)methyl]-1H-1,2,3-tria-
described.
The product quality was routinely checked by one-
zole (41): Compound 37 (103.3 mg, 0.12 mmol) was dissolved in
a mixture of MeONa (0.4 mL) and MeOH (1 mL) at pH 9. The mix-
ture was stirred at room temperature under nitrogen for 72 h.
When the deprotection was complete, the solution was neutralized
and two-dimensional gel electrophoresis and mass spectrometric
fingerprinting to exclude the occurrence of any modifications or
[39–41]
sequence deviations.
Biotinylation was performed under activi-
ty-preserving conditions with the N-hydroxysuccinimide ester de-
rivative of biotin (Sigma), and the extent and site of incorporation
+
with Dowex H resin, filtered, concentrated, and lyophilized to pro-
vide 41 as a white powder (66.0 mg, 0.13 mmol, quant.). R =0.18,
were monitored by gel electrophoretic and mass spectrometric
f
1
[67,68]
MeCN/H O 95:5; H NMR (300 MHz, D O): d=8.40 (s, 1H; H-a),
product processing as described.
In addition, Gal-1 and Gal-4
2
2
7
.93–7.77 (m, 3H; H-k,l,m), 7.58–7.35 (m, 3H; H-e,f,j), 7.22 (dd,
were fluorescently labeled with fluorescein isothiocyanate and
ALEXA 647 (Invitrogen, Darmstadt, Germany) as recommended by
the commercial supplier. Lack of impairment of lectin activity was
ascertained by solid-phase assays as also performed for analytical
purposes (please see next paragraph).
3
4
3
J =9.0, J =2.5 Hz, 1H; H-h), 5.72 (d, J =9.2 Hz, 1H; H-1), 5.34
f,h
h,l
1,2
3
(
s, 2H; H-c), 4.40 (d, J1’,2’ =7.5 Hz, 1H; H-1’), 4.04–3.41 ppm (m,
1
6
1
7
2H; H-2, H-2’, H-3, H-3’, H-4, H-4’, H-5, H-5’, H-6a, H-6b, H-6a’, H-
b’); C NMR (75 MHz, D O): d=157.2, 145.2, 135.9, 131.6, 130.7,
29.4, 128.7, 126.7, 126.2, 120.3, 109.4, 104.6, 89.0, 79.3, 79.2, 77.0,
6.4, 74.2, 73.5, 72.5, 70.2, 62.6, 62.4, 61.3, 60.3 ppm; HRMS (ESI ):
1
3
2
+
Solid-phase assay: The pan-galectin glycoprotein ligand asialofe-
tuin, obtained by chemical desialylation of fetuin at pH 2 and
+
m/z calcd for C H N O : 550.2031 [M+H] ; found: 550.2026.
2
5
32
3
11
8
08C, was the active matrix component in 96-well microtiter plates
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&12
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
for binding lectins from solution. Coating was performed overnight
at 48C with glycoprotein-containing solution (50 mL) in phosphate-
buffered saline (0.5 mg/well). The following steps, including satura-
tion of remaining sites for adsorbing protein with carbohydrate-
free bovine serum albumin, incubation with solution containing la-
beled lectin in the absence or presence of test compounds, appli-
cation of the indicator (streptavidin–peroxidase conjugate used at
Acknowledgements
The authors greatly appreciate generous funding by the National
Cancer Institute (NIH grant CA096090) for research, by the Na-
tional Science Foundation (BIR-961477), the University of Minne-
sota Medical School and the Minnesota Medical Foundation for
NMR instrumentation (to K.H.M.), the EC (FP7 grant 260600–GLY-
COHIT, ITN grant 317297–GLYCOPHARM), and the Verein zur Fçr-
derung des biologisch-technologischen Fortschritts in der Medizin
e.V. (Heidelberg, Germany; to H.-J.G.), and the Natural Sciences
and Engineering Research Council of Canada, as well as by
a Canadian Research Chair (to R.R.).
ꢁ1
0
.5 mgmL , Sigma), and signal development with the chromogenic
ꢁ1
substrates o-phenylenediamine (1 mgmL )/hydrogen peroxide
ꢁ1
[15,16]
(
1 mLmL ) were done by an established protocol.
Binding
assays were routinely performed with controls for maximum level
of binding in the absence of inhibitor (100%-value) and minimum
level of carbohydrate-dependent binding by inhibition by a mixture
ꢁ
1
of lactose (100 mm) and asialofetuin (1 mgmL , 0%-value) for nor-
malization. Assays were routinely done in triplicate with up to five
independent series and standard deviations not exceeding 13.8%.
Keywords: galectins · glycoproteins · lactose · lectins ·
structure–activity relationships
Cell assays
Cytotoxicity: Cell growth in the absence/presence of a test com-
pound was quantitated in aliquots of cell suspensions after an in-
cubation period of 48 h for human colon adenocarcinoma (SW480)
cells by use of the blue chromogen 3-(4,5-dimethylthiazol-2-yl)-2,5-
[
[
1] The Sugar Code. Fundamentals of Glycosciences (Ed.: H.-J. Gabius), Wiley-
VCH, Weinheim, 2009.
2] a) H.-J. Gabius, S. Andrꢀ, J. Jimꢀnez-Barbero, A. Romero, D. Solꢆs, Trends
Biochem. Sci. 2011, 36, 298–313; b) D. Solꢆs, N. V. Bovin, A. P. Davis, J. Ji-
mꢀnez-Barbero, A. Romero, R. Roy, K. Smetana Jr., H.-J. Gabius, Biochim.
Biophys. Acta Gen. Subj. 2014; DOI: 10.1016/j.bbagen.2014.03.016.
3] a) A. Imberty, Y. M. Chabre, R. Roy, Chem. Eur. J. 2008, 14, 7490–7499;
b) H. Leffler, U. J. Nilsson, ACS Symp. Ser. 2012, 1115, 47–59.
ꢁ
1
[69]
diphenyltetrazolium bromide (5 mgmL ) as described.
were performed in aliquots of a cell suspension in triplicate.
Assays
Cytofluorimetry: Cells of the human B-lymphoblastoid line Croco II
and the pancreatic carcinoma (Capan-1) line reconstituted for ex-
[
INK4a
pression of the tumor suppressor p16
(kindly provided by Kath-
[4] H. M. I. Osborn, A. Turkson in The Sugar Code. Fundamentals of Glyco-
sciences (Ed.: H.-J. Gabius), Wiley-VCH, Weinheim, 2009, pp. 469–483.
ꢁ5
arina M. Detjen, Berlin, Germany), as well as the Pro wild-type
Chinese hamster ovary line (kindly provided by Pamela Stanley,
Bronx, New York), were grown and processed by washing and incu-
bation steps with labeled lectin and streptavidin/R-phycoerythrin
indicator conjugate (1:40, Sigma) for first-step use of biotinylated
[
[
[
[
5] S. Andrꢀ, P. J. Cejas Ortega, M. Alamino Perez, R. Roy, H.-J. Gabius, Glyco-
biology 1999, 9, 1253–1261.
6] M. C. Galan, D. Benito-Alifonso, G. M. Watt, Org. Biomol. Chem. 2011, 9,
598–3610.
7] Y. M. Chabre, R. Roy, The Sugar Code. Fundamentals of Glycosciences
Ed.: H.-J. Gabius), Wiley-VCH, Weinheim, 2009, pp. 53–70.
8] Y. M. Chabre, R. Roy, Adv. Carbohydr. Chem. Biochem. 2010, 63, 165–393.
3
[15,16,41]
lectins in FACScan analysis as described in detail previously.
(
Data processing resulted in percentages of positive cells and mean
fluorescence intensities. Controls included omission of the incuba-
tion step with the fluorescent second-step reagent, blocking of car-
bohydrate-dependent binding as above, and application of non-
cognate sugar to detect any osmolarity effect. Assays were per-
formed in duplicate with up to four independent series with strict
use of aliquots of cell suspensions of the same or the next passage
with standard deviations for both parameters not exceeding 11.5%
after normalization of data.
[9] M. R. Hartley, J. M. Lord, Biochim. Biophys. Acta Proteins Proteomics
2004, 1701, 1–14.
[10] H. Kaltner, H.-J. Gabius, Histol. Histopathol. 2012, 27, 397–416.
11] R. T. Lee, H.-J. Gabius, Y. C. Lee, J. Biol. Chem. 1992, 267, 23722–23727.
12] S. Bharadwaj, H. Kaltner, E. Y. Korchagina, N. V. Bovin, H.-J. Gabius, A.
Surolia, Biochim. Biophys. Acta Gen. Subj. 1999, 1472, 191–196.
13] R. Mikeska, R. Wacker, R. Arni, T. P. Singh, A. Mikhailov, A. Gabdoulkha-
kov, W. Voelter, C. Betzel, Acta Crystallogr. Sect. F 2005, 61, 17–25.
14] M. Jimꢀnez, S. Andrꢀ, C. Barillari, A. Romero, D. Rognan, H.-J. Gabius, D.
Solꢆs, FEBS Lett. 2008, 582, 2309–2312.
15] a) S. Andrꢀ, D. Giguꢇre, T. K. Dam, C. F. Brewer, H.-J. Gabius, R. Roy, New
J. Chem. 2010, 34, 2229–2240; b) D. Giguꢇre, R. Patnam, M.-A. Belle-
fleur, C. St-Pierre, S. Sato, R. Roy, Chem. Commun. 2006, 2379–2381;
c) S. Sirois, D. Giguꢇre, R. Roy, Med. Chem. 2006, 2, 481–489.
16] D. Giguꢇre, S. Andrꢀ, M.-A. Bonin, M.-A. Bellefleur, A. Provencal, P. Clout-
ier, B. Pucci, R. Roy, H.-J. Gabius, Bioorg. Med. Chem. 2011, 19, 3280–
287.
[17] a) H. Ahmed, H. J. Allen, A. Sharma, K. L. Matta, Biochemistry 1990, 29,
315–5319; b) D. Giguꢇre, S. Sato, C. St-Pierre, S. Sirois, R. Roy, Bioorg.
Med. Chem. Lett. 2006, 16, 1668–1672.
[18] R. N. Knibbs, N. Agarwal, J. L. Wang, I. J. Goldstein, J. Biol. Chem. 1993,
268, 14940–14947.
[
[
[
[
[
NMR spectroscopy
Determination of binding constants: Dissociation binding constants
were determined by sigmoidal (Gaussian) fits to titration curves
1
15
15
plotted from data of H, N HSQC spectra of N-labeled galectins,
[
[43]
as a function of the ligand concentration, as described. Calcula-
tions were performed with 15 to 22 individual HSQC crosspeaks
that were chemically shifted the most during the titrations.
3
5
1
15
H, N HSQC NMR titration experiments: All heteronuclear NMR ex-
periments were performed at 308C with a Bruker AVANCE 700 MHz
spectrometer equipped with a H/C/N triple-resonance probe and
x/y/z triple-axis pulse field gradient unit. For these NMR studies,
[19] R. T. Lee, Y. Ichikawa, H. J. Allen, Y. C. Lee, J. Biol. Chem. 1990, 265,
7864–7871.
20] C. P. Sparrow, H. Leffler, S. H. Barondes, J. Biol. Chem. 1987, 262, 7383–
390.
21] R. K. Merkle, R. D. Cummings, J. Biol. Chem. 1988, 263, 16143–16149.
22] S. Sato, R. C. Hughes, J. Biol. Chem. 1992, 267, 6983–6990.
23] T. Feizi, J. C. Solomon, C.-T. Yuen, K. C. G. Jeng, L. G. Frigeri, D. K. Hsu,
F.-T. Liu, Biochemistry 1994, 33, 6342–6349.
1
5
[43–45]
uniformly N-labeled galectins
were dissolved at a concentra-
[
tion of about 100 mm in potassium phosphate buffer (20 mm) and
dithiothreitol (2 mm) at pH 7.0, with use of a 95% H O/5% D O
7
2
1
2
[
[
[
15
mixture. A gradient sensitivity-enhanced version of 2D H, N HSQC
was applied with 256 (t1)ꢅ 2048 (t2) complex data points in nitro-
gen and proton dimensions, respectively. Raw data were converted
[70]
and processed by use of NMRPipe
NMRview.
and analyzed by use of
[24] S. R. Stowell, M. Dias-Baruffi, L. Penttilꢈ, O. Renkonen, A. Kwame Nyame,
[71]
R. D. Cummings, Glycobiology 2004, 14, 157–167.
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&13
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
[
[
[
[
25] S. R. Stowell, C. M. Arthur, P. Mehta, K. A. Slanina, O. Blixt, H. Leffler, D. F.
Smith, R. D. Cummings, J. Biol. Chem. 2008, 283, 10109–10123.
26] K. Bachhawat-Sikder, C. J. Thomas, A. Surolia, FEBS Lett. 2001, 500, 75–
[53] D. Gupta, H. Kaltner, X. Dong, H.-J. Gabius, C. F. Brewer, Glycobiology
1996, 6, 843–849.
[54] T. K. Dam, H.-J. Gabius, S. Andrꢀ, H. Kaltner, M. Lensch, C. F. Brewer, Bio-
chemistry 2005, 44, 12564–12571.
79.
27] N. Ahmad, H.-J. Gabius, H. Kaltner, S. Andrꢀ, I. Kuwabara, F.-T. Liu, S.
Oscarson, T. Norberg, C. F. Brewer, Can. J. Chem. 2002, 80, 1096–1104.
28] J. Hirabayashi, T. Hashidate, Y. Arata, N. Nishi, T. Nakamura, M. Hirashi-
ma, T. Urashima, T. Oka, M. Futai, W. E. G. Mꢉller, F. Yagi, K.-i. Kasai, Bio-
chim. Biophys. Acta Gen. Subj. 2002, 1572, 232–254.
[55] a) S. Andrꢀ, H. Sanchez-Ruderisch, H. Nakagawa, M. Buchholz, J. Kopitz,
P. Forberich, W. Kemmner, C. Bçck, K. Deguchi, K. M. Detjen, B. Wieden-
mann, M. von Knebel Doeberitz, T. M. Gress, S.-I. Nishimura, S. Rosewicz,
H.-J. Gabius, FEBS J. 2007, 274, 3233–3256; b) M. Amano, H. Eriksson,
J. C. Manning, K. M. Detjen, S. Andrꢀ, S.-I. Nishimura, J. Lehtiç, H.-J.
Gabius, FEBS J. 2012, 279, 4062–4080.
[56] a) S. Martꢆn-Santamarꢆa, H.-J. Gabius, J. Jimꢀnez-Barbero, Pure Appl.
Chem. 2012, 84, 49–64; b) P. Vidal, V. Roldꢋs, C. Fernꢊndez-Alonso Mdel,
B. Vauzeilles, Y. Bleriot, F. J. CaÇada, S. Andrꢀ, H.-J. Gabius, J. Jimꢀnez-
Barbero, J. F. Espinosa, S. Martꢆn-Santamarꢆa, Chem. Eur. J. 2013, 19,
14581–14590.
[
[
29] H. Ideo, A. Seko, T. Ohkura, K. L. Matta, K. Yamashita, Glycobiology 2002,
12, 199–208.
30] E. M. Rapoport, S. Andrꢀ, O. V. Kurmyshkina, T. V. Pochechueva, V. V. Sev-
erov, G. V. Pazynina, H.-J. Gabius, N. V. Bovin, Glycobiology 2008, 18,
315–324.
[31] O. A. Vokhmyanina, E. M. Rapoport, S. Andrꢀ, V. V. Severov, I. Ryzhov,
G. V. Pazynina, E. Korchagina, H.-J. Gabius, N. V. Bovin, Glycobiology
[57] a) S. Andrꢀ, B. Liu, H.-J. Gabius, R. Roy, Org. Biomol. Chem. 2003, 1,
3909–3916; b) N. Ahmad, H.-J. Gabius, S. Andrꢀ, H. Kaltner, S. Sabesan,
R. Roy, B. Liu, F. Macaluso, C. F. Brewer, J. Biol. Chem. 2004, 279, 10841–
10847; c) S. Andrꢀ, M. Lahman, H.-J. Gabius, S. Oscarson, Mol. Pharm.
2010, 7, 2270–2279; d) P. V. Murphy, S. Andrꢀ, H.-J. Gabius, Molecules
2013, 18, 4026–4053; e) S. Andrꢀ, C. Grandjean, F. M. Gautier, S. Bernar-
di, F. Sansone, H.-J. Gabius, R. Ungaro, Chem. Commun. 2011, 47, 6126–
6128.
[58] a) V. Percec, P. Leowanawat, H. J. Sun, O. Kulikov, C. D. Nusbaum, T. M.
Tran, A. Bertin, D. A. Wilson, M. Peterca, S. Zhang, N. P. Kamat, K. Vargo,
D. Moock, E. D. Johnston, D. A. Hammer, D. J. Pochan, Y. Chen, Y. M.
Chabre, T. C. Shiao, M. Bergeron-Brlek, S. Andrꢀ, R. Roy, H.-J. Gabius,
P. A. Heiney, J. Am. Chem. Soc. 2013, 135, 9055–9077; b) S. Zhang, R.-O.
Moussodia, H.-J. Sun, P. Leowanawat, A. Muncan, C. D. Nusbaum, K. M.
Chelling, P. A. Heiney, M. L. Klein, S. Andrꢀ, R. Roy, H.-J. Gabius, V.
Percec, Angew. Chem. Int. Ed. 2014, 53, 10899–10903.
2012, 22, 1207–1217.
[
[
[
[
[
32] N. Nagae, N. Nishi, T. Murata, T. Usui, T. Nakumura, S. Wakatsuki, R. Kato,
Glycobiology 2009, 19, 112–117.
33] R. Leyden, T. Velasco-Torrijos, S. Andrꢀ, S. Gouin, H.-J. Gabius, P. V.
Murphy, J. Org. Chem. 2009, 74, 9010–9026.
34] S. Andrꢀ, D. V. Jarikote, D. Yan, L. Vincenz, G. N. Wang, H. Kaltner, P. V.
Murphy, H.-J. Gabius, Bioorg. Med. Chem. Lett. 2012, 22, 313–318.
35] G. N. Wang, S. Andrꢀ, H.-J. Gabius, P. V. Murphy, Org. Biomol. Chem.
2
012, 10, 6893–6907.
36] E. C. Beyer, S. E. Zweig, S. H. Barondes, J. Biol. Chem. 1980, 255, 4236–
239.
4
[
[
37] Y. Oda, K.-i. Kasai, Biochim. Biophy. Acta 1983, 761, 237–245.
38] Y. Sakakura, J. Hirabayashi, Y. Oda, Y. Ohyama, K.-i. Kasai, J. Biol. Chem.
1990, 265, 21573–21579.
[
39] H. Kaltner, D. Solꢆs, J. Kopitz, M. Lensch, M. Lohr, J. C. Manning, M. Mꢉrn-
seer, M. Schnçlzer, S. Andrꢀ, J. L. Sꢊiz, H.-J. Gabius, Biochem. J. 2008,
[59] I. Pelletier, T. Hashidate, T. Urashima, N. Nishi, T. Nakamura, M. Futai, Y.
Arata, K.-i. Kasai, M. Hirashima, J. Hirabayashi, S. Sato, J. Biol. Chem.
2003, 278, 22223–22230.
[60] R. Jin, A. Greenwald, M. D. Peterson, T. K. Waddell, J. Immunol. 2006,
177, 1289–1295.
409, 591–599.
[
[
40] H. Kaltner, D. Solꢆs, S. Andrꢀ, M. Lensch, J. C. Manning, M. Mꢉrnseer, J. L.
Sꢊiz, H.-J. Gabius, Biochemistry 2009, 48, 4403–4416.
41] H. Kaltner, D. Kꢉbler, L. Lꢋpez-Merino, M. Lohr, J. C. Manning, M. Lensch,
J. Seidler, W.-D. Lehmann, S. Andrꢀ, D. Solꢆs, H.-J. Gabius, Anat. Rec.
[61] C. Diehl, S. Genheden, K. Modig, U. Ryde, M. Akke, J. Biomol. NMR 2009,
45, 157–169.
2011, 294, 427–444.
[
[
[
42] F. M. Ruiz, I. S. Fernꢊndez, L. Lꢋpez-Merino, L. Lagartera, H. Kaltner, M.
Menꢀndez, S. Andrꢀ, D. Solꢆs, H.-J. Gabius, A. Romero, Acta Crystallogr.
Sect. D 2013, 69, 1665–1676.
43] M. C. Miller, J. P. Ribeiro, V. Roldꢋs, S. Martꢆn-Santamarꢆa, F. J. CaÇada,
I. A. Nesmelova, S. Andrꢀ, M. Pang, A. A. Klyosov, L. G. Baum, J. Jimꢀnez-
Barbero, H.-J. Gabius, K. H. Mayo, Glycobiology 2011, 21, 1627–1641.
44] a) A. B. Yongye, L. Calle, A. Ardꢊ, J. Jimꢀnez-Barbero, S. Andrꢀ, H.-J.
Gabius, K. Martꢆnez-Mayorga, M. Cudic, Biochemistry 2012, 51, 7278–
[62] C. Diehl, O. Engstrçm, T. Delaine, M. Hꢌkansson, S. Genheden, K. Modig,
H. Leffler, U. Ryde, U. J. Nilsson, M. Akke, J. Am. Chem. Soc. 2010, 132,
14577–14589.
[63] I. N. Lykakis, C. Efe, C. Gryparis, M. Stratakis, Eur. J. Org. Chem. 2011,
2334–2338.
[64] J. He, L. Feng, J. Li, R. Tao, F. Wang, X. Liao, Q. Sun, Q. Long, Y. Ren, J.
Wan, H. He, Bioorg. Med. Chem. 2012, 20, 1665–1670.
[65] J.-W. Yuan, L.-B. Qu, X.-L. Chen, Z.-B. Qu, X.-Q. Liu, D.-D. Ke, J. Chin.
Chem. Soc. 2011, 58, 24–30.
7289; b) M. A. Berbꢆs, S. Andrꢀ, F. J. Canada, R. Pipkorn, H. Ippel, K. H.
Mayo, D. Kꢉbler, H.-J. Gabius, J. Jimꢀnez-Barbero, Biochem. Biophys. Res.
Commun. 2014, 443, 126–131.
45] E. Ermakova, M. C. Miller, I. V. Nesmelova, L. Lꢋpez-Merino, M. A. Berbꢆs,
Y. Nesmelov, Y. V. Tkachev, L. Lagartera, V. A. Daragan, S. Andrꢀ, F. J.
CaÇada, J. Jimꢀnez-Barbero, D. Solꢆs, H.-J. Gabius, K. H. Mayo, Glycobiolo-
gy 2013, 23, 508–523.
46] Z. Gan, S. Cao, Q. Wu, R. Roy, J. Carbohydr. Chem. 1999, 18, 755–773.
47] R. Daly, T. McCabe, E. M. Scanlan, J. Org. Chem. 2013, 78, 1080–1090.
48] A. S. Rowan, N. I. Nicely, N. Cochrane, W. A. Wlassoff, A. Claiborne, C. J.
Hamilton, Org. Biomol. Chem. 2009, 7, 4029–4036.
[66] H.-J. Gabius, Anal. Biochem. 1990, 189, 91–94.
[67] T. Purkrꢊbkovꢊ, K. Smetana, Jr., B. Dvorꢊnkovꢊ, Z. Holꢆkovꢊ, C. Bçck, M.
Lensch, S. Andrꢀ, R. Pytlꢆk, F.-T. Liu, J. Klꢆma, K. Smetana, J. Motlꢆk, H.-J.
Gabius, Biol. Cell 2003, 95, 535–545.
[68] D. Kꢉbler, C.-W. Hung, T. K. Dam, J. Kopitz, S. Andrꢀ, H. Kaltner, M. Lohr,
J. C. Manning, L. He, H. Wang, A. Middelberg, C. F. Brewer, J. Reed, W.-D.
Lehmann, H.-J. Gabius, Biochim. Biophys. Acta Gen. Subj. 2008, 1780,
716–722.
[69] a) H.-J. Gabius, F. Darro, M. Remmelink, S. Andrꢀ, J. Kopitz, A. Danguy, S.
Gabius, I. Salmon, R. Kiss, Cancer Invest. 2001, 19, 114–126; b) J. Kopitz,
Z. Fꢆk, S. Andrꢀ, K. Smetana Jr., H.-J. Gabius, Mol. Pharm. 2013, 10,
2054–2061.
[
[
[
[
[
49] R. Daly, G. Vaz, A. M. Davies, M. O. Senge, E. M. Scanlan, Chem. Eur. J.
2012, 18, 14671–14679.
[
50] S. I. van Kasteren, H. B. Kramer, H. H. Jensen, S. J. Campbell, J. Kirkpa-
[70] F. Delaglio, S. Grzesiek, G. W. Vuister, G. Zhu, J. Pfeifer, A. Bax, J. Biomol.
NMR 1995, 6, 277–293.
trick, N. J. Oldham, D. C. Anthony, B. G. Davis, Nature 2007, 446, 1105–
1
109.
51] R. R. Schmidt, W. Kinzy, Adv. Carbohydr. Chem. Biochem. 1994, 50, 21–
23.
52] a) F. D. Tropper, F. O. Andersson, S. Braun, R. Roy, Synthesis 1992, 7,
18–620; b) T. C. Shiao, D. Giguꢇre, N. Galanos, R. Roy, Carbohydrate
[71] B. A. Johnson, R. A. Blevins, J. Biomol. NMR 1994, 4, 603–614.
[
[
1
6
Chemistry: Proven Synthetic Methods, Vol. 2 (Ed.: P. Kovac), CRC, Boca
Raton, 2014, pp. 251–256.
Received: August 21, 2014
Published online on && &&, 0000
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&14
&
ÞÞ
These are not the final page numbers!

FULL PAPERS
Screening small libraries of glycomi-
metics: Evaluation of biorelevant cell
assays of families of aglycone-modified
Lac and LacNAc derivatives followed by
HSQC NMR can select candidates with
finely tuned selectivities and protein
S. R. Rauthu, T. C. Shiao, S. Andrꢁ,
M. C. Miller, ꢀ. Madej, K. H. Mayo,
H.-J. Gabius, R. Roy*
&& – &&
Defining the Potential of Aglycone
Modifications for Affinity/Selectivity
Enhancement against Medically
Relevant Lectins: Synthesis, Activity
Screening, and HSQC-Based NMR
Analysis
“
hot spots”.
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 15
&15
&
ÞÞ
These are not the final page numbers!