Design, synthesis and evaluation of monovalent ligands for the asialoglycoprotein receptor (ASGP-R)

Article abstract of DOI:10.1016/j.bmc.2009.08.049

A series of novel aryl-substituted triazolyl d-galactosamine derivatives was synthesized as ligands for the carbohydrate recognition domain of the major subunit H1 (H1-CRD) of the human asialoglycoprotein receptor (ASGP-R). The compounds were biologically evaluated with a newly developed competitive binding assay, surface plasmon resonance and by a competitive NMR binding experiment. With compound 1b, a new ligand with a twofold improved affinity to the best so far known d-GalNAc was identified. This small, drug-like ligand can be used as targeting device for drug delivery to hepatocytes.

Full text of DOI:10.1016/j.bmc.2009.08.049

Bioorganic & Medicinal Chemistry 17 (2009) 7254–7264
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of monovalent ligands
for the asialoglycoprotein receptor (ASGP-R)
Daniela Stokmaier, Oleg Khorev, Brian Cutting, Rita Born, Daniel Ricklin, Thomas O. G. Ernst,
Fabienne Böni, Kathrin Schwingruber, Martin Gentner, Matthias Wittwer, Morena Spreafico,
*
Angelo Vedani, Said Rabbani, Oliver Schwardt, Beat Ernst
Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel aryl-substituted triazolyl D-galactosamine derivatives was synthesized as ligands for the
Received 14 July 2009
Revised 24 August 2009
Accepted 25 August 2009
Available online 29 August 2009
carbohydrate recognition domain of the major subunit H1 (H1-CRD) of the human asialoglycoprotein
receptor (ASGP-R). The compounds were biologically evaluated with a newly developed competitive
binding assay, surface plasmon resonance and by a competitive NMR binding experiment. With com-
pound 1b, a new ligand with a twofold improved affinity to the best so far known D-GalNAc was identi-
fied. This small, drug-like ligand can be used as targeting device for drug delivery to hepatocytes.
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Asialoglycoprotein receptor (ASGP-R)
Competitive binding assay
Competitive NMR binding experiments
Triazolyl D-galactosamine derivatives
1. Introduction
functional configuration of the native receptor.9–12 Recently, the
X-ray crystal structure of the carbohydrate recognition domain
The asialoglycoprotein receptor (ASGP-R) is a C-type,1 i.e. a
Ca2+-dependent lectin and is expressed exclusively by parenchy-
mal hepatocytes, which contain 100,000–500,000 binding sites
per cell. These receptors are randomly distributed over the sinusoi-
dal plasma membrane facing the capillaries. Their main function is
to maintain serum glycoprotein homeostasis by recognition, bind-
ing, and endocytosis of asialoglycoproteins (ASGPs). After internal-
ization via receptor-mediated endocytosis, the ASGPs dissociate in
the acidic environment of the endosomes and are finally degraded,
while the receptor is recycled back to the cell surface.2–5
(CRD) of the major H1-subunit was solved.13
Beyond the specificity for terminal D-galactose and N-acetyl-D-
galactosamine residues, binding to the ASGP receptor strongly de-
pends on the valency of the ligand. Whereas the affinity of a single
D-galactose residue is only in the millimolar range, bi-, tri- and
tetraantennary desialylated glycoproteins bind with dissociation
constants of 10ꢀ6, 5 ꢁ 10ꢀ9, and 10ꢀ9 M, respectively.14,15 Further-
more, Lee and co-workers demonstrated that the ASGP-R exhibits
an approximately 10–50-fold higher affinity for
D
-GalNAc versus
D
-Gal monosaccharides16 and for terminal
D
-GalNAc versus -Gal
D
The human ASGP-R consists of two homologous subunits, desig-
nated H1 and H2, which form a non-covalent heterooligomeric
cluster glycosides,17 respectively. As a result, research efforts for
the design of high affinity ligands were focused on oligovalent
complex by
a
-helical coiled coil domains with an estimated ratio
ligands, containing primarily D-GalNAc but also D-Gal or D-Lac as
of 2–5:1, respectively.6,7 Both subunits are single-spanning mem-
terminal recognition elements. Their use as homing devices in
liver-specific drug18,19 and gene delivery20–24 is well documented.
By contrast, only moderate research efforts were directed to-
wards the discovery of potent monovalent ligands. Lee et al.25
brane proteins with a calcium-dependent D-Gal/D-GalNAc recogni-
tion domain.8 Whereas the recognition of the carbohydrate moiety
is mediated via the H1-subunit, the H2-subunit accounts for the
showed that the replacement of the acetamido group in D-GalNAc
by a propanamido or trifluoroacetamido group did not lead to an
alteration of the inhibitory effect regarding 125I-asialo-orosomuco-
uid binding to rat hepatocytes. However, with the bulkier benz-
amido or phthalimido groups an up to 10-fold drop in affinity
was observed, indicating the impact of sterical factors on binding
affinity. Later on, Weis and co-workers26,27 provided a sophisti-
cated model based on the mannose-binding protein to explain
Abbreviations: ABTS, 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid);
AIBN, azo-bis-isobutyronitrile; ASGP, asialoglycoprotein; CRD, carbohydrate recog-
nition domain; DCM, dichloromethane;
D-Gal, D-galactose; D-GalNAc, N-acetyl-D-
galactosamine; -Lac, -lactose; DMSO, dimethyl sulfoxide; EDTA, ethylenedi-
D
D
aminetetraacetic acid; KD, dissociation constant; NMR, nuclear magnetic resonance;
PAA, polyacrylamide; SPR, surface plasmon resonance; Tf, trifluormethanesulfonyl.
* Corresponding author. Tel.: +41 61 2671550; fax: +41 61 2671552.
E-mail address: beat.ernst@unibas.ch (B. Ernst).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.049

D. Stokmaier et al. / Bioorg. Med. Chem. 17 (2009) 7254–7264
7255
the preferential binding of
as for the selectivity of different N-acyl derivatives.
For our investigation, we designed a directed library of easy
accessible and metabolically stable -galactosamine derivatives
for mono- or oligovalent applications, for example, for the incorpo-
ration into trivalent drug carriers previously developed.28 Further-
more, for the efficient evaluation of binding affinities the hitherto
used radio-labeled competitive binding assay29–31 was replaced
by a non-radioactive, polymer-based assay format. Finally, surface
plasmon resonance and NMR experiments allowed an independent
validation of the binding affinities.
D
-GalNAc compared with
D
-Gal as well
A closer look at the protein surface in the area of the 2-N-acetyl
group reveals a dumbbell-shaped cavity (Fig. 1D) that on the one
hand quickly leads to a steric clash with bulky substituents,25
but on the other hand can host a wide variety of linear substitu-
ents. Finally, the b-anomeric as well as the 6-OH point into the sol-
vent and could therefore serve as attachment points for
conjugation to oligovalent carriers, fluorescent labels, etc.
D
Based on these evidences, a directed library of small, drug-like
D
-GalNAc derivatives was designed where (i) the anomeric OH
was removed, since it could act as a metabolic ‘soft-spot’33 and
(ii) the 2-acetamido group was replaced by a 4-substituted 1,2,3-
triazole moiety. Docking of the test compound 1a to the H1-CRD
revealed a perfect accommodation of the triazole substituent by
the dumbbell-shaped hydrophobic binding pocket (Fig. 1D).
2. Results and discussion
2.1. Design of ASGP-R H1 ligands
2.2. Synthesis of triazole derivatives 1a–f, h
Docking
D-GalNAc to the crystal structure of the H1-CRD
(Fig. 1A and B, PDB code 1DV8)13 clearly reveals the pharmaco-
phoric groups involved in this carbohydrate–lectin interaction.
In anti-substituted triazoles as 1a, the substituent in the 4-posi-
tion of the heterocycle perfectly points into the dumbbell-shaped
binding pocket. Anti-substituted triazoles are obtained highly
The 3- and 4-hydroxyl groups of D
-GalNAc coordinate to the Ca2+
ion, requiring their equatorial and axial orientation (Fig. 1B).32
Due to steric reasons, methylation of these hydroxyls leads to a
substantial loss in affinity.25 The hydrophobic patch formed by
regioselectively by
cycloaddition.34,35 For the synthesis of the azido component 7 used
in the cycloaddition reaction (Scheme 1), peracetylated -GalNAc
a copper(I)-catalyzed Huisgen 1,3-dipolar
D
(2) was used as starting material. Its treatment with TiCl4 in
DCM yielded glycosyl chloride 3 in 89%. Reductive dehalogenation
using Bu3SnH and AIBN in refluxing toluene gave 4 in quantitative
yield. Deacetylation (?5), followed by amine-azide exchange36
the circular arrangement of ring C–H bonds on the a-face of D-Gal-
NAc establishes a lipophilic contact with the indole side chain of
Trp243 (Fig. 1A). The pharmacophoric requirements are summa-
rized in Figure 1C.
Figure 1. (A) Manual docking (MacYeti 7.05) of b-D-GalNAc into the H1-CRD of the ASGP-R (PDB code 1DV8).13 Illustration of the hydrophobic interactions between Trp243
and the -face of
-GalNAc and the N-acetate group with His256;27 (B) coordination of the equatorial 3- and axial 4-hydroxyl group of -GalNAc with Ca2+; (C) summary of the
important interactions of -GalNAc with the binding site of the H1-CRD; (D) a model of the H1-CRD interacting with the -GalNAc derivative 1a, with the 3- and 4-OH groups
coordinating Ca2+ (orange) and the triazole substituent well accommodated by a dumbbell-shaped hydrophobic binding pocket.
a
D
D
D
D

7256
D. Stokmaier et al. / Bioorg. Med. Chem. 17 (2009) 7254–7264
(?6) and subsequent re-acetylation gave 7 in 76% yield over three
steps. For affinity testing, a small sample of 7 was deprotected un-
der standard Zemplén conditions to yield 6 quantitatively.
Whereas acetylene 10a (Scheme 2) is commercially available,
10b–d and 10g were easily synthesized following known proce-
dures.37–40 10e was obtained quantitatively by propargylation of
4-hydroxybenzoic acid (8) and 10f in 69% by sulfonylation of 937
with triflic anhydride (Scheme 1).
Cu(I)-catalyzed click conditions,34,35 i.e. CuSO4 and sodium
ascorbate as reducing agent to obtain the catalytically active
OAc
OAc
AcO
OAc
O
AcO
AcO
AcO
a)
b)
O
O
AcO
AcO
AcHN OAc
NHAc
AcHN
Cl
2
3
4
OH
OAc
O
OH
HO
HO
AcO
AcO
HO
HO
O
c)
d)
e)
f)
O
N3
N3
NH2
5
6
7
OH
O
O
O
g)
h)
CO2Et
CO2Et
NH2
NHSO2CF3
8
10e
9
10f
Scheme 1. Synthesis of building blocks. Reagents and conditions: (a) TiCl4, DCM, rt, 3 d, 89%; (b) Bu3SnH, AIBN, PhMe, reflux, 1 h 20 min, quant.; (c) KOH, 18-crown-6,
dioxane/H2O, reflux, 6.5 h; (d) TfN3, NaHCO3, CuSO4ꢂ5H2O, H2O/PhMe/MeOH, rt, 24 h; (e) Ac2O, pyridine, rt, 24 h, 76% (three steps); (f) NaOMe/MeOH, rt, 3 h, quant.; (g)
propargyl bromide, K2CO3, acetone, reflux, 5 h, 73%; (h) Tf2O, Et3N, DCM, 0 °C?rt, 24 h, 69%.
O
R1
R1
R1
R1
OAc
O
OH
AcO
HO
O
R2
AcO
R1
HO
R1
OAc
O
AcO
AcO
N
N
N
N
b), c) or d)
10a-f
a)
R1
R1
N
N
R2
R1
R2
R1
N3
O
O
7
R1
R1
11a: R1 = H, R2 = H
11b: R1 = H, R2 = CN
11c: R1 = H, R2 = NO2
11d: R1, R2 = F
1a: R1 = H, R2 = H
O
1b: R1 = H, R2 = CN
1c: R1 = H, R2 = NO2
1d: R1, R2 = F
a)
Br
11e: R1 = H, R2 = CO2Et
1e: R1 = H, R2 = CO2Na
1f: R1 = H, R2 = NHSO2CF3
10g40
11f: R1 = H, R2 = NHSO2CF3
OAc
OAc
AcO
OH
HO
AcO
AcO
O
N
O
O
AcO
HO
f)
e)
N
N
N
N
N
N
N
N
N
N
P(Cy)2
i-Pr
Br
N
N
i-Pr
O
O
O
11g
i-Pr
1242
11h
1h
Scheme 2. Synthesis of triazoles. Reagents and conditions: (a) CuCl, Et3N, DCM, rt, 24 h, 10a (?11a, 93%), 10b38 (?11b, 98%), 10c37 (?11c, 95%), 10d39 (?11d, 73%), 10e
(?11e, 82%), 10f (?11f, 41%), or 10g40 (?11g, 84%); (b) NaOMe/MeOH, rt, 3 h (1a, 45%; 1c, 33%; 1d, 68%; 1f, 73%); (c) H2O/MeOH/Et3N (5:5:1), rt, 24 h (1b, 81%); (d) NaOH,
H2O/MeOH, rt, 24 h (1e, 74%); (e) N-methylpiperazine, Pd2(dba)3, Cs2CO3, PhMe, 80 °C, 24 h; (f) NaOMe/MeOH, rt, 24 h (44%, two steps).

D. Stokmaier et al. / Bioorg. Med. Chem. 17 (2009) 7254–7264
7257
Cu(I), led only to low yields of anti-substituted triazoles and in
some cases even failed completely. However, with an alternative
procedure, involving CuCl as the direct source of Cu(I) and triethyl-
amine as base, the cycloaddition reactions of azide 7 with the phe-
nyl propargyl ethers 10a–f yielded the protected anti-substituted
triazoles 11a–f in 41–98% yield and after deprotection the test
compounds 1a–f (Scheme 2). To further explore the dumbbell-
shaped binding site and to improve solubility of the test com-
pound, a piperazinylphenyl derivative was synthesized. Click
chemistry (?11g) and Pd-catalyzed Buchwald–Hartwig coupling
using the biaryl monophosphine ligand 12 (?11h)41 followed by
deacetylation by Zemplén conditions yielded 1h.
jugate was established analog to binding assays reported for
selectin antagonists.44,45 For this assay, a microtiter plate coated
with the H1-CRD is incubated with biotinylated
D-GalNAc-PAA
conjugated to streptavidin-horseradish peroxidase. Then, after
incubation with the analyte at various concentrations and washing
with HBS-buffer, the remaining PAA glycoconjugate is quantified
by a colorimetric reaction using the enzyme’s substrate 2,20-azi-
no-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS). PAAs con-
taining b-
efficiently bound b-
PAA (Fig. 3A). Since the interaction is Ca2+-dependent,
D
-Glc or sLea are not recognized, whereas H1-CRD
-GalNAc-PAA and to a lesser extent b- -Gal-
-GalNAc-
D
D
D
polymer binding is completely inhibited in the presence of 1 mM
EDTA in the assay buffer (Fig. 3B).
2.3. Expression of H1-CRD
For a further validation of the binding assay, the IC50’s of a num-
ber of monosaccharides were compared with KD values derived
from surface plasmon resonance (SPR, Biacore) measurements,
were the H1-CRD was immobilized on a CM5 chip by amine cou-
pling, and IC50’s reported in literature.16
Interestingly, consistently negative sensorgrams in the surface
plasmon resonance experiments, that is, a net decrease in reso-
nance units, were obtained with the monosaccharides reported
in Table 1. When fitted to a binding isotherm, these negative sen-
sorgrams appear to clearly result from specific receptor–ligand
interactions. Numerous factors such as the buffer capacity, the
ion-strength of the buffer and matrix type of the sensor chip could
be excluded as direct cause of the negative sensorgrams. A possible
explanation is a ligand-induced conformational change of the
A truncated form of the H1-subunit of the human hepatic ASGP-
R consisting of the whole CRD domain (amino acid residues 147–
291) was recombinantly expressed in Escherichia coli and purified
to homogeneity by affinity chromatography.43 The monomeric
and dimeric forms were subsequently purified by ion exchange
chromatography. The purified proteins were analyzed by SDS–
PAGE and immuno-blot (Fig. 2).
2.4. Competitive binding assay
For the characterization of the H1-CRD ligands, a cell-free, com-
petitive binding assay based on a polyacrylamide (PAA) glycocon-
Figure 2. SDS–PAGE and immuno-blot analysis. (A) Monomeric and dimeric forms of H1-CRD were separated on a non-reducing 15% SDS–PAGE and visualized by coomassie
staining. M: molecular weight marker, 1 and 2: H1-CRD monomer, 3 and 4: H1-CRD monomer and dimer; (B) Immuno-blot analysis was performed with IgY polyclonal anti-
H1-CRD antibodies and alkaline phosphatase coupled rabbit anti-chicken IgG. 5: H1-CRD monomer, 6: H1-CRD dimer, 7 and 8: H1-CRD monomer and dimer.
Figure 3. (A) Specificity of the binding assay. Comparison of H1-CRD binding of b-
effect of EDTA on b- -GalNAc-PAA binding to H1-CRD.
D-GalNAc-PAA and b-D-Gal-PAA and the negative controls b-D
-Glc-PAA and sLea-PAA; (B)
D

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D. Stokmaier et al. / Bioorg. Med. Chem. 17 (2009) 7254–7264
Table 1
Table 2
Affinity data obtained by the competitive binding assay (IC50) and surface plasmon
resonance (KD)
Determination of the relative IC50 (rIC50) values for the triazolyl D-galactosamine
derivatives
Monosaccharides
Competitive
binding assaya
(human H1)
Surface plasmon
resonance
(human H1)
Literature
data16 (rabbit
ASGP-R)
Entry
Compound
rIC50 SD
OH
HO
O
1
1.0
HO
IC50 SD
M]
rIC50 KD SD
M]
rKD
IC50
M]
rIC50
AcHN
OH
[
l
[
l
[
l
OH
HO
D-GalNAc
113 13
1
150 0.9
1460 10
2200 40
2760 60
>10,000
1
9.7
14.7 1000
18
90
1
O
D-Gal
1049 101
9.3
1700
18.9
11.1
17.8
667
2
3
13.7 4.3
HO
b-
D
-GalOMe
-GalOMe
-Glc
1828 169 16
N3
6
a-
D
2831 140
>30,000
25
n.d.
1600
60,000
OH
HO
D
n.d.
O
HO
IC50 defines the molar concentration of the test compound that reduces the maxi-
mal specific binding of -GalNAc-PAA to H1-CRD by 50%. The relative IC50 (rIC50) is
N
N
D
1.1 0.2
N
the ratio of the IC50 of the test compound to the IC50 of the reference compound D-
GalNAc. SD: standard deviation. Literature data describe the inhibition of 125I-asi-
aloorosomucoid binding to isolated rabbit hepatic lectin.16
O
a
1a
Compounds tested in triplicates, each at six different concentrations. The
coefficient of variation, which is defined as the standard deviation divided by the
mean, was 13.8% for unspecific binding and 4.8% for specific binding. These results
demonstrate the low variability of the assay, making it suitable for the character-
ization of H1-CRD ligands.
OH
HO
HO
O
N
N
4
5
6
7
0.5 0.1
0.8 0.2
1.4 0.3
8.7
N
NC
immobilized receptor leading to a decrease of its hydrodynamic ra-
dius and, as a consequence, yielding a negative refraction index.46
Since the negative refraction index correlates with the analyte con-
centration, we mirrored the negative sensorgrams by multiplica-
tion of each data point with ꢀ1. The KDs obtained correlate
nicely with affinity data derived from the competitive binding as-
say and from the literature values (Table 1).
O
1b
OH
HO
HO
O
N
N
N
O2N
All three assay formats revealed a 10–20-fold higher affinity for
O
D
-GalNAc compared to
binds with slightly higher affinity than the corresponding
mer, although according to docking studies (see Fig. 1B) both agly-
cons point to the solvent. Furthermore, -Glc-binding was beyond
D
-Gal. Interestingly, methyl b-
D
-galactoside
1c
a-ano-
OH
HO
HO
O
N
D
N
F
the detection limit, supporting the importance of the coordination
of the 3- and 4-hydroxyls to Ca2+. Overall, the three assay formats
gave consistently comparable results, confirming the reliability of
our new competitive binding assay.
The affinities of the triazoles are summarized in Table 2 and Fig-
ure 4. Compounds that were not soluble in HBS-buffer were dis-
solved in DMSO prior to dilution with buffer. The final DMSO
concentration in the diluted samples did not exceed 5% and was
well tolerated in the assay (data not shown).
F
N
F
O
F
F
1d
OH
HO
O
HO
N
N
N
NaO2C
O
Not unexpectedly, the azide scaffold 6 (Table 2, entry 2) exhib-
ited an approximately 14-fold decrease in affinity compared to D-
1e
GalNAc (entry 1), because the proposed hydrophobic contact of
the N-acetate group with His25627 is no longer present. On the
other hand, compound 1a (entry 3), bearing a phenyloxymethyl-
OH
HO
HO
O
substituted triazole, had an affinity close to the one of D-GalNAc
N
N
a
8
—
(rIC50 1.1), indicating that the rather bulky substituent in the 2-po-
sition is tolerated. Only the p-cyano derivative 1b (entry 4) and the
p-nitro-derivative 1c (entry 5) showed clearly better affinities than
N
F3CO2SHN
O
1f
D-GalNAc. Despite its bulkiness, the N-methylpiperazine derivative
1h exhibited a slightly improved rIC50 compared to -GalNAc. This
D
result further supports our hypothesis that compounds extended
at the 2-position of the amino pyranose ring can interact with
the dumbbell-shaped binding pocket (see Fig. 1D).
OH
HO
HO
O
N
N
9
0.9 0.1
N
N
2.5. Competitive NMR binding experiments
N
O
For an independent assessment of the binding affinities, com-
petitive binding experiments by NMR47,48 were performed with
1h
SD: standard deviation.
a
D-GalNAc as a reference compound and 1b as competitor. In a first
Compound not soluble in the 10% DMSO/buffer stock solution used in the assay.
step, binding of -GalNAc to the H1-CRD was demonstrated by the
D
large difference in transverse relaxation times in the absence and
presence of the lectin (Fig. 5A).

D. Stokmaier et al. / Bioorg. Med. Chem. 17 (2009) 7254–7264
7259
Figure 4. (A) Relative IC50s derived from the competitive solid-phase binding assay; (B) Plot of
binding to H1-CRD by GalNAc and derivatives. Microtiter plates were coated with a 3
compound solution and 0.5 g/mL of streptavidin-peroxidase coupled -GalNAc-PAA were added to the protein. After 2 h incubation the plates were washed and the
colorimetric reaction was developed and quantified as described in experimental section. (j) GalNAc, (.) 1b, (N) 1h.
D-GalNAc-PAA inhibition versus concentration. Inhibition of
D-GalNAc-PAA
lg/mL solution of monomeric H1-CRD. After washing steps a serial dilution of the test
l
D
Figure 5. (A) Transverse relaxation of
H1-CRD with -GalNAc and the corresponding T1rho time constants. The leftmost point, at 400
Additional points correspond to the identical analysis with increasing -GalNAc concentrations.
D
-GalNAc, either in the presence (circles) or in the absence (triangles) of ASGP-R H1-CRD. T1rho experiments;47 (B) Titration of ASGP-R
-GalNAc, is the T1rho time constant determined for the data in Figure 5B.
D
lM D
D
For the quantitative evaluation of the relative affinity of 1b rel-
ative to -GalNAc, a titration curve documenting the changes in the
transverse relaxation times of -GalNAc was required. The T1rho
time constants were therefore measured for different concentra-
tions of -GalNAc and fit to a one-site binding model (Fig. 5B).
azole derivatives was synthesized and biologically evaluated. For
that purpose, a competitive binding assay allowing the determina-
tion of Ca2+-dependent binding of potential ligands to the H1-CRD
was developed. The reliability of this sensitive, rapid and simple
assay was confirmed by a comparison with IC50’s reported in liter-
ature and KD values derived from SPR measurements.
D
D
D
The relative affinities were then determined in a two-step process.
In a first step, a NMR sample with H1-CRD and the concentration of
The presented triazolyl galactosamine derivatives which are
synthetically easily accessible and hydrolytically stable have
D
-GalNAc corresponding to the initial point of the titration curve
(400 M) was investigated, indicating a high degree of reproduc-
l
drug-like properties. Thus, 1b may be used instead of D-GalNAc
ibility according to the nearly identical T1rho time constants for
the first and second sample (282 2 ms and 280 3 ms, respec-
tively). The second step consisted of mixing a small amount of
for oligovalent drug-carriers directed to the ASGP-R, for example,
with the scaffold we recently published.28 Eventually, improved
affinity may be realized by further modifications of the substitu-
ents of the triazole ring. These follow-up experiments are currently
being performed.
the competitor with the second sample (H1-CRD, 400
NAc) and measuring the resulting T1rho time constant, from which
the apparent concentration of -GalNAc was determined by means
of the titration curve. When 200 M of 1b were added, the T1rho
lM of D-Gal-
D
It remains to be seen whether the new ligands selectively bind
to the H1-CRD of the ASGP-R or bind equally well to the three other
l
time constant increased to 401 2 ms. Interpolating this value on
D-Gal/D
-GalNAc-receptors of the C-type lectin family,49 the Kupffer
the titration curve in Figure 5B indicates an apparent concentration
cell receptor, the macrophage galactose lectin, and the scavenger
of
-GalNAc from its apparent concentration and dividing through the
concentration of 1b a relative affinity of 1.9 with respect to -Gal-
D-GalNAc of 785 lM. By subtracting the actual concentration for
receptor C-type lectin (SRCL).50–53
D
D
4. Experimental
NAc was obtained, a result almost identical to that received by the
competitive solid-phase assay.
4.1. General methods
3. Conclusion
NMR spectra were recorded on a Brucker Avance DRX-500
(500 MHz) spectrometer. Assignment of 1H and 13C NMR spectra
was achieved using 2D methods (COSY, HSQC, TOCSY). Chemical
shifts are expressed in ppm using residual CHCl3, CHD2OD and
Based on docking studies of
the H1-CRD of the ASGP-R, a directed library of aryl-substituted tri-
D-GalNAc to the crystal structure of

7260
D. Stokmaier et al. / Bioorg. Med. Chem. 17 (2009) 7254–7264
HDO as references. NMR solvents were purchased from Sigma–Al-
drich (CDCl3), Armar Chemicals (MeOH-d4, D2O) and Cambridge
Isotope Laboratories (Tris-d11, DMSO-d6). Optical rotations were
measured using a Perkin–Elmer Polarimeter Model 341. The LC/
HR-MS analysis were carried out using a Agilent 1100 LC equipped
with a photodiode array detector and a Micromass QTOF I. Reac-
tions were monitored by TLC using glass plates coated with Silica
Gel 60 F254 (Merck). Carbohydrate-containing compounds were
visualized by charring with a molybdate solution (0.02 M solution
of ammonium cerium sulfate dihydrate and ammonium molybdate
tetrahydrate in aqueous 10% H2SO4). All other compounds were
visualized with KMnO4 solution (2% KMnO4 and 4% NaHCO3 in
water). Column chromatography was performed on Silica Gel 60
(Fluka, 0.040–0.060 mm). Methanol (MeOH) was dried by refluxing
with sodium methoxide and distilled immediately before use. Pyr-
idine was freshly distilled under argon over CaH2. Dichlorometh-
ane (DCM) was dried by filtration over Al2O3 (Fluka, type 5016 A
basic). Dioxane and toluene were dried by refluxing with sodium
and benzophenone. Molecular sieves (4 Å) were activated in vacuo
at 500 °C for 2 h immediately before use.
was purified by flash chromatography on silica gel (toluene/EtOAc
(1:4?1:9?0:1), yielding 4 (2.03 g, quant).
1H NMR (500 MHz, CDCl3): d 1.93 (s, 3H, NHAc), 2.02, 2.04, 2.14
(3s, 9H, 3 OAc), 3.15 (t, J = 11.1 Hz, 1H, H-1ax), 3.76 (m, 1H, H-5),
4.06 (m, 2H, H-6), 4.18 (dd, J = 5.2, 11.3 Hz, 1H, H-1eq), 4.40 (m,
1H, H-2), 4.92 (dd, J = 3.3, 11.2 Hz, 1H, H-3), 5.35 (m, 1H, H-4),
5.69 (d, J = 8.0 Hz, 1H, NH); 13C NMR (125 MHz, CDCl3): d 20.6,
20.7, 20.8 (3 OCOCH3), 23.2 (NHCOCH3), 46.5 (C-2), 62.2 (C-6),
67.1 (C-4), 68.6 (C-1), 71.5 (C-3), 75.0 (C-5), 170.25, 170.3, 170.5,
171.4 (4 CO); Anal. Calcd for C14H21NO8: C, 50.75; H, 6.39; N,
4.23. Found: C, 51.07; H, 6.49; N, 4.12.
4.1.3. 2-Amino-1,5-anhydro-D-galactitol (5)
To a solution of 4 (3.17 g, 9.58 mmol) in dioxane (8.5 mL) were
subsequently added water (6.5 mL), 18-crown-6 (21.1 mg) and
potassium hydroxide (840 mg, 15.0 mmol). The reaction mixture
was refluxed for 5 h. Another 5 equiv (2.10 g) of potassium hydrox-
ide was then added, and the mixture was refluxed for another
1.5 h. The solvent was removed in vacuo and the crude product 5
(3.91 g) was used without further purification in the next step.
The vector pET3b encoding the amino acid residues 147–291 of
H1-subunit, was kindly provided by M. Spiess (University of Basel,
Switzerland). E. coli strain AD494 (DE3) was purchased from Nova-
gen (Lucerne, Switzerland). Bacto-Yeast extract, Bacto-Agar and
Bacto-Tryptone for LB (Luria-Bertani) and TB (Terrific Broth) cul-
ture media were purchased from Becton Dickinson (Allschwil,
4.1.4. 3,4,6-Tri-O-acetyl-1,5-anhydro-2-azido-2-deoxy-D-
galactitol (7)
Triflyl azide stock solution preparation: Sodium azide (3.49 g,
53.7 mmol) was dissolved in water (8.7 mL). Toluene was added,
and the mixture was cooled to 0 °C with stirring. Then triflic anhy-
dride (5.8 mL, 34.4 mmol) was added dropwise. The biphasic reac-
tion mixture was stirred vigorously at 0 °C for 1 h and at 10 °C for
another 2 h. The reaction mixture was neutralized with satd aq
NaHCO3. The phases were separated, and the aqueous phase ex-
tracted with toluene (2 ꢁ 8 mL). The organic layers were combined
to give the triflyl azide stock solution.
Switzerland). IPTG (isopropyl b-D-thiogalactopyranoside) was from
AppliChem. Sepharose-4B column material, kanamycin, carbenicil-
lin and protein-standard BSA solution were obtained from Sigma
(Buchs, Switzerland). The Shodex IEC-DEAE column was from Bre-
chtbühler (Schlieren, Switzerland). Biotinylated polyacrylamide
polymers (
(Moscow, Russia). HEPES [4-(2-hydroxyethyl)-piperazine-1-eth-
anesulfonic acid], oxalic acid, CaCl2 and methyl b- -glucopyrano-
side were from Fluka (Buchs, Switzerland). Methyl - and b-
galactopyranoside and bovine serum albumin (BSA) were from Sig-
ma (Buchs, Switzerland). Lactose, N-acetyl- -galactosamine and D-
D-GalNAc-, D-Gal-PAA) were obtained from Lectinity
Amine-azide exchange: Compound 5 (1.56 g, 9.56 mmol), NaH-
CO3 (319 mg, 38.0 mmol) and CuSO4ꢂH2O (95.0 mg, 380
D
lmol)
a
D
-
were dissolved in water (6.4 mL). The triflyl azide stock solution
(21 mL, 9.56 mmol) was added and the biphasic reaction mixture
was made homogenous by the dropwise addition of MeOH. The
mixture was stirred at rt overnight during that time the color of
the mixture turned from blue to green. The solvent was removed
in vacuo and the residue containing 6 (1.81 g) was taken up in
dry pyridine (36 mL, 447 mmol), and acetic anhydride (8.2 mL,
86.2 mmol) was added. The reaction mixture was stirred at rt un-
der argon overnight. The solvent was removed in vacuo and the
crude product was purified by flash chromatography (petrol
ether/EtOAc 20:1?9:1) to give 7 (2.31 g, 76%).
D
galactosamine were obtained from Pfanstiehl Laboratories (Ohio,
USA). Streptavidin-peroxidase conjugate was from Roche Applied
Science (Switzerland). Fetal bovine serum (FBS) was from Invitro-
gen. The peroxidase substrate kit ABTS [2,20-azino-di-(3-ethyl-
benzthiazoline-6-sulfonic acid)] was obtained from BioRad
(California, USA) and MaxiSorp 96-well microtiter plates were
from Nunc (Roskilde, Denmark). Anti-H1-CRD polyclonal antibod-
ies were produced in chicken and purified from their eggs after
PEG-precipitation56 by affinity chromatography on a agarose-
immobilized H1-CRD column. Surface plasmon resonance experi-
ments were performed on a Biacore 3000 machine using CM5 chips
(Biacore AB, Uppsala, Sweden).
[a]
+0.04 (c 1.00, CHCl3); 1H NMR (500 MHz, CDCl3): d 2.09,
D
2.16, 2.22 (3s, 9H, 3 OAc), 3.24 (t, J = 11.3 Hz, 1H, H-1ax), 3.79
(dt, J = 0.9, 6.5 Hz, 1H, H-5), 3.93 (m, 1H, H-2), 4.08 (d, J = 6.5 Hz,
2H, H-6), 4.12 (dd, J = 5.4, 11.6 Hz, 1H, H-1eq), 4.91 (dd, J = 3.3,
10.4 Hz, 1H, H-3), 5.40 (d, J = 0.9 Hz, 1H, H-4); 13C NMR
(125 MHz, CDCl3): d 20.6, 20.8, 22.1 (3 COCH3), 55.9 (C-2), 61.9
(C-6), 67.1 (C-4), 68.1 (C-1), 73.4 (C-3), 74.9 (C-5); Anal. Calcd for
C12H17N3O7: C, 45.72; H, 5.43; N, 13.33. Found: C, 45.90; H, 5.44;
N, 13.18.
4.1.1. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-
galactopyranosyl chloride (3)
Titanium tetrachloride (185 lL, 1.69 mmol) was added to a sus-
pension of 2 (500 mg, 1.28 mmol) in dry DCM (5 mL). The mixture
was stirred at rt under argon for 3 d. The solvent was removed in
vacuo, and the residue was purified by flash chromatography on
silica gel (petrol ether/EtOAc 2:3?1:4), yielding 3 (419 mg, 89%).
The analytical data were identical to those found in the literature.54
4.1.5. 1,5-Anhydro-2-azido-2-deoxy-D-galactitol (6)
Compound 7 (20.0 mg, 63.4 mol) was dissolved in MeOH
l
(2 mL) and sodium metal (10 mg) was added. The resulting solu-
tion was stirred overnight, the solvent was removed in vacuo
and the residue was purified on an RP-C18 column (H2O/MeOH
20:0?9:1, stepwise gradient) yielding 6 (12.0 mg, quant).
4.1.2. 2-Acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-
galactitol (4)
A solution of 3 (2.24 g, 6.12 mmol) in dry toluene (33 mL) was
degassed in an ultrasound bath under a steady flow of argon for
30 min. Tributyltin hydride (2.14 g, 7.35 mmol) and AIBN (ca.
25 mg) were added and the mixture was refluxed under argon
for 80 min. The solvent was removed in vacuo, and the residue
[
a
]
ꢀ0.12 (c 1.00, MeOH); 1H NMR (500 MHz, CD3OD): d 3.07
D
(t, J = 11.1 Hz, 1H, H-1ax), 3.38 (m, 1H, H-5), 3.50 (dd, J = 3.3,
9.9 Hz, 1H, H-3), 3.62–3.72 (m, 3H, H-2, H-6), 3.83 (d, J = 3.0 Hz,
1H, H-4), 3.95 (dd, J = 5.4, 11.2 Hz, 1H, H-1eq); 13C NMR
(125 MHz, CDCl3): d 60.6 (C-2), 62.9 (C-6), 69.1 (C-1), 70.6 (C-4),

D. Stokmaier et al. / Bioorg. Med. Chem. 17 (2009) 7254–7264
7261
75.3 (C-3), 81.0 (C-5); HR-MS Calcd for C6H11N3NaO4 [M+Na]+:
212.0647; Found 212.0650.
(dd, J = 5.0, 11.6 Hz, 1H, H-1eq), 4.92 (dt, J = 4.9, 10.7 Hz, 1H, H-
2), 5.22 (s, 2H, CH2OAr), 5.48–5.52 (m, 2H, H-3, H-4), 7.01, 7.56
(m, 4H, C6H4), 7.67 (s, 1H, H-5 triazole); 13C NMR (125 MHz,
CDCl3): d 20.2, 20.5, 20.6 (3 COCH3), 56.1 (C-2), 61.7, 61.9 (C-6,
CH2OAr), 66.9 (C-4), 68.6 (C-1), 71.3 (C-3), 75.2 (C-5), 115.4 (2C,
2 C-ortho), 118.8 (C-para), 123.0 (C-5 triazole), 134.0 (2C, 2 C-
meta), 142.9 (C-4 triazole), 161.2 (C-ipso), 169.2, 169.8, 170.4 (3
CO); HR-MS Calcd for C22H24N4O8 [M+H]+: 473.1672; Found:
473.1667.
4.1.6. Ethyl 4-(2-propynyloxy)-benzoate (10e)
Ethyl 4-hydroxybenzoate (8, 500 mg, 3.01 mmol) was dissolved
in dry acetone (5 mL), and K2CO3 (580 mg, 4.21 mmol) was added,
followed by propargyl bromide (651 lL, 6.02 mmol), and the mix-
ture was refluxed for 3 h. The mixture was then diluted with DCM
(50 mL), washed with H2O (25 mL) and brine (25 mL), dried
(Na2SO4), and the solvent was removed in vacuo. The residue
was purified by flash chromatography on silica gel (petrol ether/
EtOAc 20:1?4:1), yielding 10e (452 mg, 73%).
4.1.10. 1-(3,4,6-Tri-O-acetyl-1,5-anhydro-2-deoxy-D-galactitol-
2-yl)-4-(4-nitrophenyloxy) methyl-1,2,3-triazole (11c)
1H NMR (500 MHz, CDCl3): d 1.36 (t, J = 7.2 Hz, 3H, OCH2CH3),
2.55 (t, J = 2.4 Hz, 1H, CH), 4.33 (q, J = 7.2 Hz, 2H, OCH2CH3), 4.73
(d, J = 2.4 Hz, 2H, CH2, propynyl), 6.98, 8.00 (AA0, BB0 of AA0BB0,
J = 9.0 Hz, 4H, C6H4); 13C NMR (125 MHz, CDCl3): d 14.3 (OCH2CH3),
55.7 (OCH2), 60.5 (OCH2CH3), 76.0 (CH), 77.8 (CQ), 114.4, 123.7,
131.4, 161.0 (6C, C6H4), 166.2 (CO); Anal. Calcd for C12H12O3: C,
70.58; H, 5.92. Found: C, 70.58; H, 5.93.
Prepared according to the procedure described for 11a, using 7
(50.0 mg, 158 l lmol). The com-
mol) and 10c37 (49.7 mg, 316
pound was purified by flash chromatography (petrol ether/EtOAc
1:1) to give 11c (74.0 mg, 95%).
1H NMR (500 MHz, CDCl3): d 1.80, 2.03, 2.15 (3s, 9H, 3
COCH3), 3.99–4.04 (m, 2H, H-1ax, H-5), 4.12 (d, J = 6.4 Hz, 2H,
H-6), 4.30 (dd, J = 5.0, 11.6 Hz, 1H, H-1eq), 4.93 (m, 1H, H-2),
5.26 (s, 2H, CH2OAr), 5.49–5.52 (m, 2H, H-3, H-4), 7.02 (AA0
of AA0BB0, J = 9.3 Hz, 2H, C6H4), 7.70 (s, 1H, H-5 triazole), 8.15
(BB0 of AA0BB0, J = 9.3 Hz, 2H, C6H4); 13C NMR (125 MHz, CDCl3):
4.1.7. 1-Trifluoromethylsulfonamido-4-(2-
propynyloxy)benzene (10f)
Compound 937 (100 mg, 679
l
mol) was dissolved in dry DCM
L, 747 mol) was added, followed by
the dropwise addition of triflic anhydride (123 L, 747 mol) at
d
20.3, 20.6, 20.7 (3 COCH3), 56.2 (C-2), 61.9, 62.3 (C-6,
CH2OAr), 67.0 (C-4), 68.6 (C-1), 71.4 (C-3), 75.3 (C-5), 114.8
(2C, C-ortho), 123.2 (C-5 triazole), 125.9 (2C, C-meta),
(3 mL). Triethylamine (103
l
l
l
l
2
2
0 °C. The solution was allowed to reach rt and stirred under argon
overnight. The solvent was removed in vacuo and the residue was
purified by flash chromatography on silica gel (petrol ether/EtOAc
19:1?9:1), yielding 10f (130 mg, 69%).
141.9 (C-para), 142.8 (C-4 triazole), 163.0 (C-ipso), 169.3,
170.0, 170.5 (3 CO); HR-MS Calcd for C21H24N4O10 [M+H]+:
493.1571; Found: 493.1579.
1H NMR (500 MHz, CDCl3): d 2.54 (t, J = 2.4 Hz, 1H, CH); 4.69 (d,
J = 2.4 Hz, 2H, OCH2); 6.97, 7.23 (AA0, BB0 of AA0BB0, J = 8.9 Hz, 4H,
C6H4); 13C NMR (125 MHz, CDCl3): d 56.1 (OCH2), 76.1 (CH), 77.9
(CQ), 119.8 (J = 322.8 Hz, CF3), 115.8, 126.6, 126.9, 157.2 (C6H4);
Anal. Calcd for C10H8NO3F3S: C, 43.01; H, 2.89; N, 5.02. Found: C,
42.95; H, 3.00; N, 4.97.
4.1.11. 1-(3,4,6-Tri-O-acetyl-1,5-anhydro-2-deoxy-D-galactitol-
2-yl)-4-(pentafluorophenoxy) methyl-1,2,3-triazole (11d)
Prepared according to the procedure described for 11a, using 7
(50.0 mg, 158 l lmol). The com-
mol) and 10d39 (70.2 mg, 316
pound was purified by flash chromatography (petrol ether/EtOAc
4:1?1:1) to give 11d (62.0 mg, 73%).
1H NMR (500 MHz, CDCl3): d 1.85, 2.05, 2.18 (3s, 9H, 3
COCH3), 3.97–4.02 (m, 2H, H-1ax, H-5), 4.13 (d, J = 6.4 Hz,
2H, H-6), 4.29 (dd, J = 5.0, 11.6 Hz, 1H, H-1eq), 4.95 (dt, J =
5.0, 11.0 Hz, 1H, H-2), 5.27 (s, 2H, CH2OAr), 5.49–5.53 (m,
2H, H-3, H-4), 7.74 (m, 1H, H-5 triazole); 13C NMR
(125 MHz, CDCl3): d 20.1, 20.5, 20.6 (3 COCH3), 56.1 (C-2),
61.8 (C-6), 66.9 (C-4), 67.6 (CH2OAr), 68.6 (C-1), 71.3 (C-3),
75.2 (C-5), 123.6 (C-5 triazole), 136.9, 139.0, 141.1, 142.9
(6C, C6F5), 142.5 (C-4 triazole), 169.3, 169.9, 170.4 (3 CO);
HR-MS Calcd for C21H21F5N3O8 [M+H]+: 538.1249; Found:
538.1252.
4.1.8. 1-(3,4,6-Tri-O-acetyl-1,5-anhydro-2-deoxy-D-galactitol-2-
yl)-4-phenoxymethyl-1,2,3-triazole (11a)
Compound 7 (50.0 mg, 158
(3 mL). The mixture was degassed in an ultrasound bath under a
flow of argon for 20 min. Copper(I) chloride (31.2 mg, 316 mol),
DIPEA (54.4 L, 316 mol) and 10a (40.6 L, 316 mol) were
lmol) was dissolved in dry DCM
l
l
l
l
l
added. The mixture was stirred under argon at rt for 24 h. The sol-
vent was removed in vacuo, and the crude mixture was purified by
flash chromatography (petrol ether/EtOAc 3:2) to yield compound
11a (66.5 mg, 93%).
1H NMR (500 MHz, CDCl3): d 1.81, 2.08, 2.19 (3s, 9H, 3 OAc),
4.00–4.07 (m, 2H, H-1ax, H-5), 4.15 (d, J = 6.4 Hz, 2H, H-6), 4.33
(dd, J = 5.1, 11.6 Hz, 1H, H-1eq), 4.92 (dt, J = 5.0, 11.0 Hz, 1H, H-
2), 5.22 (s, 2H, CH2OPh), 5.50 (dd, J = 3.2, 11.0 Hz, 1H, H-3), 5.54
(d, J = 2.5 Hz, 1H, H-4), 6.95–6.99, 7.26–7.30 (m, 5H, C6H5), 7.62
(s, 1H, H-5 triazole); 13C NMR (125 MHz, CDCl3): d 20.3, 20.7,
20.7 (3 COCH3), 56.0 (C-2), 61.8 (2C, CH2OPh, C-6), 67.0 (C-4),
68.7 (C-1), 71.5 (C-3), 75.3 (C-5), 114.7, 121.4, 122.7, 129.6 (6C,
C6H5), 118.5 (C-5 triazole), 144.3 (C-4 triazole), 169.3, 170.0,
170.5 (3 CO); HR-MS Calcd for C21H26N3O8 [M+H]+: 448.1720;
Found: 448.1725.
4.1.12. 1-(3,4,6-Tri-O-acetyl-1,5-anhydro-2-deoxy-D-galactitol-
2-yl)-4-(4-ethoxycarbonylphenoxy)methyl-1,2,3-triazole (11e)
Prepared according to the procedure described for 11a, using 7
(50.0 mg, 160 lmol) and 10e (64.5 mg, 316 lmol). The compound
was purified by flash chromatography (hexane/EtOAc 3:2?2:3) to
give 11e (62.0 mg, 82%).
1H NMR (500 MHz, CDCl3): d 1.35 (t, J = 7.1 Hz, 3H, CH2CH3),
1.79, 2.05, 2.16 (3s, 9H, 3 COCH3), 3.99–4.10 (m, 2H, H-1ax, H-
5), 4.13 (d, J = 6.4 Hz, 2H, H-6), 4.29–4.34 (m, 3H, H-1eq,
CH2CH3), 4.92 (dt, J = 4.9, 10.9 Hz, 1H, H-2), 5.23 (s, 2H, CH2OAr),
5.49–5.53 (m, 2H, H-3, H-4), 6.95 (AA0 of AA0BB0, J = 8.9 Hz, 2H,
C6H4), 7.66 (s, 1H, H-5 triazole), 7.96 (BB0 of AA0BB0, J = 8.9 Hz,
2H, C6H4); 13C NMR (125 MHz, CDCl3): d 14.3 (CH2CH3), 20.2,
20.5, 20.6 (3 COCH3), 56.0 (C-2), 60.6 (CH2CH3), 61.8 (2C, C-6,
CH2OAr), 66.9 (C-4), 68.6 (C-1), 71.4 (C-3), 75.2 (C-5), 114.2
(2C, 2 C-ortho), 122.9 (C-5 triazole), 131.5 (2C, 2 C-meta), 131.6
(C-para), 143.5 (C-4 triazole), 161.5 (C-ipso), 169.2, 169.9, 170.4
(3 CO); HR-MS Calcd for C24H30N3O10 [M+H]+: 520.1931; Found:
520.1937.
4.1.9. 1-(3,4,6-Tri-O-acetyl-1,5-anhydro-2-deoxy-
yl)-4-(4-cyanophenoxy)methyl-1,2,3-triazole (11b)
Prepared according to the procedure described for 11a, using 7
(50.0 mg, 158 mol). The compound
mol) 10b38 (49.7 mg, 316
D-galactitol-2-
l
l
was purified by flash chromatography (petrol ether/EtOAc
1:1?0:1) to give 11b (73.0 mg, 98%).
1H NMR (500 MHz, CDCl3): d 1.79, 2.04, 2.16 (3s, 9H, 3 COCH3),
3.99–4.04 (m, 2H, H-1ax, H-5), 4.12 (d, J = 6.4 Hz, 2H, H-6), 4.30

7262
D. Stokmaier et al. / Bioorg. Med. Chem. 17 (2009) 7254–7264
4.1.13. 1-(3,4,6-Tri-O-acetyl-1,5-anhydro-2-deoxy-
2-yl)-4-(trifluoromethylsulfonamidophenyloxy)methyl-1,2,3-
triazole (11f)
Prepared according to the procedure described for 11a, using 7
(50.0 mg, 158 lmol) and 10f (88.2 mg, 316 lmol). The compound
D
-galactitol-
60.7 (C-2), 62.6 (CH2OAr), 62.9 (C-6), 69.7 (C-1), 70.3 (C-4), 73.5
(C-3), 80.5 (C-5), 105.3 (CN), 116.8 (2C, 2 C-ortho), 120.0 (C-para),
126.2 (C-5 triazole), 135.2 (2C, 2 C-meta), 143.5 (C-4 triazole),
163.2 (C-ipso); HR-MS Calcd for C16H19N4O5 [M+H]+: 347.1355;
Found: 347.1358.
was purified by flash chromatography (petrol ether/EtOAc
3:1?1:1) to give 11f (39.0 mg, 41%).
4.1.17. 1-(1,5-Anhydro-2-deoxy-
nitrophenoxy)methyl-1,2,3-triazole (1c)
Compound 11c (74.0 mg, 150 mol) was deacetylated accord-
ing to the procedure described for 1a. The final product was puri-
fied by LC-MS to give 1c (18.0 mg, 33%).
D-galactitol-2-yl)-4-(4-
1H NMR (500 MHz, CDCl3): d 1.81, 2.06, 2.18 (3s, 9H, 3 COCH3),
4.00–4.05 (m, 2H, H-1ax, H-5), 4.14 (d, J = 6.4 Hz, 2H, H-6), 4.32
(dd, J = 5.0, 11.6 Hz, 1H, H-1eq), 4.95 (m, 1H, H-2), 5.12 (s, 2H,
CH2OAr), 5.50–5.53 (m, 2H, H-3, H-4), 6.88, 7.22 (AA0, BB0 of AA0BB0,
l
J = 8.8 Hz, 4H, C6H4), 7.68 (s, 1H, H-5 triazole), 8.56 (s, 1H, NH); 13
C
1H NMR (500 MHz, CD3OD): d 3.76–3.87 (m, 2H, H-5, H-6a),
3.90 (t, J = 11.2 Hz, 1H, H-1ax), 4.10 (m, 2H, H-6b, H-4), 4.24 (dd,
J = 5.1, 11.2 Hz, 1H, H-1eq), 4.28 (dd, J = 3.2, 10.6 Hz, 1H, H-3),
4.87 (m, 1H, H-2), 5.38 (s, 2H, CH2OAr), 7.23 (AA0 of AA0BB0,
J = 9.3 Hz, 2H, C6H4), 8.29 (m, 3H, C6H4, H-5 triazole); 13C NMR
(125 MHz, CD3OD): d 60.3 (C-2), 62.5, 62.6 (C-6, CH2OAr), 69.3
(C-1), 69.8 (C-4), 72.9 (C-3), 81.0 (C-5), 116.1 (2C, 2 C-ortho),
126.5 (C-5 triazole), 127.0 (2C, 2 C-meta), 142.6 (C-para), 143.3
(C-4 triazole), 164.5 (C-ipso); HR-MS Calcd for C15H19N4O7
[M+H]+: 367.1254; Found: 367.1255.
NMR (125 MHz, CDCl3): d 20.2, 20.6, 20.7 (3 COCH3), 56.3 (C-2),
61.7, 61.9 (C-6, CH2OAr), 67.0 (C-4), 68.6 (C-1), 71.4 (C-3), 75.2
(C-5), 115.4 (2C, 2 C-ortho), 119.84 (q, J = 323.1 Hz, CF3), 123.2
(C-5 triazole), 126.6 (2C, 2 C-meta), 127.1 (C-para), 143.5 (C-4 tria-
zole), 157.3 (C-ipso), 169.5, 170.1, 170.6 (3 CO); HR-MS Calcd for
C22H25F3N4NaO10 [M+Na]+: 617.1141; Found: 617.1150.
4.1.14. 1-(3,4,6-Tri-O-acetyl-1,5-anhydro-2-deoxy-D-galactitol-
2-yl)-4-(4-bromophenoxy) methyl-1,2,3-triazole (11g)
Prepared according to the procedure described for 11a, using 7
(200 mg, 634
l
mol) and 10g40 (267 mg, 1.07 mmol). The com-
4.1.18. 1-(1,5-Anhydro-2-deoxy-
(pentafluorophenoxy)methyl-1,2,3-triazole (1d)
Compound 11d (60.0 mg, 111 mol) was deacetylated accord-
D-galactitol-2-yl)-4-
pound was purified by flash chromatography (hexane/EtOAc 1:1)
to give 11g (279 mg, 84%).
l
1H NMR (500 MHz, CDCl3): d 1.74, 2.00, 2.11 (3s, 9H, 3 COCH3),
3.93–4.00 (m, 2H, H-1ax, H-5), 4.07 (d, J = 6.4 Hz, 2H, H-6), 4.24
(dd, J = 5.0, 11.6 Hz, 1H, H-1eq), 4.86 (dt, J = 5.0, 11.0 Hz, 1H, H-
2), 5.10 (s, 2H, CH2OAr), 5.43–5.47 (m, 2H, H-3, H-4), 6.77, 7.30
(AA0, BB0 of AA0BB0, J = 8.9 Hz, 4H, C6H4), 7.57 (s, 1H, H-5 triazole);
13C NMR (125 MHz, CDCl3): d 20.2, 20.5, 20.6 (3 COCH3), 56.0 (C-
2), 61.8, 61.9 (C-6, CH2OAr), 66.9 (C-4), 68.6 (C-1), 71.4 (C-3),
75.2 (C-5), 113.5 (C-Br), 116.5 (2C, 2 C-ortho), 122.8 (C5 triazole),
132.3 (2C, 2 C-meta), 143.7 (C-4 triazole), 157.0 (C-ipso), 169.2,
169.9, 170.4 (3 CO); HR-MS Calcd for C21H25BrN3O8 [M+H]+:
526.0825; Found: 526.0818.
ing to the procedure described for 1a. The crude product was puri-
fied by LC-MS to give 1d (31.0 mg, 68%).
1H NMR (500 MHz, CD3OD): d 3.59 (m, 1H, H-5), 3.69 (dd,
J = 4.9, 11.4 Hz, 1H, H-6a), 3.75-3.90 (m, 2H, H-1ax, H-6b), 3.97
(m, 1H, H-4), 4.10-4.14 (m, 2H, H-1eq, H-3), 4.78 (dt, J = 5.0,
10.8 Hz, 1H, H-2), 5.20 (m, 2H, CH2OAr), 8.10 (m, 1H, H-5 triazole);
13C NMR (125 MHz, CD3OD):
d 60.7 (C-2), 62.9 (C-6), 68.4
(CH2OAr), 66.5 (C-1), 70.4 (C-4), 73.5 (C-3), 81.5 (C-5), 126.9 (C-5
triazole), 142.9 (C-4 triazole); HR-MS Calcd for C15H15F5N3O5
[M+H]+: 412.0932; Found: 412.0934.
4.1.19. 1-(1,5-Anhydro-2-deoxy-
carboxyphenoxy)methyl-1,2,3-triazole (1e)
Compound 11e (62.0 mg, 119 mol) was dissolved in MeOH/
D-galactitol-2-yl)-4-(sodium 4-
4.1.15. 1-(1,5-Anhydro-2-deoxy-
phenoxymethyl-1,2,3-triazole (1a)
Compound 11a (65.0 mg, 145
D
-galactitol-2-yl)-
l
lmol) was dissolved in dry MeOH
H2O (4 mL, 1:1), and NaOH (170 mg, 4.72 mmol) was added. The
solution was stirred at rt for 24 h, after which the solvent was re-
moved in vacuo, and the residue was purified by flash chromatog-
raphy (DCM/MeOH 5:1?2:1) to give 1e (32.0 mg, 74%).
1H NMR (500 MHz, CD3OD): d 3.62 (m, 1H, H-5), 3.73 (m, 2H, H-
6), 3.81 (t, J = 11.3 Hz, 1H, H-1ax), 3.99 (d, J = 3.0 Hz, 1H, H-4), 4.11-
4.17 (m, 2H, H-3, H-1eq), 4.80 (m, 1H, H-2), 5.20 (s, 2H, CH2OAr),
7.02, 7.93 (AA0, BB0 of AA0BB0, J = 8.8 Hz, 4H, C6H4), 8.19 (s, 1H, H-
5 triazole); 13C NMR (125 MHz, CD3OD): d 60.6 (C-2), 62.4
(CH2OAr), 62.9 (C-6), 69.7 (C-4), 70.5 (C-1), 73.3 (C-3), 81.2 (C-5),
115.1 (2C, 2 C-ortho), 126.0 (C-5 triazole), 127.5 (C-para), 132.5
(2C, 2 C-meta), 144.1 (C-4 triazole), 162.8 (C-ipso), 173.7 (CO);
HR-MS Calcd for C16H19N3NaO7 [M+H]+: 388.1121; Found:
388.1128.
(5 mL) and sodium metal (20.0 mg, 869 lmol) was added. The
solution was stirred at rt under argon for 3 h, after which the sol-
vent was removed in vacuo and the residue was purified by pre-
parative LC-MS to give 1a (21.0 mg, 45%).
[a]
D +36.4 (c 0.17, MeOH); 1H NMR (500 MHz, CD3OD): d 3.31 (t,
J = 1.6 Hz, 1H, H-5), 3.61–3.81 (m, 2H, H-6), 3.84 (t, J = 11.1 Hz, 1H,
H-1ax), 3.99 (d, J = 3.0 Hz, 1H, H-4), 4.14–4.18 (m, 2H, H-1eq, H-3),
4.81 (dt, J = 5.0, 10.8 Hz, 1H, H-2), 5.16 (s, 2H, CH2OPh), 6.93–7.01,
7.26–7.30 (m, 5H, C6H5), 8.14 (s, 1H, H-5 triazole); 13C NMR
(125 MHz, CD3OD): d 60.7 (C-2), 62.3 (CH2OPh), 62.9 (C-6), 69.8
(C-1), 70.4 (C-4), 73.5 (C-3), 81.5 (C-5), 122.2, 125.8, 130.6, 159.8
(6C, C6H5), 115.9 (C-5 triazole), 144.53 (C-4 triazole); HR-MS Calcd
for C15H20N3O5 [M+H]+: 322.1403; Found: 322.1401.
4.1.16. 1-(1,5-Anhydro-2-deoxy-
cyanophenoxy)methyl-1,2,3-triazole (1b)
Compound 11b (73.0 mg, 155 mol) was dissolved in H2O/
D
-galactitol-2-yl)-4-(4-
4.1.20. 1-(1,5-Anhydro-2-deoxy-
trifluoromethylsulfonamidophenoxy) methyl-1,2,3-triazole (1f)
Compound 11f (39.0 mg, 61.6 mol) was deacetylated accord-
D-galactitol-2-yl)-4-(4-
l
l
MeOH/Et3N (5:5:1, 5.5 mL) and stirred at rt overnight. The solvent
was removed in vacuo and the residue was purified by flash chro-
matography (DCM/MeOH 9:1) to give 1b (44.0 mg, 81%).
ing to the procedure described for 1a. The final product was puri-
fied by flash chromatography on silica gel (DCM/MeOH 10:1) to
give 1f (21.2 mg, 73%).
1H NMR (500 MHz, CD3OD): d 3.62 (m, 1H, H-5), 3.73 (dd,
J = 4.9, 11.4 Hz, 1H, H-6a), 3.80 (dd, J = 7.1, 11.4 Hz, 1H, H-6b),
3.84 (t, J = 11.1 Hz, 1H, H-1ax), 3.99 (d, J = 3.0 Hz, 1H, H-4), 4.14–
4.18 (m, 2H, H-1eq, H-3), 4.82 (dt, J = 5.0, 10.9 Hz, 1H, H-2), 5.36
(s, 2H, CH2OAr), 7.17, 7.67 (AA0, BB0 of AA0BB0, J = 9.0 Hz, 4H,
C6H4), 8.19 (s, 1H, H-5 triazole); 13C NMR (125 MHz, CD3OD): d
1H NMR (500 MHz, CD3OD): d 3.58 (m, 1H, H-5), 3.68 (dd,
J = 4.8, 11.4 Hz, 1H, H-6a), 3.73–3.81 (m, 2H, H-1ax, H-6b), 3.95
(d, J = 3.0 Hz, 1H, H4), 4.10–4.13 (m, 2H, H-1eq, H-3), 4.77 (dt,
J = 5.1, 10.8 Hz, 1H, H-2), 5.11 (s, 2H, CH2OAr), 6.94, 7.13 (AA0 of
AA0BB0, J = 9.0 Hz, 2H, C6H4), 8.11 (s, 1H, H-5 triazole); 13C NMR
(125 MHz, CD3OD): d 60.7 (C-2), 62.6 (CH2OAr), 62.9 (C-6), 69.7

D. Stokmaier et al. / Bioorg. Med. Chem. 17 (2009) 7254–7264
7263
(C-1), 70.4 (C-4), 73.4 (C-3), 81.4 (C-5), 116.3 (2C, 2 C-ortho), 121.9
well of the test compound solution (6
a 0.5 g/mL of streptavidin-peroxidase coupled PAA-glycopoly-
mers ( -GalNAc-, -Gal-PAA) were added. The plates were incu-
bated for 2 h at rt and 250 rpm. The plates were then carefully
washed twice with 150 L/well HBS-buffer. After the addition of
100 L/well of ABTS-substrate, the colorimetric reaction was al-
lM to 2 mM) and 50 lL of
(q, J = 323.9 Hz, CF3), 125.9 (C-5 triazole), 126.8 (2C, 2 C-meta),
131.4 (C-para), 144.3 (C-4 triazole), 158.0 (C-ipso); HR-MS Calcd
for C16H19F3N4O7SNa [M+Na]+: 491.0824; Found: 491.0828.
l
D
D
l
4.1.21. 1-(3,4,6-Tri-O-acetyl-1,5-anhydro-2-deoxy-
D-galactitol-
l
2-yl)-4-[(4-methyl-piperazin-1-yl)-phenoxymethyl]-1,2,3-
triazole (11h)
lowed to develop for 2 min. The reaction was stopped by the addi-
tion of 2% aqueous oxalic acid and the optical density (OD) was
measured at 415 nm on a microplate-reader (Spectramax 190,
Molecular Devices, California, USA). The IC50 values of the com-
pounds tested in triplicates in three independent experiments
were calculated with PRISM software (GraphPad Software, Inc, La Jol-
la, USA). The data set was normalized to percentage of binding by
Compound 11g (75.0 mg, 142
199 mol) were azeotropically dried with toluene, and Pd2dba3
(3.00 mg, 2.89 mol), N-meth-
mol), X-phos42 (12, 1.35 mg, 2.84
ylpiperazine (17.0 L, 157 mol) and dry toluene (3 mL) were
lmol) and Cs2CO3 (64.8 mg,
l
l
l
l
l
added. The resultant mixture was stirred at 80 °C under argon for
24 h. The solvent was removed in vacuo, and the residue purified
by flash chromatography (EtOAc/MeOH 9:1?4:1) to give 11h,
which was still slightly impure according to NMR. The compound
was used without further purification in the next step.
setting background binding (D-GalNAc-PAA binding to wells with-
out H1-CRD) as 0% and control binding (maximal
D-GalNAc-PAA
binding to H1-CRD) as 100%. The nonlinear fit of the normalized
data was calculated with a four-parameter logistic equation with
the bottom of the curve constraint to zero and the top to 100.
The IC50 defines the molar concentration of the test compound that
4.1.22. 1-(1,5-Anhydro-2-deoxy-
piperazin-1-yl)-phenoxymethyl]-1,2,3-triazole (1h)
Compound 11h (35.1 mg, 64.3 mol) was deacetylated accord-
D-galactitol-2-yl)-4-[(4-methyl-
reduces the maximal specific binding of
by 50%. The relative IC50 (rIC50) is the ratio of the IC50 of the test
compound to the IC50 of -GalNAc.
D-GalNAc-PAA to H1-CRD
l
ing to the procedure described for 1a. The final product was puri-
fied by flash chromatography (DCM/MeOH 9:1?7:3) to give 1h
(12.0 mg, 44%).
D
4.4. Surface Plasmon Resonance Experiments (Biacore)
1H NMR (500 MHz, CD3OD): d 2.63 (s, 3H, NCH3), 3.02 (m, 4H,
(CH2)2NCH3), 3.18 (m, 4H, (CH2)2NPh), 3.59 (m, 1H, H-5), 3.69
(dd, J = 4.9, 11.4 Hz, 1H, H-6a), 3.74–3.82 (m, 2H, H-1ax, H-6b),
3.95 (d, J = 3.0 Hz, 1H, H-4), 4.10-4.13 (m, 2H, H-1eq, H-3), 4.02
(dd, J = 3.0, 10.5 Hz, 1H, H-3), 4.76 (m, 1H, H-2), 5.07 (s, 2H,
CH2OAr), 6.92 (m, 4H, C6H4), 8.09 (s, 1H, H-5 triazole); 13C NMR
(125 MHz, CD3OD): d 44.7 (NCH3), 49.5 (2C, (CH2)2NCH3), 55.4
(2C, (CH2)2NPh), 60.7 (C-2), 62.8 (2C, C-6, CH2OAr), 69.7 (C-1),
70.4 (C-4), 73.4 (C-3), 81.4 (C-5), 116.7 (2C, 2 C-ortho), 119.9 (2C,
2 C-meta), 125.8 (C-5 triazole), 144.6 (C-para), 146.4 (C-4 triazole),
154.7 (C-ipso); HR-MS Calcd for C20H30N5O5 [M+H]+: 420.2247;
Found: 420.2252.
The analyses were performed on a Biacore 3000 machine (Bia-
core AB, Uppsala, Sweden) using CM5 chips. After activating the
surface of the chip for 5–10 min at a flow rate of 5
a standard amine coupling procedure, H1-CRD, diluted in 10 mM
acetate buffer pH 4.5 to a final concentration of 20 g/mL, was in-
lL/min using
l
jected for 5–15 min and the surface was deactivated for a time cor-
responding to the activation phase. The obtained surface densities
ranged from 1800 to 2500 RUs. HBS-N buffer (10 mM HEPES pH
7.4, 50 mM CaCl2) was used as running buffer. The screening of
the compounds was performed by the injection of twofold serial
dilutions between 5 mM and 5
sample was injected for 30 s with an undisturbed dissociation
phase of 20 s and 50 L/min. No regeneration or washing steps
lM as randomized triplicates. Each
4.2. Expression and purification of recombinant H1-CRD
l
were applied. Five buffer blanks were injected at the beginning
and one between the triplicate series. Signals of an untreated flow
cell and averaged blank injections were subtracted from the sam-
ple sensorgrams. Since referenced sensorgrams showed negative
SPR signals, the whole data set was mirrored by multiplication of
each data point with ꢀ1. Mirrored steady state data were evalu-
ated between 10 and 20 s of the injection period and were fitted
to a single site-binding model. Data processing and equilibrium
binding constant determinations were accomplished with SCRUBBER
software Version 2.
The vector pET3b encoding the amino acid residues 147–291 of
the H1-subunit was transformed into E. coli strain AD494 (DE3).
Following IPTG (0.4 mM) induction, the protein was accumulated
as inclusion bodies. After collection and lysis of the cells the pro-
tein extract was denatured under reducing conditions and dialyzed
against Tris/HCl buffer (20 mM TrisCl, 120 mM NaCl, 20 mM CaCl2,
pH 7.8). The correctly folded H1-CRD was purified by affinity chro-
matography on a D-Gal-Sepharose column attached to an FPLC-sys-
tem (Bio-Rad). In a second step the monomer and dimer fractions
were separated by ion exchange HPLC (Agilent 1100) on a DEAE
column. The monomeric fraction was quantified by a Bradford as-
say55 and immediately used for the binding assay or stored at
ꢀ20 °C. The purity and identity of the purified protein were veri-
fied by SDS–PAGE and immuno-blot analysis, respectively. For
the immuno-blot analysis, polyclonal (IgY) anti-H1-CRD antibodies
were used. The antibodies were produced in chicken and purified
from their eggs after PEG-precipitation56 by affinity chromatogra-
phy on a agarose-immobilized H1-CRD column.
4.5. NMR experiments
Shigemi NMR tubes were used to reduce the sample volume,
and therefore protein consumption, needed for measurement to
250 lL. The ASGP-R protein was present at 17 lM, as determined
by a Bradford assay. The buffer consisted of 1 mM CaCl2 in D2O (Ar-
mar Chemicals), maintained at pH 7.5 with 25 mM Tris-d11. A stock
solution of D-GalNAc was prepared in D2O at 50 mM. A stock solu-
tion of 1b in DMSO-d6 was prepared at 50 mM.
4.3. Competitive solid-phase binding assay
All NMR experiments were carried out at 300 K on a Bruker
DRX500 spectrometer, equipped with Z-gradient SEI probe. The
pulse sequence used for the T1rho relaxation was slightly modified
from that described by Hajduk.57 Following the 90° non-selective
pulse, a continuous-wave spin-lock of 2 kHz was applied for a var-
ious duration to monitor the T1rho relaxation. The pulse sequence
terminated with the DPFGSE water suppression sequence to
suppress the magnetization from the residual protons in the D2O
Flat-bottom 96-well microtiter plates were coated with 100
lL/
well of a 3 g/mL solution of monomeric H1-CRD in 20 mM HEPES,
l
150 mM NaCl and 1 mM CaCl2, pH 7.4 (HBS-buffer) overnight at
4 °C. The coating solution was discarded and the wells were
blocked with 150
After three washing steps with 150
l
L/well of 1% BSA in HBS-buffer for 2 h at 4 °C.
L/well of HBS-buffer, 50 L/
l
l

7264
D. Stokmaier et al. / Bioorg. Med. Chem. 17 (2009) 7254–7264
solvent.58 For each T1rho time constant measurement, 15 experi-
ments were performed with different durations of the spin-lock
which started at 20 ms to a final value of 300 ms, in intervals of
18. Biessen, E. A.; Beuting, D. M.; Vietsch, H.; Bijsterbosch, M. K.; Van Berkel, T. J. J.
Hepatol. 1994, 21, 806.
19. Biessen, E. A.; Vietsch, H.; Rump, E. T.; Fluiter, K.; Kuiper, J.; Bijsterbosch, M. K.;
van Berkel, T. J. Biochem. J. 1999, 340, 783.
20. Wu, G. Y.; Wu, C. H. J. Biol. Chem. 1991, 263, 14621.
21. Hara, T.; Aramaki, Y.; Takada, S.; Koike, K.; Tsuchiya, S. Gene Ther. 1995, 2, 784.
22. Hara, T.; Aramaki, Y.; Takada, S.; Koike, K.; Tsuchiya, S. Gene 1995, 159, 167.
23. Hara, T.; Kawasawa, H.; Aramaki, Y.; Takada, S.; Koike, K.; Ishidate, K.; Kato, H.;
Tsuchiya, S. Biochim. Biophys. Acta 1996, 1278, 51.
24. Kawakami, S.; Yamashita, F.; Nishikawa, M.; Takakura, Y.; Hashida, M. Biochem.
Biophys. Res. Commun. 1998, 252, 78.
25. Wong, T. C.; Townsend, R. R.; Lee, Y. C. Carbohydr. Res. 1987, 170, 27.
26. Kolatkar, A. R.; Weis, W. I. J. Biol. Chem. 1996, 271, 6679.
27. Kolatkar, A. R.; Leung, A. K.; Isecke, R.; Brossmer, R.; Drickamer, K.; Weis, W. I. J.
Biol. Chem. 1998, 273, 19502.
28. Khorev, O.; Stokmaier, D.; Schwardt, O.; Cutting, B.; Ernst, B. Bioorg. Med. Chem.
2008, 16, 5216; and references cited therein.
29. Hudgin, R. L.; Pricer, W. E.; Ashwell, G.; Stockert, R. J.; Morell, A. G. J. Biol. Chem.
1974, 249, 5536.
30. Quesenberry, M. S.; Drickamer, K. Glycobiology 1991, 1, 615.
31. Park, E. I.; Manzella, S. M.; Baenziger, J. U. J. Biol. Chem. 2003, 278, 4597.
32. Lee, R. T. Biochem. 1982, 21, 1045.
33. Trunzer, M.; Faller, B.; Zimmerlin, A. J. Med. Chem. 2009, 52, 329.
34. (a) Tornøe, C. W.; Meldal, M. In Peptides: The Wave of the Future: Proceedings of
the Second International and the Seventeenth American Peptide Symposium; Lebl,
M., Houghten, R. A., Eds.; Springer, 2001; p 263; (b) Tornøe, C. W.; Christensen,
C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.
20 ms. For concentrations of
600 M, 16 scans were measured, preceded by eight dummy scans,
and a recovery delay of 10 s between successive scans. For concen-
trations of -GalNAc that were 600 M or greater, the identical
procedure was followed, except that only eight scans were mea-
sured. Prior to the measurement upon addition of a competing
compound, a 1 h incubation time for equilibration was allowed.
The NMR data were analyzed using XWINNMR version 3.5 oper-
ating on a PC running under Linux OS. The spectra were apodized
with an exponential decay function with 2 Hz line broadening.
The inversion recovery data, as well as the one-site binding model,
were fit using PRISM 4 (GraphPad Software Inc., San Diego, USA).
D-GalNAc that were less than
l
D
l
Acknowledgment
The authors would like to thank the Swiss National Science
Foundation (SNF) for funding the research.
35. Kolb, H. C.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004.
36. Titz, A.; Radic, Z.; Schwardt, O.; Ernst, B. Tetrahedron Lett. 2006, 47,
2383.
Supplementary data
37. Li, H.; Cheng, F.; Duft, A. M.; Adronov, A. J. Am. Chem. Soc. 2005, 127, 14518.
38. Argentini, M.; dos Santos, D. F.; Weinreich, R.; Hansen, H.-J. Inorg. Chem. 1998,
37, 6018.
39. Brooke, G. M. J. Chem. Soc., Perkin Trans. 1 1974, 233.
40. Kotsuki, H.; Araki, T.; Miyazaki, A.; Iwasaki, M.; Datta, P. K. Org. Lett. 1999, 1,
499.
41. Wolfe, J. P.; Wagaw, S.; Buchwald, S. L. J. Am. Chem. Soc. 1996, 118, 7215.
42. Huang, X.; Anderson, K. W.; Zim, D.; Jiang, L.; Klapars, A.; Buchwald, S. L. J. Am.
Chem. Soc. 2003, 125, 6653.
43. Fornstedt, N.; Porath, J. FEBS Lett. 1975, 57, 187.
44. Weitz-Schmidt, G.; Stokmaier, D.; Scheel, G.; Nifant’ev, N. E.; Tuzikov, A. B.;
Bovin, N. V. Anal. Biochem. 1996, 238, 184.
45. Thoma, G.; Magnani, J. L.; Oehrlein, R.; Ernst, B.; Schwarzenbach, F.; Duthaler,
R. O. J. Am. Chem. Soc. 1997, 119, 7414.
46. Gestwicki, J. E.; Hsieh, H. V.; Pitner, J. B. Anal. Chem. 2001, 73, 5732.
47. Dalvit, C.; Flocco, M.; Knapp, S.; Mostardini, M.; Perego, R.; Stockman, B. J.;
Veronesi, M.; Varasi, M. J. Am. Chem. Soc. 2002, 124, 7702.
48. Lepre, C. A.; Moore, J. M.; Peng, J. W. Chem. Rev. 2004, 104, 3641.
49. Coombs, P. J.; Taylor, M. E.; Drickamer, K. Glycobiology 2006, 16, 1C.
50. Li, M.; Kurata, H.; Itoh, N.; Yamashina, I.; Kawasaki, T. J. Biol. Chem. 1990, 265,
11295.
51. Nakamura, K.; Funakoshi, H.; Miyamoto, K.; Tokunaga, F.; Nakamura, T.
Biochem. Biophys. Res. Commun. 2001, 280, 1028.
52. Hoyle, G. W.; Hill, R. L. J. Biol. Chem. 1988, 263, 7487.
53. Wu, J.; Nantz, M. H.; Zern, M. A. Front. Biosci. 2002, 7, d717.
54. Tarasiejska, Z.; Jeanloz, R. W. J. Am. Chem. Soc. 1958, 80, 6325.
55. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
Supplementary data (1H NMR spectra of target compounds 1a–
f, 1h and 6) associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2009.08.049.
References and notes
1. Drickamer, K.; Taylor, M. E. Annu. Rev. Cell. Biol. 1993, 9, 237.
2. Hubbard, A. L.; Sturenbrok, H. J. Cell Biol. 1979, 83, 56.
3. Spiess, M. Biochem. 1990, 29, 10009.
4. Geffen, I.; Spiess, M. Int. Rev. Cytol. 1992, 137B, 181.
5. Weigel, P. H.; Yik, J. H. Biochim. Biophys. Acta 2002, 1572, 341.
6. Yik, J. H.; Saxena, A.; Weigel, P. H. J. Biol. Chem. 2002, 277, 23076.
7. Henis, Y. I.; Katzir, Z.; Shia, M. A.; Lodish, H. F. J. Cell. Biol. 1990, 111, 1409.
8. Bider, M. D.; Wahlberg, J. M.; Kammerer, R. A.; Spiess, M. J. Biol. Chem. 1996,
271, 31996.
9. Tozawa, R.; Ishibashi, S.; Osuga, J.; Yamamoto, K.; Yagyu, H.; Ohashi, K.;
Tamura, Y.; Yahagi, N.; Iizuka, Y.; Okazaki, H.; Harada, K.; Gotoda, T.; Shimano,
H.; Kimura, S.; Nagai, R.; Yamada, N. J. Biol. Chem. 2001, 276, 12624.
10. Ishibashi, S.; Hammer, R. E.; Herz, J. J. Biol. Chem. 1994, 269, 27803.
11. Bider, M. D.; Spiess, M. FEBS Lett. 1998, 434, 37.
12. Shia, M. A.; Lodish, H. F. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 158.
13. Meier, M.; Bider, M. D.; Malashkevich, V. N.; Spiess, M.; Burkhard, P. J. Mol. Biol.
2000, 300, 857.
14. Lee, Y. C.; Townsend, R. R.; Hardy, M. R.; Lonngren, J.; Arnarp, J.; Haraldsson,
M.; Lönn, H. J. Biol. Chem. 1983, 258, 199.
56. Polson, P.; Coetzer, T.; Kruger, J.; von Maltzahn, E.; van der Merwe, K. J. Immin.
Invest. 1985, 14, 323.
57. Hajduk, P. J.; Olejniczak, E. T.; Fesik, S. W. J. Am. Chem. Soc. 1997, 119, 12257.
58. Hwang, T.-. L.; Shaka, A. J. J. Magn. Reson. Ser. A 1995, 112, 275.
15. Lee, R. T.; Lee, Y. C. Glycoconj. J. 2000, 17, 543.
16. Connolly, D. T.; Townsend, R. R.; Kawaguchi, K.; Bell, W. R.; Lee, Y. C. J. Biol.
Chem. 1982, 257, 939.
17. Lee, R. T.; Lee, Y. C. Bioconj. Chem. 1997, 8, 762.