Kinetic and thermodynamic properties of MAG antagonists

Article abstract of DOI:10.1016/j.carres.2010.03.010

Paraplegia is caused by injuries of the central nervous system (CNS) and especially young people suffer from these severe consequences as, for example, the loss of motor functions. The lack of repair of the injured nerve strands originates from the inhibitory environment for axon regeneration in the CNS. Specific inhibitory proteins block the regrowth of nerve roots. One of these neurite outgrowth inhibitors is the myelin-associated glycoprotein (MAG), which is a member of the Siglec family (sialic acid-binding immunoglobulin-like lectin). In previous studies, we identified potent small molecule MAG antagonists. In this communication, we report new neuraminic acid derivatives modified in the 4- and 5-position, and the influence of various structural modifications on their kinetic and thermodynamic binding properties.

Full text of DOI:10.1016/j.carres.2010.03.010

Carbohydrate Research 345 (2010) 1348–1359
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Kinetic and thermodynamic properties of MAG antagonists
Stefanie Mesch a, Katrin Lemme a, Hendrik Koliwer-Brandl b, Daniel S. Strasser a, Oliver Schwardt a,
Soerge Kelm b, Beat Ernst a,
*
a Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstr. 50, 4056 Basel, Switzerland
b Center for Biomolecular Interactions Bremen, Glycobiochemistry, University of Bremen, POB 330440, D-28334 Bremen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Paraplegia is caused by injuries of the central nervous system (CNS) and especially young people suffer
from these severe consequences as, for example, the loss of motor functions. The lack of repair of the
injured nerve strands originates from the inhibitory environment for axon regeneration in the CNS. Spe-
cific inhibitory proteins block the regrowth of nerve roots. One of these neurite outgrowth inhibitors is
the myelin-associated glycoprotein (MAG), which is a member of the Siglec family (sialic acid-binding
immunoglobulin-like lectin). In previous studies, we identified potent small molecule MAG antagonists.
In this communication, we report new neuraminic acid derivatives modified in the 4- and 5-position, and
the influence of various structural modifications on their kinetic and thermodynamic binding properties.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 21 January 2010
Received in revised form 4 March 2010
Accepted 9 March 2010
Available online 16 March 2010
Keywords:
Carbohydrate mimetics
Siglecs
Myelin-associated glycoprotein (MAG)
Thermodynamics of carbohydrate–protein
interactions
Kinetics of carbohydrate–lectin interactions
1. Introduction
initiated by sialidase treatment, suggesting that the sialic acid-
mediated interactions of MAG predominantly contribute to the
Paraplegia is caused by injuries of the central nervous system
(CNS). A therapy for full regeneration of injured nerve strands is
not yet available. The lack of regeneration originates from the
inhibitory environment in the CNS,1,2 that is, specific inhibitors
on residual myelin and on astrocytes, which are recruited to the
site of injury.3–5 In the last decade, several inhibitor proteins have
been identified, one of them being the myelin-associated glycopro-
tein (MAG).6 MAG is a transmembrane glycoprotein, belonging to
the Siglec family (sialic acid-binding immunoglobulin-like lec-
tin).7,8 On the surface of neurons, MAG interacts with two classes
of targets: proteins of the Nogo receptor family9,10 and ganglio-
sides, primarily the gangliosides GD1a and GT1b.11–14 Although
the relative role of Nogo receptors and gangliosides as MAG ligands
has yet to be resolved, in some systems, neurite outgrowth can be
inhibitory process.15 Therefore, blocking MAG with potent glyco-
mimetic antagonists may be a valuable therapeutic approach to
enhance axon regeneration. Based on the best known natural
ligand of MAG identified to date, the ganglioside GQ1b
a (Fig. 1),
different series of antagonists have been developed.16–21
With neuraminic acid derivatives such as 1,16 Kelm et al. re-
ported a remarkable simplification of the relevant tetrasaccharide
binding epitope of GQ1ba. Further reported modifications are re-
lated to lipophilic interactions. Thus, antagonists with a lipophilic
core, for example, the biphenyl derivatives 218 or a lipophilic
replacement of the
synthesized (Fig. 1).
a
-(2?6)-linked Neu5Ac, for example, 319 were
The concept of drug discovery is based upon selectively
addressing particular biological targets preferably by low molecu-
lar weight compounds. In vitro determined drug–target interac-
tions are classically rated in terms of binding parameters such as
IC50’s and KD’s. An alternative perspective on drug optimization is
the residence time of the drug–target binary complex,22 as quanti-
fied by the dissociation half-life (t1/2). Potential advantages of a
long residence time are extended duration of the pharmacological
effect and target selectivity.22,23 Especially in the field of carbohy-
drate–lectin interactions, this is a crucial point to address. As a re-
sult of the shallow and water accessible binding sites of lectins,
carbohydrates bind with only low affinity and show very fast dis-
sociation off-rates, leading to t1/2 in the range of seconds. Examples
Abbreviations: AIBN,
a,
a0-azodiisobutyronitrile; aq, aqueous; BnBr, benzyl
bromide; DCM, dichloromethane; DMAP, 4-dimethylaminopyridine; DMF, N,N-
dimethylformamide; FAc, fluoro-acetyl; HBS-E, HEPES/NaCl/EDTA buffer; HBS-EP,
HEPES–NaCl–EDTA–P20 buffer; ITC, isothermal titration calorimetry; KD, dissocia-
tion constant; MS, mass spectrometry; Neu5Ac, N-acetylneuraminic acid; NgR,
Nogo receptor; NMR, nuclear magnetic resonance; PDC, pyridinium dichromate;
PPTS, pyridinium p-toluenesulfonate; RP, reversed phase; SPR, surface plasmon
resonance; STD-NMR, saturation transfer difference nuclear magnetic resonance
spectroscopy; THF, tetrahydrofuran.
* Corresponding author. Tel.: +41 267 15 51; fax: +41 267 15 52.
E-mail address: beat.ernst@unibas.ch (B. Ernst).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.03.010

S. Mesch et al. / Carbohydrate Research 345 (2010) 1348–1359
1349
HO
OH
FAcHN
OMe
O
C13H27
HO
CO2Na
OH
HO
O
C17H35
O
116
OH
N
H
OH
OH
OH
OH
HO
AcHN
HO
AcHN
O
CO2Na
O
CO2H
O
O
HO
OH
HO
HO
O
OH
O
HO
HO
O
HO
O
O
HO
O
HO
AcHN
OH
O
O
O
O
AcHN
O
OH
OH
HO
NaO2C
OH
AcHN
OH
O
HO
CO2H
218
OH
CO2H
O
HO
AcHN
OH
CO2H
O
OH
HO
HO
O
O
O
OH
HO
HO
OH
OH
OH
O
O
AcHN
RHN
O
O
O
O
O
O
HO
F
NHAc
OH
OH
N
H
OH
N
H
CO2Na
CO2Na
F
OH
OH
GQ1b
α
319
421: R = Ac
21: R = FAc
Cl
Cl
5
Figure 1. MAG antagonists 116, 218, 319 and 4, 521derived from the tetrasaccharide core structure (highlighted in box) of GQ1b
a.
Table 1
Carbohydrate–protein interactions: thermodynamic and kinetic binding parameters
Protein
Ligand
KD
(
lM)
kon (Mꢀ1 sꢀ1
)
koff (sꢀ1
)
t1/2 (s)
P-Selectin24
E-Selectin25
GSLA-2 mAb26
MAG19
PSGL-1
ESL-1
0.3
62
4.3
2.8
4ꢁ106
1.4
3.0
0.48
0.8
0.5
0.2
1.5
0.9
4ꢁ104
Sialyl Lewisa
Neu5Ac derivative 319
1.1ꢁ105
3.5ꢁ105
of thermodynamic and kinetic parameters for carbohydrate–pro-
tein interactions are summarized in Table 1.
the binding affinity,16,21 we investigated the impact of this position
on the thermodynamic properties and also examined its influence
on the dissociation half-life time. Molecular modeling studies with
a homology model of MAG30 suggested that the hydroxy group in
the 4-position is not directly involved in the binding process19 and
therefore provides a possibility for derivatization. Because addi-
tional hydrophobic contacts based on the 4-position and an in-
verted configuration at C-4 are expected to alter the
thermodynamic and kinetic behavior, we synthesized a small li-
brary of antagonists and analyzed their binding properties by sur-
face plasmon resonance.
For medical applications, an improved t1/2 of the drug–protein
complex is beneficial, because the therapeutic effect can be
reached with a lower dose. Zanamivir is one of the prominent
examples, where a carbohydrate-based lead was optimized to yield
a drug with a dramatically improved kinetic behavior, showing a
half-life of 33 min of its complex with the B/Memphis/3/89
(H3N2) influenza virus.27 In this communication, we present vari-
ous MAG antagonists modified at the 4- and 5-position with the
aim to modulate their kinetic properties. In general, lead optimiza-
tion is often achieved by additional lipophilic contacts and thereby
improving the binding entropy. As a result of the increased lipo-
philicity, the dissociation half-life (t1/2) of the drug–target complex
is extended.28,29 The starting point for our investigation was MAG
antagonist 5,21 a result of an extended optimization program
focusing exclusively on the improvement of its thermodynamic
binding properties.17–19,21
2.1. A MAG antagonist modified in the 5-position of the Neu5Ac
scaffold
With isothermal titration calorimetry, we determined the ther-
modynamic parameters
DH, DS, and DG
31 of antagonist 5 interact-
ing with a recombinant protein consisting of the three N-terminal
domains of MAG and the Fc part of human IgG (MAGd1-3-Fc).32 For
the ITC experiment, a solution of 5 (500 lM, HBS-E buffer) was in-
2. Results and discussion
jected into a solution of MAGd1-3-Fc (48.35 lM, HBS-E buffer) at
25 °C (Fig. 2).
Recently, we reported the synthesis and biological evaluation of
a series of MAG antagonists with affinities in the low micromolar
range.21 Furthermore, pharmacokinetic parameters such as stabil-
ity and membrane penetration indicated that the antagonists 4 and
5 (Fig. 1) fulfill the basic requirements for lead compounds. As hal-
ogenated acetates at the 5-position led to a drastic improvement of
The experimental data were fitted to a theoretical titration
curve (one site binding model) using Origin version 7 software
(MicroCal) and the thermodynamic parameters calculated accord-
ing to the equation shown in Table 2. The ITC experiment con-
firmed the high potency of 5, having a KD in the nanomolar

1350
S. Mesch et al. / Carbohydrate Research 345 (2010) 1348–1359
Figure 2. Enthalpogram (left) and corresponding fit (right) of the titration of MAGd1-3-Fc with antagonist 5. For the fit, the first injection was not taken into account.
Table 2
Thermodynamic parameters of antagonist 5
2.2. MAG antagonists modified in the 4-position of the Neu5Ac
scaffold
Ligand
5
N
KD (nM)
D
G (kJ/mol)*
ꢀ39.1
calculated
D
H (kJ/mol)*
TDS (kJ/mol)*
1.03
142
ꢀ39.2
ꢀ0.14
As an increased lipophilicity often leads to prolonged residence
times,35 hydrophobic substituents were introduced in the 4-posi-
tion. Furthermore, we planned to investigate the influence of the
configuration at this position on the kinetic binding behavior.
Starting from the Neu5Ac donor 6,36 compound 8 was obtained
according to published procedures (Scheme 1).21 After deacetyla-
tion under Zemplén conditions (?9), the acetonide37 10 was
formed with the two hydroxy groups in the 7- and 8-position, per-
mitting selective modifications of the 4-position. To obtain the 4-
deoxy compound 14, the 4-hydroxy group in 10 was transformed
into the corresponding thiocarbonate 12, a precursor for a Barton
deoxygenation using tributyltin hydride.38 Cleavage of the aceto-
D
D
H,
G =
D
H ꢀ T
S,
and
D
G
were
according
to
the
equation
D
D
S = RTln KA = ꢀRTln KD;
N represents the stoichiometry, * values’
accuracy 5%.
range.21 Interestingly, the interaction is exclusively enthalpy
driven.33,34
In a next step, the kinetic binding properties of 5 were deter-
mined by surface plasmon resonance (SPR) (Fig. 3). Because the
off-rate at 25 °C turned out to be very fast, the Biacore experiment
was repeated at lower temperature. At 5 °C, a clear slowdown of
koff for 5 (R: FAc) compared to that for 4 (R: Ac) was observed.
Figure 3. Kinetic fits of compounds 4 and 5 at 5 °C. In the case of 5, an increase of the dissociation half-life (t1/2) by a factor of 2 was observed. For the fitting of the
sensorgrams Scrubber 2.0c was applied.

S. Mesch et al. / Carbohydrate Research 345 (2010) 1348–1359
1351
RO
AcO
OAc
HO
O
AcHN
AcHN
O
AcHN
O
SMe
O
O
O
O
O
F
O
F
Ref 21
b)
OR
COOMe
F
COOMe
COOR
F
N
H
N3
N
H
OR
OAc
Cl
Cl
636
8, R = Ac
9, R = H
10, R = CH3
11, R = Na
a)
c)
PhO
S
O
AcHN
AcHN
O
O
O
O
O
O
O
F
F
e, f, c)
d)
OH
O
10
11
COONa
F
COOMe
F
N
H
N
H
OH
14
12
Cl
Cl
R1
R1
AcHN
O
AcHN
O
O
O
O
O
O
F
F
g)
f)
O
OH
COONa
F
COONa
F
N
H
N
H
OH
16a-h
15a-h
Cl
Cl
Scheme 1. Reagents and conditions: (a) NaOMe, MeOH (61%); (b) MeO2C(CH3)2, PPTS, MeCN (60%); (c) 10% NaOH (aq), MeOH (42%); (d) C7H5ClO2S, DCM, pyridine (83%); (e)
n-Bu3SnH, AIBN, toluene, 100 °C (?13, 20%); (f) 80% AcOH (aq), 60 °C (?16a–h, 10–80%); (g) (i) (R1O)2C@O, DMAP, pyridine (?15a, 80%, 15b, 61%); or (ii) BnBr, KOH (aq,
50%), 18-crown-6, DCM, 60 °C (?15c, 60%); or (iii) R1NCO, DMAP, pyridine (?15d–h, 20–37%).
nide under acidic conditions followed by hydrolysis of the methyl
ester yielded test compound 14 in excellent overall yield.
Starting from 11, the hydroxy group in the 4-position was either
acylated with the corresponding anhydrides (?15a,b), the corre-
sponding isocyanides (?15d–h), or reacted with benzyl bromide
under phase-transfer catalysis conditions (?15c). Finally, the ace-
tonide was cleaved under acidic conditions to yield the test com-
pounds 16a–h (Scheme 1, Table 3).
Test compound 22 was obtained by reduction of 17 with
BH3ꢁNH3, yielding the 4-hydroxy compound 20 [(R)-stereoisomer]
with the inverted configuration at C-4 compared to Neu5Ac.43 Fi-
nally, removal of the acetonide and hydrolysis of the methyl ester
gave compound 22.
2.3. Biological evaluation and kinetic studies
The 4-disubstituted antagonist 19 was obtained in a two-
step procedure (Scheme 2). Oxidation of 10 with pyridinium
dichromate under acidic conditions39 yielded 17; however, due
to the instability of the acetonide only in moderate 30% yield.
Various other conditions, for example, using molecular sieves
instead of acetic anhydride40 did not lead to notably improved
yields. Then, 17 was reacted with the tetramethylzirconium
complex41 followed by the cleavage of the acetonide to yield
18 with an acceptable stereoselectivity (11% of the S-stereoiso-
mer was formed). The zirconium complex was chosen in order
to avoid undesired side reactions (e.g., enolization) as reported
earlier by Hartmann et al.42 Final deprotection gave compound
19.
First, the affinity of the test compounds 14, 16a–h, 19, and 22
toward MAG was determined by
a
surface plasmon
resonance based biosensor (Biacore) experiment.21 Fc-MAGd1-3-Fc32
was immobilized on a dextran chip containing a surface of
covalently bound protein A. A reference cell providing only protein
A was used to compensate unspecific binding to the matrix.
Dilution series of the compounds were prepared either in pure
HEPES-buffer or in buffer containing 3% DMSO and passed over the
flow cells. As reported earlier,21 negative sensorgrams were ob-
tained (after subtraction of the reference cell). After their mirror-
ing, they could be fitted to a one-to-one binding model using
Scrubber 2.0c (Table 4). The kinetic parameters kon and koff were
obtained by applying a global fit (Scrubber 2.0c).
Table 3
Substituents R1 in antagonist 16
16a
16b
16c
16d
16e
O
16f
16g
O
16h
O
O
O
O
O
O
O
O
O
O
O
O
S
O
HN
HN
HN
HN
HN
S
O

1352
S. Mesch et al. / Carbohydrate Research 345 (2010) 1348–1359
O
AcHN
O
O
O
OH
AcHN
O
O
O
F
O
O
F
O
COOMe
F
e)
a)
N
H
O
COOMe
F
N
H
10
Cl
Cl
17
20
b, c)
c)
Me
OH
AcHN
AcHN
O
O
O
OH
O
OH
O
O
F
F
OH
COOMe
F
COOMe
F
N
H
N
H
OH
OH
Cl
Cl
18
21
d)
f)
Me
OH
AcHN
AcHN
O
O
O
OH
O
OH
O
O
F
F
OH
COONa
F
COONa
F
N
H
N
H
OH
OH
Cl
Cl
19
22
Scheme 2. Reagents and conditions: (a) PDC, Ac2O, DCM, rt (30%); (b) Me4Zr, THF, ꢀ78 °C, 50% NH4Cl (aq); (c) 80% AcOH (aq), 60 °C (?18, 32 + 11% (S)-stereoisomer, two
steps; ?21, 90%); (d) LiOH (aq), THF (34%); (e) BH3ꢁNH3, MeOH, 0 °C (36%); (f) 10% NaOH (aq) (34%).
Table 4
The deoxy compound 14 showed a decrease in affinity by a fac-
tor of six. The inversion of the configuration at the 4-position
Affinities and kinetic parameters of Neu5Ac derivatives modified in the 4-position
(?22) or the introduction of a methyl group in equatorial position
(?19) led also to a drop in binding affinity (factor of 4 and 15,
R1
AcHN
O
respectively). Originally, we assumed based on docking studies to
a homology model of MAG30 that the hydroxy group in the 4-posi-
tion is not directed toward the protein and can therefore be mod-
ified. Our results, however, suggest that the equatorial 4-hydroxy
contributes to binding, maybe by hydrogen bonding, as the change
of the configuration at C-4 also leads to a decreased affinity. Fur-
thermore, a steric clash of the methyl group in 19 could be respon-
sible for a further reduction of affinity.
O
O
F
R2
OH
COONa
F
N
H
OH
Cl
Compd R1
R2
KD
kon
koff
(sꢀ1
t1/2
(s)
(
l
M)
(Mꢀ1 sꢀ1
)
)
In case of antagonists 16a,b and 16d–h, a pronounced drop in
the binding affinity or even a complete abolishment of binding
was observed. In contrast, 16c showed the same binding affinity
as reference compound 4. The loss in potency for 16a,b and 16d–
h is probably the consequence of the rigid geometry of ester and
carbamate substituents, leading to a steric clash with the protein,
whereas the benzyl ether in 16c seems to adapt a favorable spatial
arrangement compensating the impact of the 4-hydroxy to binding
affinity.
Beside the decreased binding affinity, no substantial improve-
ment of the kinetic properties could be achieved. All compounds
showed fast dissociation rate constants, leading to residence times
in the range of seconds (Table 4). Despite the increased lipophilic-
ity, even 16c does not show an extended dissociation half-life.
421
14
16a
16b
16c
OH
H
OAc
OBz
OBn
O
H
H
H
H
H
2.0
1.5 ꢂ 105
2.8 ꢂ 104
4.6 ꢂ 104
1.6 ꢂ 104
1.6 ꢂ 104
0.54
0.28
0.52
0.41
0.33
1.2
2.5
1.3
1.7
2.1
11.4
11.5
26.0
2.1
2.7 ꢂ 104
7.00
0.1
NHPh
16d
16e
H
H
257
114
O
O
Ph
5.3 ꢂ 106
>100
<0.01
N
H
O
O
S
16f
H
H
15.6
n.b.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
N
H
O
O
S
16g
N
H
O
O
O
16h
H
49.4
2.2 ꢂ 104
1.10
0.6
3. Conclusion
N
H
O
Me
H
19
22
OH 30.0
OH 9.0
2.4 ꢂ 10ꢀ4
3.6 ꢂ 10ꢀ4
0.53
0.33
1.3
2.1
A small library of MAG antagonists modified in the 4-position of
the Neu5Ac scaffold was synthesized with the goal to improve the
half-life of the antagonist-MAG complex. Although all modifica-
n.d., not determined; n.b., not binding.

S. Mesch et al. / Carbohydrate Research 345 (2010) 1348–1359
1353
tions in the 4-position were not successful, the investigation of
modifications in the 5-position led to a reduction of the dissocia-
tion rate constant koff, although only by a factor of 2. In conclusion,
the prolongation of the residence time remains a challenge and
will be a critical issue in further studies on the kinetic properties
of glycomimetics.
of the reaction, IRA-93 was added and stirring was continued for
30 min. After filtration, the solvent was evaporated and the pure
product 10 (13 mg, 60%) was obtained by chromatography on silica
gel (1% gradient of MeOH in DCM). 1H NMR (500 MHz, CD3OD): d
1.33, 1.51 (2s, 6H, 2 C(CH3)2), 1.75 (t, J = 12.4 Hz, 1H, H-3a), 1.99
(s, 3H, NHAc), 2.67 (dd, J = 4.3, 12.6 Hz, 1H, H-3b), 3.56–3.65 (m,
1H, H-4), 3.83 (s, 3H, OMe), 3.86–4.00 (m, 3H, H-5, H-6, H-9a),
4.03 (dd, J = 3.1, 14.1 Hz, 1H, H-9b), 4.26 (d, J = 6.8 Hz, 1H, H-7),
4.48 (ddd, J = 3.3, 7.1, 8.7 Hz, 1H, H-8), 4.56–4.69 (m, 1H, CH2Ar),
4.96 (B of AB, J = 11.6 Hz, 1H, CH2Ar), 7.09–7.27 (m, 4H, NH, CHar),
7.46, 7.83 (AA0, BB0 of AA0BB0, J = 8.6 Hz, 4H, CHar); 13C NMR
(126 MHz, CD3OD): d 23.0 (NHAc), 25.8, 27.1 (C(CH3)2), 41.6, 41.8
(2C, C-3, C-9), 53.2 (OMe), 54.3 (C-5), 61.0 (CH2Ar), 68.4 (C-4),
74.3 (C-6), 75.8 (C-7), 77.7 (C-8), 100.5 (C-2), 110.2 (C(CH3)2),
117.9, 118.0, 125.4, 126.7, 129.7, 130.1, 130.2, 134.3, 138.7 (12C,
C-Ar), 169.3, 170.4, 173.9 (3CO). ESI-MS calcd for C29H33ClF2N2O9
[M+Na]+: 649.18; found m/z 649.15.
4. Experimental
4.1. General methods
NMR spectra were recorded on a Bruker Avance DMX-500
(500 MHz) spectrometer. Assignment of 1H and 13C NMR spectra
was achieved using 2D methods (COSY, HSQC, TOCSY, ROESY,
and NOESY). Chemical shifts are expressed in ppm using residual
CHCl3, CHD2OD, CHD2CN, and HDO as references. Optical rotations
were measured using Perkin–Elmer Polarimeters 241 and 341. MS
analyses were carried out using a Waters Micromass ZQ Detector
system. The spectra were recorded in positive or negative ESI
mode. Reactions were monitored by TLC using glass plates coated
with Silica Gel 60 F254 (Merck) and visualized by using UV light
and/or by charring with a molybdate solution (a 0.02 M solution
of ammonium cerium sulfate dihydrate and ammonium molybdate
tetrahydrate in aq 10% H2SO4). Column chromatography was per-
formed on silica gel (Fluka, 40–60 mesh). MeOH was dried by heat-
ing at reflux with sodium methoxide and distilled immediately
before use. Pyridine was freshly distilled under argon over CaH2.
Dichloromethane (DCM), dichloroethane (DCE), acetonitrile
(MeCN), toluene, and benzene were dried by filtration over Al2O3
(Fluka, type 5016 A basic). Molecular sieves (3 or 4 Å) were acti-
vated in vacuo at 500 °C for 2 h immediately before use. Com-
pounds 6–8 were prepared according to a published procedure.21,36
4.2.3. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-7,8-O-isopropylidene-
glycero- -galacto-2-nonulopyranosid]onate (11)
Compound 10 (20.0 mg, 30 mol) was dissolved in MeOH
D-
a-D
l
(2 mL) and 10% aq NaOH (0.1 mL) was added. After 2 h, the solution
was neutralized with 7% aq HCl. Then the solvent was evaporated
and the pure product 11 (13 mg, 42%) was obtained by chromatog-
raphy on RP-8 (10% of MeOH in water). ½a D20
ꢀ33.4 (c 0.37, MeOH);
ꢃ
1H NMR (500 MHz, CD3OD): d 1.27, 1.41 (2s, 6H, 2 (CH3)2), 1.65 (t,
J = 12.0 Hz, 1H, H-3a), 1.92 (s, 3H, NHAc), 2.75 (dd, J = 4.3, 12.0 Hz,
1H, H-3b), 3.57 (ddd, J = 4.3, 10.3, 12.0 Hz, 1H, H-4), 3.82–3.99 (m,
3H, H-5, H-9a, H-9b), 4.11 (d, J = 10.3 Hz, 1H, H-6), 4.18 (d,
J = 6.8 Hz, 1H, H-7), 4.35 (dd, J = 7.1, 12.5 Hz, 1H, H-8), 4.61, 4.89
(A, B of AB, J = 10.6 Hz, 2H, CH2Ar), 7.00–7.16 (m, 2H, CHar), 7.30
(t, J = 6.8 Hz, 1H, CHar), 7.40, 7.91 (AA0, BB0 of AA0BB0, J = 8.6 Hz,
4H, CHar); 13C NMR (126 MHz, CD3OD): d 22.9 (NHAc), 25.4, 27.4
(C(CH3)2), 41.9, 42.1 (2C, C-3, C-9), 54.7 (C-5), 60.3 (CH2Ar), 69.75
(C-4), 73.1 (C-6), 75.8 (C-7), 76.5 (C-8), 102.8 (C-2), 109.3
(C(CH3)2), 117.0, 117.1, 125.2, 126.5, 129.6, 130.6, 134.1, 138.6
(12C, C-Ar), 169.5, 173.9, 174.4 (3CO). ESI-MS calcd for
4.2. Synthesis and characterization of compounds 9–22
4.2.1. Methyl [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-
nonulopyranosid]onate (9)
D-glycero-a-D-galacto-2-
C
28H30ClF2N2NaO9 [M+H]+: 634.15; found m/z 635.16.
Compound 821 (217 mg, 42.0 mmol) was dissolved in dry MeOH
(8.0 mL) and treated with methanolic NaOMe (1 M, 1.0 mL) for 2 h.
The reaction mixture was neutralized with Amberlyst 15, filtered
over a pad of Celite, and the Celite was washed thoroughly with
MeOH. The solvent was evaporated under reduced pressure and
the crude product was purified by chromatography on silica gel
(1% gradient of MeOH in DCM) to yield 9 (90.0 mg, 61%) as a white
foam. 1H NMR (500 MHz, CD3OD) d 1.80 (t, J = 12.3 Hz, 1H, H-3a),
1.97 (s, 3H, NHAc), 2.72 (dd, J = 4.5, 12.8 Hz, 1H, H-3b), 3.45 (d,
J = 8.8 Hz, 1H, H-7), 3.55 (dd, J = 7.3, 13.8 Hz, 1H, H-9a), 3.61–
3.74 (m, 2H, H-4, H-6), 3.75–3.90 (m, 5H, OMe, H-5, H-9), 4.04
(td, J = 3.2, 8.8 Hz, 1H, H-8), 4.68, 4.86 (A, B of AB, J = 12.1 Hz, 2H,
CH2Ar), 7.06–7.24 (m, 3H, CHar), 7.46, 7.82 (AA0, BB0 of AA0BB0,
J = 8.4 Hz, 4H, CHar); 13C NMR (126 MHz, CD3OD): d 22.7 (NHAc),
41.6 (C-3), 45.1 (C-9), 53.4 (OMe), 53.8 (C-5), 68.5 (C-4), 70.8 (C-
8), 72.1 (C-7), 74.9 (C-6), 117.8, 125.4, 126.4, 128.0, 129.7, 130.1,
131.3, 133.0, 134.4, 138 (12C, C-Ar), 169.6, 170.6, 175.0 (3CO).
ESI-MS calcd for C26H29ClF2N2O9 [M+Na]+: 609.14; found m/z
609.19.
4.2.4. Methyl [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-7,8-O-isopropylidene-4-O-
(phenoxycarbonothioyl)-
D-glycero-a-D-galacto-2-
nonulopyranosid]onate (12)
Compound 10 (96 mg, 0.15 mmol) was dissolved in dry pyri-
dine/DCM (2/1; 1.5 mL). After cooling to 0 °C phenyl chlorothiofor-
mate (162 lL, 1.20 mmol) was added dropwise. Stirring was
continued for 2 h at rt and then MeOH (2 mL) was added. After
evaporation of the solvents, the residue was dissolved in DCM
(5 mL) and washed with 0.5 M aq CuSO4 (1 mL) and water
(1 mL). The organic layer was dried over Na2SO4, filtered, and con-
centrated. The crude product was purified by chromatography on
silica gel (1% gradient of MeOH in DCM) to yield 12 (14 mg, 83%).
1H NMR (500 MHz, CD3OD): d 1.37, 1.54 (2s, 6H, C(CH3)2), 1.97–
2.00 (m, 4H, NHAc, H-3a), 3.01 (dd, J = 4.6, 12.3 Hz, 1H, H-3b),
3.87 (s, 3H, OMe), 3.96–4.02 (m, 1H, H-9a), 4.05 (dd, J = 3.2,
14.2 Hz, 1H, H-9b), 4.15 (d, J = 10.3 Hz, 1H, H-6), 4.27–4.37 (m,
2H, H-5, H-7), 4.53 (td, J = 3.4, 8.6 Hz, 1H, H-8), 4.66, 5.02 (A, B of
AB, J = 11.5 Hz, 2H, CH2Ar), 5.59 (td, J = 4.6, 12.0 Hz, 1H, H-4),
7.07 (d, J = 8.3 Hz, 2H, CHar), 7.13–7.18 (m, 1H, CHar), 7.21–7.27
(m, 2H, CHar), 7.30 (t, J = 7.4 Hz, 1H, CHar), 7.43 (t, J = 7.9 Hz, 2H,
CHar), 7.48, 7.84 (AA0, BB0 of AA0BB0, J = 8.6 Hz, 4H, CHar); 13C NMR
(126 MHz, CD3OD): d 23.0 (NHAc), 25.8, 27.0 (C(CH3)2), 37.9 (C-
3), 41.7 (C-9), 51.3 (C-5), 53.4 (OMe), 61.3 (CH2Ar), 73.9 (C-6),
75.5 (C-7), 77.7 (C-8), 80.1 (C-4), 100.1 (C-2), 110.4 (C(CH3)2),
118.0, 122.1, 122.9, 125.5, 126.8, 126.9, 127.3, 127.7, 129.7,
4.2.2. Methyl [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-7,8-O-isopropylidene-
glycero- -galacto-2-nonulopyranosid]onate (10)
Compound 9 (20.0 mg, 30 mol) was dissolved in dry MeCN
(250 L) and 2,2-dimethoxypropane (17 L, 0.12 mmol) was
added. After cooling to 0 °C PPTS (3.0 mg, 10 mol) was added.
The reaction mixture was stirred at rt overnight. After completion
D-
a
-D
l
l
l
l

1354
S. Mesch et al. / Carbohydrate Research 345 (2010) 1348–1359
130.1, 130.6, 134.2, 138.8, 152.6, 154.6, 154.9 (18C, C-Ar), 169.3,
3.86–4.07 (m, 2H, H-5, H-9a), 4.15 (dd, J = 4.0, 14.5 Hz, 1H, H-9b),
4.20–4.28 (m, 2H, H-6, H-7), 4.39–4.46 (m, 1H, H-8), 4.63, 4.93 (A,
B of AB, J = 11.9 Hz, 2H, CH2Ar), 5.05–5.17 (m, 1H, H-4), 7.00–7.14
(m, 2H, CHar), 7.16–7.27 (m, 1H, CHar), 7.37, 7.84 (AA0, BB0 of AA0BB0,
J = 7.9 Hz, 4H, CHar); 13C NMR (126 MHz, CD3OD): d 20.9 (OAc), 22.9
(NHAc), 25.8, 27.2 (C(CH3)2), 39.2, 40.2 (C-3, C-9), 52.4 (C-5), 60.4
(CH2Ar), 71.5, 72.7, 75.8, 76.9 (C-4, C-6, C-7, C-8), 101.3 (C-2),
110.1 (C(CH3)2), 117.2, 117.3, 125.3, 126.4, 129.6, 130.5, 138.9,
140.1 (12C, C-Ar), 170.6, 172.3, 173.5, 177.9 (4CO). ESI-MS calcd
for C30H32ClF2N2NaO10 [M+H]+: 677.16; found m/z 677.21.
169.8, 173.5 (3CO), 196.0 (CS). ESI-MS calcd for C36H37ClF2N2O10
S
[M+Na]+: 785.17; found m/z 785.27.
4.2.5. Methyl [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,4,5,9-tetradeoxy-7,8-O-isopropylidene-
D-
glycero- -galacto-2-nonulopyranosid]onate (13)
a-D
Compound 12 (96 mg, 0.13 mmol) was dissolved in dry toluene
(4 mL). After the sequential addition of freshly distilled n-Bu3SnH
(340 lL, 1.3 mmol) and AIBN (29 mg, 0.18 mmol), the reaction
mixture was stirred at 100 °C for 1 h. After evaporation of the sol-
vent, the crude product was purified by chromatography on silica
gel (1% gradient MeOH in DCM) to yield 13 (15.3 mg, 20%) as a
white foam. 1H NMR (500 MHz, CD3OD): d 1.33 (s, 3H, CH3),
1.35–1.42 (m, 1H, H-4a), 1.51 (s, 3H, CH3), 1.81 (td, J = 4.0,
13.8 Hz, 1H, H-3a), 1.94 (s, 3H, NHAc), 2.00–2.07 (m, 1H, H-4b),
2.37 (dt, J = 3.4, 13.1 Hz, 1H, H-3b), 3.81 (s, 3H, OMe), 3.92–4.02
(m, 2H, H-9a, H-9b), 4.02–4.08 (m, 2H, H-5, H-6), 4.26 (d,
J = 7.6 Hz, 1H, H-7), 4.48 (ddd, J = 3.3, 7.0, 8.6 Hz, 1H, H-8), 4.56,
4.96 (A, B of AB, J = 11.6 Hz, 2H, CH2Ar), 7.10–7.20 (m, 3H, CHar),
7.46, 7.83 (AA0, BB0 of AA0BB0, J = 8.7 Hz, 4H, CHar); 13C NMR
(126 MHz, CD3OD): d 22.8 (NHAc), 25.8, 27.1 (C(CH3)2), 27.5 (C-
4), 32.8 (C-3), 41.7 (C-9), 46.2 (C-5), 53.0 (OMe), 61.0 (CH2Ar),
75.9, 76.4, 77.7 (C-6, C-7, C-8), 100.9 (C-2), 110.2 (C(CH3)2),
117.8, 125.4, 126.7, 129.7, 130.1, 134.3, 138.7 (12C, C-Ar), 169.3,
170.8, 172.9 (3CO). ESI-MS calcd for C29H33ClF2N2O8 [M+Na]+:
633.18; found m/z 633.12.
4.2.8. Sodium [(2,3-difluorobenzyl) 5-acetamido-4-O-benzoyl-9-
(4-chlorobenzamido)-3,5,9-trideoxy-7,8-O-isopropylidene-
glycero- -galacto-2-nonulopyranosid]onate (15b)
Compound 11 (10.0 mg, 20 mol) was dissolved in dry pyri-
dine (0.5 mL) at rt. After cooling to 0 °C, benzoic anhydride
(100 mg, 0.44 mmol) and DMAP (1.0 mg, 8.0 mol) were added.
D-
a-D
l
l
The reaction mixture was allowed to reach rt and stirring was
continued for 24 h. After completion of the reaction, the solvent
was removed by co-evaporation with toluene. Then DCM (5 mL)
was added and the organic layer was washed with 0.5 M aq
CuSO4 (2 mL), satd aq NaHCO3 (3 ꢂ 2 mL), and H2O (2 mL). Final-
ly, the crude product was purified by chromatography on silica
gel (10% gradient of MeOH in DCM) to yield 15b (7.1 mg, 61%).
1H NMR (500 MHz, CD3OD): d 1.31, 1.47 (2s, 6H, C(CH3)2), 1.76
(s, 3H, NHAc), 1.82–1.96 (m, 1H, H-3a), 2.93 (dd, J = 4.2,
11.8 Hz, 1H, H-3b), 3.97 (dd, J = 6.0, 13.4 Hz, 1H, H-9a), 4.10–
4.20 (m, 1H, H-9b), 4.25 (d, J = 6.5 Hz, 1H, H-7), 4.27–4.37 (m,
2H, H-5, H-6), 4.46 (dd, J = 6.1, 12.4 Hz, 1H, H-8), 4.67, 4.98 (A,
B of AB, J = 11.7 Hz, 2H, CH2Ar), 5.23–5.40 (m, 1H, H-4), 6.98–
7.15 (m, 2H, CHar), 7.27 (t, J = 6.5 Hz, 1H, CHar), 7.42 (d,
J = 7.9 Hz, 2H, CHar), 7.48 (t, J = 7.4 Hz, 2H, CHar), 7.55 (t,
J = 7.5 Hz, 1H, CHar), 7.88 (d, J = 8.3 Hz, 2H, CHar), 7.92 (d,
J = 7.5 Hz, 2H, CHar); 13C NMR (126 MHz, CD3OD): d 22.8 (NHAc),
25.7, 27.3 (C(CH3)2), 30.8 (C-3), 39.3 (C-9), 42.3 (C-5), 52.0
(CH2Ar), 72.6, 72.9, 75.9, 76.8 (C-4, C-6, C-7, C-8), 110.0 (C-
2), 111.4 (C(CH3)2), 117.3, 129.1, 129.5, 129.6, 130.5, 130.6,
130.7, 131.2, 133.0, 134.3, 135.1 (18C, C-Ar), 172.0 (4C, 4CO).
4.2.6. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,4,5,9-tetradeoxy-D-glycero-a-D-galacto-2-
nonulopyranosid]onate (14)
Compound 13 (15.0 mg, 20 lmol) was dissolved in 80% aq AcOH
(2.5 mL) and heated for 3 h at 60 °C. After TLC showed completion
of the reaction, the reaction mixture was cooled to rt. The pH was
set to 10 by addition of 10% aq NaOH. Stirring was continued for
4 h and then the reaction mixture was neutralized with 7% aq
HCl. After evaporation of the solvents, the crude product was puri-
fied by chromatography on RP-8 (5% gradient of MeOH in water) to
yield 14 (4.5 mg, 34%) as a white solid. ½a D20
ꢃ
ꢀ29.7 (c 0.13, MeOH);
ESI-MS calcd for C
35H34ClF2N2NaO10 [M+Na]+: 739.18; found
m/z 739.27.
1H NMR (500 MHz, D2O): d 1.47 (q, J = 11.2 Hz, 1H, H-4a), 1.68 (td,
J = 3.7, 13.3 Hz, 1H, H-3a), 1.94 (s, 3H, NHAc), 2.00–2.09 (m, 1H, H-
4b), 2.35–2.47 (m, 1H, H-3b), 3.40–3.58 (m, 2H, H-7, H-9a), 3.70–
3.81 (m, 3H, H-6, H-8, H-9b), 3.87 (td, J = 4.0, 11.1 Hz, 1H, H-5),
4.61, 4.79 (A, B of AB, J = 11.8 Hz, 2H, CH2Ar), 7.00–7.27 (m, 3H,
CHar), 7.53, 7.75 (AA0, BB0 of AA0BB0, J = 8.5 Hz, 4H, CHar); 13C NMR
(126 MHz, D2O): d 21.8 (C-4), 26.9 (NHAc), 31.7 (C-3), 44.5 (C-9),
51.7 (C-5), 60.5 (CH2Ar), 68.2, 70.1 (C-7, C-8), 75.8 (C-6), 99.3 (C-
2), 117.0, 124.3, 125.8, 126.5, 128.7, 128.8, 132.1, 137.5 (12C, C-
Ar), 174.2, 186.3 (3C, 3CO). HRMS calcd for C25H26ClF2N2NaO11
[M]+: 601.1141; found m/z 601.1154.
4.2.9. Sodium [(2,3-difluorobenzyl) 5-acetamido-4-O-benzyl-9-
(4-chlorobenzamido)-3,5,9-trideoxy-7,8-O-isopropylidene-
glycero- -galacto-2-nonulopyranosid]onate (15c)
Compound 11 (10 mg, 20 mol) was dissolved in DCM/50% aq
KOH (1/3, 0.5/1.5 mL). After adding 18-crown-6 (1.0 mg) and ben-
zyl bromide (60 L, 0.5 mmol), the reaction mixture was heated
D
-
a-D
l
l
to 60 °C and stirring was continued for 18 h. The solvents were
removed under reduced pressure and the crude product was puri-
fied by chromatography on silica gel (10% gradient of MeOH in
DCM) to yield 15c (6.8 mg, 60%). 1H NMR (500 MHz, CD3OD): d
1.32, 1.47 (2s, 6H, C(CH3)2), 1.64–1.78 (m, 1H, H-3a), 1.94 (s,
3H, NHAc), 3.02 (dd, J = 4.0, 12.0 Hz, 1H, H-3b), 3.56–3.64 (m,
1H, H-4), 3.97 (dd, J = 6.1, 13.3 Hz, 1H, H-9a), 4.00–4.11 (m, 2H,
H-5, H-9b), 4.22 (d, J = 10.5 Hz, 1H, H-6), 4.27 (d, J = 6.7 Hz, 1H,
H-7), 4.44 (dd, J = 6.4, 12.8 Hz, 1H, H-8), 4.49 (A of AB,
J = 12.0 Hz, 1H, CH2Ar), 4.69 (A0, B0 of A0B0, J = 11.9 Hz, 2H, CH2Ar),
4.99 (B of AB, J = 11.8 Hz, 1H, CH2Ar), 7.08–7.19 (m, 3H, CHar),
7.22–7.28 (m, 5H, CHar), 7.46, 7.92 (AA0, BB0 of AA0BB0, J = 8.4 Hz,
4H, CHar); 13C NMR (126 MHz, CD3OD): d 21.5 (NHAc), 24.1,
25.8 (C(CH3)2), 37.8 (C-3), 40.5 (C-9), 51.7 (C-5), 58.8 (CH2Ar),
70.2 (C-6), 71.7 (CH2Ar), 74.4 (C-4), 75.3, 75.4 (C-7, C-8), 100.7
(C-2), 108.1 (C(CH3)2), 115.6, 115.7, 123.8, 124.9, 127.0, 127.1,
127.8, 128.1, 128.9, 129.0, 132.6, 137.1, 138.6 (18C, C-Ar), 167.8,
171.9, 173.4 (3CO). ESI-MS calcd for C35H36ClF2N2NaO9 [MꢀH]ꢀ:
701.21; found m/z 701.52.
4.2.7. Sodium [(2,3-difluorobenzyl) 5-acetamido-4-O-acetyl-9-
(4-chlorobenzamido)-3,5,9-trideoxy-7,8-O-isopropylidene-
glycero- -galacto-2-nonulopyranosid]onate (15a)
Compound 11 (13.2 mg, 20 mol) was dissolved in dry pyridine
(0.5 mL). After cooling to 0 °C, acetic anhydride (0.25 mL,
2.64 mmol) and DMAP (1.0 mg, 8.0 mol) were added. The reaction
D-
a-D
l
l
mixture was allowed to reach rt and stirring was continued for
24 h. After completion of the reaction, the solvent was removed
by co-evaporation with toluene. Then DCM (5 mL) was added
and the organic layer was washed with 0.5 M aq CuSO4 (2 mL), satd
aq NaHCO3 (3 ꢂ 2 mL), and H2O (2 mL). Finally, the crude product
was purified by chromatography on silica gel (10% gradient MeOH
in DCM) to yield 15a (11.2 mg, 80%). 1H NMR (500 MHz, CD3OD): d
1.29, 1.43 (2s, 6H, C(CH3)2), 1.70 (t, J = 11.5 Hz, 1H, H-3a), 1.86 (s,
3H, NHAc), 1.93 (s, 3H, OAc), 2.75 (dd, J = 4.5, 11.5 Hz, 1H, H-3b),

S. Mesch et al. / Carbohydrate Research 345 (2010) 1348–1359
1355
4.2.10. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-7,8-O-isopropylidene-4-O-
J = 4.3, 12.8 Hz, 1H, H-3b), 3.48–3.63 (m, 1H, H-9a), 3.81–3.94 (m,
1H, H-9b), 4.10 (d, J = 10.4 Hz, 1H, H-7), 4.27–4.36 (m, 3H, H-5,
H-6, H-8), 4.58, 4.70 (A, B of AB, J = 11.6 Hz, 2H, CH2Ar), 5.15–
5.29 (m, 1H, H-4), 6.93 (d, J = 5.4 Hz, 1H, CHar), 7.02–7.08 (m, 1H,
CHar), 7.09–7.19 (m, 2H, CHar), 7.20–7.25 (m, 2H, CHar), 7.31 (AA0,
BB0 of AA0BB0, J = 8.5 Hz, 4H, CHar); 13C NMR (126 MHz, CD3OD): d
22.8 (NHAc), 25.5, 27.7 (C(CH3)2), 38.3 (C-3), 42.5 (C-9), 52.1 (C-
5), 60.7 (CH2Ar), 70.9, 71.8 (C-4, C-7), 76.1, 76.8 (C-6, C-8), 101.8
(C-2), 108.1 (C(CH3)2), 109.3, 117.5, 117.6, 121.8, 125.3, 125.4,
125.6, 126.0, 129.5, 130.6, 134.1, 138.0, 138.6, 150.5, 152.5 (16C,
C-Ar), 155.2 (NC(O)O), 169.4, 173.6, 173.9 (3CO). ESI-MS calcd for
phenylcarbamoyl-
D
-glycero-
a
-D
-galacto-2-nonulopyranosid]
onate (15d)
Compound 11 (30.0 mg, 50
(1.0 mL). Phenyl isocyanate (24
8.0 mol) were added. The reaction mixture was stirred for 24 h
lmol) was dissolved in dry pyridine
lL, 0.24 mmol) and DMAP (1.0 mg,
l
and then water (0.1 mL) was added. After removal of the solvents
under reduced pressure, the crude product was purified by chro-
matography on silica gel (10% gradient of MeOH in DCM) to yield
15d (10 mg, 29%). 1H NMR (500 MHz, CD3OD): d 1.25, 1.38 (2s,
6H, C(CH3)2), 1.85–1.92 (m, 4H, NHAc, H-3a), 2.76 (dd, J = 4.8,
13.0 Hz, 1H, H-3b), 3.50–3.64 (m, 1H, H-9a), 3.79–3.94 (m, 1H,
H-9b), 4.10 (d, J = 10.4 Hz, 1H, H-7), 4.28–4.35 (m, 3H, H-5, H-6,
H-8), 4.58, 4.70 (A, B of AB, J = 11.9 Hz, 2H, CH2Ar), 5.21 (td,
J = 4.7, 11.0 Hz, 1H, H-4), 6.96 (t, J = 7.3 Hz, 1H, CHar), 7.00–7.13
(m, 2H, CHar), 7.21 (dd, J = 7.7, 8.3 Hz, 2H, CHar), 7.23–7.28 (m,
1H, CHar), 7.31 (AA0 of AA0BB0, J = 8.6 Hz, 2H, CHar), 7.35 (d,
J = 8.0 Hz, 2H, CHar), 7.84 (BB0 of AA0BB0, J = 8.6 Hz, 2H, CHar); 13C
NMR (126 MHz, CD3OD): d 22.9 (NHAc), 25.5, 27.6 (C(CH3)2), 38.3
(C-3), 42.5 (C-9), 52.1 (C-5), 60.7 (CH2Ar), 70.9, 71.6 (C-4, C-7),
76.1, 76.8 (C-6, C-8), 101.8 (C-2), 109.3 (C(CH3)2), 117.5, 119.7,
124.0, 125.6, 126.0, 128.6, 128.7, 129.4, 129.5, 129.8, 130.6,
134.0, 138.6, 140.1, 150.4, 150.5, 152.4 (18C, C-Ar), 155.1 (NC(O)O),
C
33H33ClF2N3O10S [MꢀH]ꢀ: 736.15; found m/z 736.26.
4.2.13. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-7,8-O-isopropylidene-4-O-(2-
(2-thienyl)ethylcarbamoyl)-
nonulopyranosid]onate (15g)
Compound 11 (28.0 mg, 40
(0.5 mL). 2-(2-Thienyl)ethyl isocyanate (27.5 mg, 0.18 mmol) and
DMAP (1.0 mg, 8.0 mol) were added. The reaction mixture was
D-glycero-a-D-galacto-2-
l
mol) was dissolved in dry pyridine
l
stirred for 24 h at 45 °C, then cooled to rt, and treated with water
(0.1 mL). After removal of the solvents under reduced pressure,
the crude product was purified by chromatography on silica gel
(10% gradient of MeOH in DCM) to yield 15g (16 mg, 37%). 1H
NMR (500 MHz, CD3OD): d 1.20, 1.27 (2s, 6H, C(CH3)2), 1.71 (t,
J = 12.3 Hz, 1H, H-3a), 1.81 (s, 3H, NHAc), 2.62 (d, J = 4.8, 12.3 Hz,
1H, H-3b), 2.81–2.91 (m, 2H, CH2), 3.15–3.22 (m, 2H, CH2), 3.95–
4.04 (m, 3H, H-7, H-9a, H-9b), 4.16–4.25 (m, 2H, H-5, H-8), 4.46–
4.55 (m, 1H, H-6), 4.64, 4.88 (A, B of AB, J = 12.8 Hz, 2H, CH2Ar),
4.98–5.08 (m, 1H, H-4), 6.64–6.74 (m, 1H, CHar), 6.75–6.81 (m,
1H, CHar), 6.84–6.93 (m, 1H, CHar), 6.94–7.00 (m, 1H, CHar), 7.07
(d, J = 4.9 Hz, 1H, CHar), 7.10–7.20 (m, 1H, CHar), 7.23, 7.74 (AA0,
BB0 of AA0BB0, J = 8.3 Hz, 4H, CHar); 13C NMR (126 MHz, CD3OD): d
23.0 (NHAc), 25.9, 27.7 (C(CH3)2), 31.0 (CH2), 38.6 (C-3), 43.5 (C-
9), 49.9 (CH2), 52.1 (C-5), 60.8 (CH2Ar), 71.0 (C-7), 75.8 (C-4),
76.1 (C-8), 76.9 (C-6), 101.6 (C-2), 109.4 (C(CH3)2), 117.7, 124.7,
126.3, 127.9, 129.5, 130.4, 130.6, 134.0, 138.7, 139.0, 140.0,
142.4, 142.5, 148.6, 150.5, 152.4 (16 C-Ar), 158.4 (NC(O)O),
169.4, 173.7, 173.9 (3CO). ESI-MS calcd for
C35H35ClF2N3O10
[MꢀH]ꢀ: 730.20; found m/z 730.33.
4.2.11. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-7,8-O-isopropylidene-4-O-
phenylethylcarbamoyl-
nonulopyranosid]onate (15e)
Compound 11 (30.0 mg, 50
(1.5 mL). Phenylethyl isocyanate (50
(3.0 mg, 24.0 mol), and NEt3 (20 L) were added. The reaction
D-glycero-a-D-galacto-2-
lmol) was dissolved in dry pyridine
l
L, 0.42 mmol), DMAP
l
l
mixture was stirred at 60 °C for 24 h and then water (0.2 mL)
was added. After removal of the solvents under reduced pressure,
the crude product was purified by chromatography on silica gel
(10% gradient of MeOH in DCM) to yield 15e (7.3 mg, 20%). 1H
NMR (500 MHz, CD3OD): d 1.30, 1.35 (2s, 6H, (CH3)2), 1.79 (t,
J = 12.3 Hz, 1H, H-3a), 1.88 (s, 3H, NHAc), 2.61–2.78 (m, 3H, H-
3b, CH2), 3.18–3.26 (m, 2H, CH2), 3.53–3.70 (m, 1H, H-9a), 3.87
(d, J = 12.1 Hz, 1H, H-9b), 4.07 (d, J = 10.6 Hz, 1H, H-7), 4.18–4.26
(m, 2H, H-5, H-6), 4.27–4.36 (m, 1H, H-8), 4.56, 4.69 (A, B of AB,
J = 11.6 Hz, 2H, CH2Ar), 5.08 (td, J = 4.8, 11.0 Hz, 1H, H-4), 7.01–
7.09 (m, 2H, CHar), 7.14 (d, J = 7.5 Hz, 4H, CHar), 7.19–7.27 (m,
2H, CHar), 7.31, 7.83 (AA0, BB0 of AA0BB0, J = 8.5 Hz, 4H, CHar); 13C
NMR (CD3OD) d 23.1 (NHAc), 25.8, 27.7 (C(CH3)2), 36.8 (CH2),
38.4 (C-3), 42.6 (C-9), 43.4 (CH2), 52.1 (C-5), 60.7 (CH2Ar), 70.9
(C-7), 71.5 (C-4), 75.7 (C-6), 76.8 (C-8), 101.7 (C-2), 109.2
(C(CH3)2), 117.4, 125.5, 125.2, 126.0, 127.4, 128.7, 129.5, 129.9,
130.0, 130.4, 130.6, 134.1, 140.5, 150.5 (18C, C-Ar), 158.2 (COONH),
169.4, 173.5, 173.6 (3CO). ESI-MS calcd for C35H38ClF2N3O10
S
[M+Na]+: 788.19; found m/z 788.42.
4.2.14. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-7,8-O-isopropylidene-4-O-
((2-methylfuran-3-yl)carbamoyl)-D-glycero-a-D-galacto-2-
nonulopyranosid]onate (15h)
Compound 11 (37.0 mg, 50
(0.5 mL). 2-Methylfuran-3-yl isocyanate (22.0 mg, 0.18 mmol) and
DMAP (1.0 mg, 8.0 mol) were added. The reaction mixture was
lmol) was dissolved in dry pyridine
l
stirred for 24 h and then water was added. After removal of the sol-
vents under reduced pressure, the crude product was purified by
chromatography on silica gel (10% gradient of MeOH in DCM) to
yield 15h (11.0 mg, 28%). 1H NMR (500 MHz, CD3OD): d 1.20,
1.33 (2s, 6H, C(CH3)2), 1.72–1.82 (m, 1H, H-3a), 1.85 (s, 3H, NHAc),
2.08 (s, 3H, CH3), 2.68 (dd, J = 4.4, 12.8 Hz, 1H, H-3b), 3.40–3.53 (m,
1H, H-9a), 3.84 (d, J = 12.8 Hz, 1H, H-9b), 4.04 (d, J = 10.2 Hz, 1H, H-
7), 4.20–4.29 (m, 3H, H-5, H-6, H-8), 4.51, 4.63 (A, B of AB,
J = 11.7 Hz, 2H, CH2Ar), 5.11 (td, J = 4.2, 10.7, 11.1 Hz, 1H, H-4),
6.36 (s, 1H, CHar), 6.97–7.20 (m, 4H, CHar), 7.27, 7.78 (AA0, BB0 of
AA0BB0, J = 8.4 Hz, 4H, CHar); 13C NMR (126 MHz, CD3OD): d 11.2
(CH3), 22.9 (NHAc), 25.6, 27.6 (C(CH3)2), 38.3 (C-3), 42.5 (C-9),
52.2 (C-5), 60.7 (CH2Ar), 70.9 (C-7), 71.9 (C-4), 76.1 (C-8), 76.9
(C-6), 101.8 (C-2), 109.3 (C(CH3)2), 111.4, 117.5, 117.6, 120.4,
125.5, 126.0, 126.4, 129.5, 130.6, 134.2, 138.6, 138.9, 140.8, 150.5
(16C, C-Ar), 156.4 (NC(O)O), 169.4, 173.6 (3C, 3CO). ESI-MS calcd
for C34H36ClF2N3O11 [M+Na]+: 758.18; found m/z 758.42.
169.4, 173.5, 173.6 (3CO). ESI-MS calcd for
C37H39ClF2N3O10
[MꢀH]ꢀ: 758.23; found m/z 758.37.
4.2.12. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-7,8-O-isopropylidene-4-O-(3-
thienylcarbamoyl)-D-glycero-a-D-galacto-2-
nonulopyranosid]onate (15f)
Compound 11 (20.0 mg, 30
(0.5 mL). 3-Thienyl isocyanate (12
l
mol) was dissolved in dry pyridine
L, 0.1 mmol) and DMAP
mol) were added. The reaction mixture was stirred
l
(1.0 mg, 8.0
l
for 24 h and then water (0.1 mL) was added. After removal of the
solvents under reduced pressure, the crude product was purified
by chromatography on silica gel (10% gradient of MeOH in DCM)
to yield 15f (8.0 mg, 35%). 1H NMR (500 MHz, CD3OD): d 1.25,
1.38 (2s, 6H, C(CH3)2), 1.87–1.90 (m, 4H, H-3a, NHAc), 2.76 (dd,

1356
S. Mesch et al. / Carbohydrate Research 345 (2010) 1348–1359
4.2.15. Sodium [(2,3-difluorobenzyl) 5-acetamido-4-O-acetyl-9-
water) yielded 16d (1.5 mg, 15%). ½a D20
ꢃ
ꢀ1.0 (c 0.1, MeOH); 1H NMR
(4-chlorobenzamido)-3,5,9-trideoxy-
D
-glycero-
a
-D
-galacto-2-
(500 MHz, CD3OD): d 1.78 (s, 3H, NHAc), 1.88–1.99 (m, 1H, H-3a),
2.68 (d, J = 9.1 Hz, 1H, H-3b), 3.33–3.49 (m, 1H, H-9a), 3.60 (d,
J = 10.7 Hz, 1H, H-9b), 3.74 (d, J = 5.4 Hz, 1H, H-7), 4.14 (d,
J = 2.6 Hz, 1H, H-8), 4.19–4.46 (m, 2H, H-5, H-6), 4.57, 4.79 (A, B
of AB, J = 11.2 Hz, 2H, CH2Ar), 5.26 (td, J = 4.9, 10.7 Hz, 1H, H-4),
6.95 (t, J = 7.2 Hz, 1H, CHar), 7.13 (d, J = 6.0 Hz, 2H, CHar), 7.19 (t,
J = 7.7 Hz, 3H, CHar), 7.28–7.35 (m, 2H, CHar), 7.41, 7.82 (AA0, BB0
of AA0BB0, J = 8.3 Hz, 4H, CHar); 13C NMR (126 MHz, CD3OD): d
22.8 (NHAc), 38.9 (C-3), 44.2 (C-9), 51.2 (C-5), 60.4 (CH2Ar), 69.4
(C-7), 70.8 (C-4), 72.5 (C-8), 73.3 (C-6), 103.0 (C-2), 117.6, 117.7,
119.8, 124.2, 125.6, 126.3, 128.1, 129.4, 129.8, 130.3, 133.4,
139.1, 139.9, 150.6 (18C, C-Ar), 154.9 (NC(O)O), 169.9, 174.3,
177.6 (3CO). ESI-MS calcd for C32H32ClF2N3O10 [M+Na]+: 714.16;
found m/z 714.30.
nonulopyranosid]onate (16a)
Compound 15a (11.0 mg, 20 lmol) was dissolved in 80% aq
AcOH (1.5 mL) and stirred for 3 h at 60 °C. Then the reaction mix-
ture was cooled to rt and neutralized with 10% aq NaOH. After re-
moval of the solvents under reduced pressure the crude product
was purified on RP-18 (10% gradient of MeOH in water) to yield
16a (8.5 mg, 80%). ½a D20
ꢃ
ꢀ32.8 (c 0.24, MeOH); 1H NMR (500 MHz,
D2O): d 1.79 (t, J = 12.1 Hz, 1H, H-3a), 1.91 (s, 3H, NHAc), 2.03 (s,
3H, OAc), 2.75 (dd, J = 4.8, 12.4 Hz, 1H, H-3b), 3.42 (dd, J = 7.9,
14.2 Hz, 1H, H-9a), 3.52 (d, J = 10.0 Hz, 1H, H-7), 3.69–3.80 (m,
2H, H-8, H-9b), 3.91 (d, J = 11.7 Hz, 1H, H-6), 4.04 (t, J = 10.3 Hz,
1H, H-5), 4.62, 4.87 (A, B of AB, J = 11.8 Hz, 2H, CH2Ar), 4.91 (td,
J = 4.9, 11.5 Hz, 1H, H-4), 7.08 (dt, J = 8.4, 13.1 Hz, 2H, CHar), 7.17
(t, J = 6.7 Hz, 1H, CHar), 7.49, 7.72 (AA0, BB0 of AA0BB0, J = 8.5 Hz,
4H, CHar); 13C NMR (126 MHz, D2O): d 20.2 (OAc), 21.8 (NHAc),
37.3 (C-3), 42.6 (C-9), 49.3 (C-5), 60.6 (CH2Ar), 69.6, 70.2, 70.8,
72.1 (C-4, C-6, C-7, C-8), 101.1 (C-2), 117.0, 124.3, 125.8, 126.5,
128.6, 128.8, 132.1, 137.5, 138.0, 147.5, 149.0, 149.5 (12 C-Ar),
167.0, 172.8, 173.1, 174.5 (4CO). HRMS calcd for C27H29ClF2N2O9
[M+Na]+: 637.1379; found m/z 637.1362.
4.2.19. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-4-O-phenylethylcarbamoyl-
glycero- -galacto-2-nonulopyranosid]onate (16e)
Prepared from 15e (7.3 mg, 10 mol) according to the procedure
D-
a-D
l
described for 16a. Purification on RP-18 (10% gradient of MeOH in
water) yielded 16e (1.5 mg, 20%). ½a D20
ꢃ
ꢀ5.6 (c 0.11, MeOH); 1H
NMR (500 MHz, CD3OD): d 1.71–1.83 (m, 1H, H-3a), 1.87 (s, 3H,
NHAc), 2.38–2.51 (m, 1H, H-3b), 2.53–2.68 (m, 2H, CH2), 3.05–3.18
(m, 2H, CH2), 3.31–3.47 (m, 2H, H-8, H-9a), 3.48–3.65 (m, 2H, H-7,
H-9b), 4.05–4.29 (m, 2H, H-5, H-6), 4.46 (A, B of AB, J = 11.4 Hz, 2H,
CH2Ar), 5.04 (dt, J = 5.3, 10.5, 11.0 Hz, 1H, H-4), 6.93–7.19 (m, 8H,
CHar), 7.32, 7.72 (AA0, BB0 of AA0BB0, J = 8.2 Hz, 4H, CHar); 13C NMR
(126 MHz, CD3OD): d 22.8 (NHAc), 37.0 (C-3), 39.0, 43.4 (2 CH2),
44.6 (C-9), 51.2 (C-5), 60.4 (CH2Ar), 70.0 (C-4), 70.9, 72.1 (C-7, C-8),
72.3 (C-6), 102.8 (C-2), 117.6, 125.3, 126.3, 126.6, 127.2, 127.3,
129.5, 129.8, 130.2, 130.3, 133.7, 134.2, 138.7, 139.0, 140.4, 152.5
(18C, C-Ar), 158.0 (NC(O)O), 169.9, 174.0, 175.9 (3CO). HRMS calcd
for C34H36ClF2N3O10 [M+Na]+: 764.1774; found m/z 764.1791.
4.2.16. Sodium [(2,3-difluorobenzyl) 5-acetamido-4-O-benzoyl-
9-(4-chlorobenzamido)-3,5,9-trideoxy-D-glycero-a-D-galacto-2-
nonulopyranosid]onate (16b)
Prepared from 15b (7.1 mg, 10 lmol) according to the procedure
described for 16a. Purification on RP-18 (10% gradient of MeOH in
water) yielded 16b (4.1 mg, 60%). ½a D20
ꢃ
ꢀ7.4 (c 0.28, MeOH); 1H
NMR (500 MHz, D2O): d 1.80 (s, 3H, NHAc), 1.96 (t, J = 12.0 Hz, 1H,
H-3a), 2.90 (dd, J = 4.9, 12.4 Hz, 1H, H-3b), 3.43 (dd, J = 8.0, 14.3 Hz,
1H, H-9a), 3.56 (d, J = 8.7 Hz, 1H, H-7), 3.73–3.83 (m, 2H, H-8, H-
9b), 4.00 (dd, J = 1.8, 10.6 Hz, 1H, H-6), 4.24 (t, J = 10.3 Hz, 1H, H-5),
4.66, 4.81 (A, B of AB, J = 11.9 Hz, 2H, CH2Ar), 5.13 (ddd, J = 4.8,
10.2, 11.6 Hz, 1H, H-4), 7.01–7.17 (m, 2H, CHar), 7.20 (t, J = 6.9 Hz,
1H, CHar), 7.46–7.56 (m, 4H, CHar), 7.65 (t, J = 7.5 Hz, 1H, CHar),
7.74, 7.96 (AA0, BB0 of AA0BB0, J = 8.6 Hz, 4H, CHar); 13C NMR
(126 MHz, D2O): d 21.7 (NHAc), 37.4 (C-3), 42.6 (C-9), 49.4 (C-5),
60.7 (CH2Ar), 69.6 (C-7), 70.2 (C-8), 71.7 (C-4), 72.2 (C-6), 100.0 (C-
2), 117.0, 117.2, 125.8, 128.7, 128.8, 128.9, 129.4, 132.1, 133.9,
137.5 (18C, C-Ar), 172.9, 174.3 (4C, 4CO). HRMS calcd for
4.2.20. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-4-O-(3-thienylcarbamoyl)-
glycero- -galacto-2-nonulopyranosid]onate (16f)
Prepared from 15f (8.0 mg, 10 mol) according to the procedure
D-
a-D
l
described for 16a. Purification on RP-18 (10% gradient of MeOH in
water) yielded 16f (2.1 mg, 31%). ½a D20
ꢃ
ꢀ17.8 (c 0.5, MeOH); 1H
C
32H30ClF2N2NaO10 [M+Na]+: 721.1345; found m/z 721.1353.
NMR (500 MHz, CD3OD): d 1.76 (t, J = 11.9 Hz, 1H, H-3a), 1.88 (s,
3H, NHAc), 3.02 (dd, J = 5.0, 12.1 Hz, 1H, H-3b), 3.44 (dd, J = 1.7,
9.0 Hz, 1H, H-7), 3.48 (dd, J = 7.7, 13.7 Hz, 1H, H-9a), 3.76 (dd,
J = 3.1, 13.7 Hz, 1H, H-9b), 3.84 (dd, J = 1.3, 10.5 Hz, 1H, H-6),
3.97–4.08 (m, 2H, H-5, H-8), 4.66, 4.92 (A, B of AB, J = 12.2 Hz,
2H, CH2Ar), 4.98 (td, J = 5.0, 11.0 Hz, 1H, H-4), 6.94 (d, J = 4.9 Hz,
1H, CHar), 7.04–7.11 (m, 2H, CHar), 7.17 (s, 1H, CHar), 7.23 (dd,
J = 3.2, 5.1 Hz, 1H, CHar), 7.25–7.32 (m, 1H, CHar), 7.43, 7.80 (AA0,
BB0 of AA0BB0, J = 8.6 Hz, 4H, CHar); 13C NMR (126 MHz, CD3OD): d
22.6 (NHAc), 39.7 (C-3), 44.4 (C-9), 51.7 (C-5), 60.3 (CH2Ar), 71.4
(C-8), 72.2 (C-7), 72.5 (C-4), 74.0 (C-6), 108.0 (C-2), 108.1, 117.0,
121.8, 122.4, 125.2, 125.4, 126.3, 129.7, 130.1, 134.4, 138.6 (16C,
C-Ar), 152.4 (NC(O)O), 174.9 (3C, 3CO). HRMS calcd for
4.2.17. Sodium [(2,3-difluorobenzyl) 5-acetamido-4-O-benzyl-9-
(4-chlorobenzamido)-3,5,9-trideoxy-D-glycero-a-D-galacto-2-
nonulopyranosid]onate (16c)
Prepared from 15c (6.8 mg, 10 lmol) according to the procedure
described for 16a. Purification on RP-18 (10% gradient of MeOH in
water) yielded 16c (2.0 mg, 36%). ½a D20
ꢃ
ꢀ8.4 (c 0.16, MeOH); 1H
NMR (500 MHz, D2O): d 1.55 (t, J = 12.0 Hz, 1H, H-3a), 1.77 (s, 3H,
NHAc), 2.86 (dd, J = 4.6, 12.4 Hz, 1H, H-3b), 3.27–3.39 (m, 2H, H-7,
H-9a), 3.42–3.51 (m, 1H, H-4), 3.60–3.69 (m, 3H, H-6, H-8, H-9b),
3.79 (t, J = 10.2 Hz, 1H, H-5), 4.39, 4.55 (A, B of AB, J = 11.8 Hz, 2H,
CH2Ar), 4.61, 4.70 (A0, B0 of A0B0, J = 11.6 Hz, 2H, CH2Ar), 7.00–7.12
(m, 3H, CHar), 7.20–7.35 (m, 5H, CHar), 7.41, 7.62 (AA0, BB0 of AA0BB0,
J = 8.6 Hz, 4H, CHar); 13C NMR (126 MHz, D2O): d 21.9 (NHAc), 37.8
(C-3), 49.5 (C-9), 50.0 (C-5), 69.7, 70.2, 71.2 (C-7, C-8, CH2Ar), 72.6
(C-6), 75.6 (C-4), 101.4 (C-2), 126.0, 128.3, 128.6, 128.7, 128.8,
132.1, 136.7 (18C, C-Ar), 170.0, 172.9, 174.5 (3CO). HRMS calcd for
C
30H30ClF2N3O10S [MꢀH]ꢀ: 720.1206; found m/z 720.1209.
4.2.21. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-4-O-(2-(2-
thienyl)ethylcarbamoyl)-
D
-glycero-
a-
D-galacto-2-
C
32H32ClF2N2NaO9 [M+H]+: 685.1741; found m/z 685.1745.
nonulopyranosid]onate (16g)
Prepared from 15g (16.0 mg, 20
lmol) according to the proce-
4.2.18. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-4-O-phenylcarbamoyl-
dure described for 16a. Purification on RP-18 (10% gradient of
D-
MeOH in water) yielded 16g (1.5 mg, 10%). ½a D20
ꢀ5.9 (c 0.18,
ꢃ
glycero-
Prepared from 15d (10 mg, 10
described for 16a. Purification on RP-18 (10% gradient of MeOH in
a-
D
-galacto-2-nonulopyranosid]onate (16d)
MeOH); 1H NMR (500 MHz, CD3OD): d 1.73–1.83 (m, 4H, H-3a,
NHAc), 2.54 (dd, J = 5.0, 13.0 Hz, 1H, H-3b), 2.85 (t, J = 7.0 Hz, 2H,
CH2), 3.16–3.22 (m, 2H, CH2), 3.28 (dd, J = 6.4, 14.0 Hz, 1H, H-9a),
lmol) according to the procedure

S. Mesch et al. / Carbohydrate Research 345 (2010) 1348–1359
1357
3.45 (dd, J = 5.0, 14.0 Hz, 1H, H-9b), 3.66 (d, J = 5.6 Hz, 1H, H-7),
4.00–4.13 (m, 2H, H-5, H-8), 4.20 (d, J = 10.6 Hz, 1H, H-6), 4.50,
4.69 (A, B of AB, J = 11.7 Hz, 2H, CH2Ar), 5.07 (td, J = 4.9, 10.9 Hz,
1H, H-4), 6.71 (d, J = 3.0 Hz, 1H, CHar), 6.79–6.85 (m, 1H, CHar),
7.05–7.19 (m, 4H, CHar), 7.36, 7.78 (AA0, BB0 of AA0BB0, J = 8.6 Hz,
4H, CHar); 13C NMR (126 MHz, CD3OD): d 22.9 (NHAc), 30.9
(CH2), 43.5 (C-3), 44.0 (C-9), 46.7 (CH2), 51.2 (C-5), 60.5 (CH2Ar),
69.2, 70.6, 72.3 (C-4, C-7, C-8), 73.9 (C-6), 103.0 (C-2), 117.6,
117.7, 124.7, 125.7, 126.1, 126.3, 127.9, 129.8, 130.4, 133.3,
139.2, 142.4 (16C, C-Ar), 157.9 (NC(O)O), 169.8, 174.2, 174.6
(3CO). ESI-MS calcd for C32H34ClF2N3O10S [M+Na]+: 748.14; found
m/z 748.29.
was added dropwise and the pale yellow solution was warmed up
to 0 °C and stirring was continued for 30 min. After cooling to
ꢀ78 °C, a solution of 17 (20.0 mg, 30
lmol) in THF (0.5 mL) was
added. The reaction mixture was stirred for 3 h and then allowed
to warm to 0 °C. After addition of semi-satd aq NH4Cl (0.5 mL),
the reaction mixture was extracted with DCM (5 mL). The com-
bined organic layers were dried over Na2SO4, filtered, and the sol-
vents were evaporated. Afterwards the crude mixture was
dissolved in 80% aq AcOH (1 mL) and stirred for 3 h at 60 °C. After
removal of the solvents under reduced pressure, the pure product
18 was obtained by chromatography on silica gel (1% gradient of
MeOH in DCM) (6.0 mg, 32% + 2.0 mg, 11% (S)-stereoisomer). 1H
NMR (500 MHz, CDCl3): d 1.24 (s, 3H, CH3), 2.07 (s, 3H, NHAc),
2.96 (d, J = 15.0 Hz, 1H, H-3a), 3.35 (d, J = 15.0 Hz, 1H, H-3b), 3.48
(d, J = 8.9 Hz, 1H, H-7), 3.73 (dd, J = 6.9, 13.1 Hz, 1H, H-9a), 3.79
(s, 3H, OMe), 3.82–3.90 (m, 2H, H-6, H-9b), 4.09–4.18 (m, 1H, H-
8), 4.68–4.80 (m, 2H, H-5, CH2Ar), 4.95 (B of AB, J = 11.7 Hz, 1H,
CH2Ar), 6.55 (d, J = 6.6 Hz, 1H, NHAc), 7.00–7.18 (m, 4H, NH, CHar),
7.39, 7.74 (AA0, BB0 of AA0BB0, J = 8.5 Hz, 4H, CHar); 13C NMR
(126 MHz, CDCl3): d 23.0 (NHAc), 29.0 (CH3), 45.2 (C-9), 47.5 (C-
3), 52.8 (C-5), 53.6 (OMe), 69.7 (C-8), 70.5 (C-7), 71.5 (C-6), 98.1
(C-2), 122.8, 128.5, 128.6, 129.0, 129.2 (12C, C-Ar), 178.1 (3C,
3CO). ESI-MS calcd for C30H35ClF2N2O9 [M+Na]+: 663.19; found
m/z 663.05.
4.2.22. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-4-O-((2-methylfuran-3-
yl)carbamoyl)-
(16h)
D
-glycero-
a
-D
-galacto-2-nonulopyranosid]onate
Prepared from 15h (11.0 mg, 10
l
mol) according to the proce-
dure described for 16a. Purification on RP-18 (10% gradient of
MeOH in water) yielded 16h (2.4 mg, 25%). ½a D20
ꢀ2.4 (c 0.37,
ꢃ
MeOH); 1H NMR (500 MHz, CD3OD): d 1.71 (dd, J = 7.9, 11.6 Hz,
1H, H-3a), 1.82 (s, 3H, NHAc), 2.07 (s, 3H, CH3), 2.50 (dd, J = 5.1,
12.3 Hz, 1H, H-3b), 3.31 (d, J = 9.4 Hz, 1H, H-7), 3.44 (dd, J = 6.5,
13.8 Hz, 1H, H-9a), 3.71 (dd, J = 2.6, 13.6 Hz, 1H, H-9b), 3.84–3.97
(m, 1H, H-8), 4.06 (t, J = 11.5 Hz, 1H, H-6), 4.18 (t, J = 10.6 Hz, 1H,
H-5), 4.45, 4.69 (A, B of AB, J = 11.2 Hz, 2H, CH2Ar), 5.11–5.21 (m,
1H, H-4), 6.35 (s, 1H, CHar), 7.00–7.06 (m, 2H, CHar), 7.11 (s, 1H,
CHar), 7.18 (s, 1H, CHar), 7.37, 7.74 (AA0, BB0 of AA0BB0, J = 8.4 Hz,
4H, CHar); 13C NMR (126 MHz, CD3OD): d 11.3 (CH3), 22.9 (NHAc),
39.3 (C-3), 44.1 (C-9), 60.4 (CH2Ar), 67.5, 70.2, 71.0 (C-4, C-7, C-8),
72.0 (C-6), 101.9 (C-2), 111.4, 112.2, 117.2, 125.4, 126.7, 129.7,
130.2, 130.3, 134.1, 138.6, 138.8, 140.8, 143.5 (16C, C-Ar), 156.4
(NC(O)O), 169.8, 176.7, 180.5 (3CO). HRMS calcd for
4.2.25. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-4-methyl-D-glycero-a-D-talo-
nonulopyranosid]onate (19)
Compound 18 (6.0 mg, 10 lmol) was dissolved in THF (1.0 mL)
and LiOH (9.0 mg, 0.4 mmol) in water (1.0 mL) was added. The
mixture was stirred at rt for 4 h and neutralized with 7% aq HCl.
After removal of the solvents under reduced pressure, the pure
product 19 (2.0 mg, 34%) was obtained by chromatography on
RP-8 (5% gradient of MeOH in water) followed by Dowex 50X8
C
31H32ClF2N3O11 [M+Na]+: 718.1592; found m/z 718.1584.
ion-exchange and P2 size exclusion chromatography. ½a D20
ꢀ6.3 (c
ꢃ
4.2.23. Methyl [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-7,8-O-isopropylidene-
manno-2,4-nonudiulopyranosid]onate (17)
0.26, MeOH); 1H NMR (500 MHz, D2O): d 1.19 (s, 3H, CH3), 1.79
(d, J = 14.1 Hz, 1H, H-3a), 2.02 (s, 3H, NHAc), 2.60 (d, J = 14.1 Hz,
1H, H-3b), 3.45 (dd, J = 7.3, 13.6 Hz, 1H, H-9a), 3.50 (dd, J = 1.9,
8.6 Hz, 1H, H-7), 3.65–3.83 (m, 2H, H-8, H-9b), 3.88 (d,
J = 10.6 Hz, 1H, H-5), 4.31 (dd, J = 2.0, 10.6 Hz, 1H, H-6), 4.56,
4.76 (A, B of AB, J = 12.0 Hz, 2H, CH2Ar), 7.03–7.23 (m, 3H, CHar),
7.53, 7.76 (AA0, BB0 of AA0BB0, J = 8.6 Hz, 4H, CHar); 13C NMR
(126 MHz, D2O): d 21.8 (NHAc), 26.0 (CH3), 42.6 (C-9), 44.8 (C-3),
52.1 (C-5), 60.1 (CH2Ar), 70.3, 70.5, 70.6, 71.4 (C-4, C-6, C-7, C-8),
100.4 (C-2), 117.0, 124.3, 125.9, 126.7, 128.7, 128.8, 132.2, 137.5
(12C, C-Ar), 170.0, 174.4, 174.6 (3CO). HRMS calcd for
a-D-
Compound 10 (63.0 mg, 10
(2.5 mL). Pyridinium dichromate (26.0 mg, 70
followed by the addition of acetic anhydride (28
l
mol) was dissolved in dry DCM
mol) was added
L, 0.3 mmol).
l
l
The reaction mixture was stirred at rt for 4 h. After addition of 2-
propanol (1 mL) the mixture was co-evaporated three times with
toluene. Purification by chromatography on silica gel (EtOAc)
yielded 17 (20 mg, 30%) as a white foam. 1H NMR (500 MHz,
CDCl3): d 1.37, 1.52 (2s, 6H, C(CH3)2), 2.17 (s, 3H, NHAc), 2.94 (d,
J = 15.2 Hz, 1H, H-3a), 3.29 (d, J = 15.0 Hz, 1H, H-3b), 3.82 (s, 3H,
OMe), 3.85–3.96 (m, 1H, H-9a), 4.02 (dt, J = 6.4, 13.0 Hz, 1H, H-
9b), 4.27 (d, J = 6.4 Hz, 1H, H-7), 4.38–4.52 (m, 2H, H-5, H-6),
4.53–4.59 (m, 1H, H-8), 4.64, 5.07 (A, B of AB, J = 11.5 Hz, 2H,
CH2Ar), 6.06 (d, J = 6.9 Hz, 1H, NH), 6.97–7.21 (m, 4H, NH, CHar),
7.40, 7.76 (AA0, BB0 of AA0BB0, J = 8.5 Hz, 4H, CHar); 13C NMR
(126 MHz, CDCl3): d 23.1 (NHAc), 25.4, 26.9 (C(CH3)2), 44.3 (C-3),
47.6 (C-9), 53.7 (C-5), 56.3 (OMe), 60.4 (CH2Ar), 73.2 (C-7), 74.6
(C-8), 75.3 (C-6), 99.8 (C-2), 109.1 (C(CH3)2), 117.2, 124.1, 124.2,
125.2, 125.9, 126.0, 128.5, 128.6, 128.8, 132.7, 137.8 (12C, C-Ar),
167.2, 170.7, 173.0, 200.1 (4CO). ESI-MS calcd for C29H31ClF2N2O9
[M+Na]+: 647.16; found m/z 647.13.
C
26H30ClF2N2O9 [M+Na]+: 585.1427; found m/z 585.1439.
4.2.26. Methyl [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-7,8-O-isopropylidene-D-
glycero-
BH3ꢁNH3 (7.0 mg, 0.22 mmol) was dissolved in MeOH (0.5 mL)
at 0 °C followed by the addition of 17 (30.0 mg, 50 mol) in MeOH
a-D-talo-nonulopyranosid]onate (20)
l
(0.5 mL). After stirring for 2 h at 0 °C, the solvent was evaporated
under reduced pressure and the crude product was purified by
chromatography on silica gel (2% gradient of 2-PrOH in DCM/
MeOH 10:1) to yield 20 (11.0 mg, 36%) as a white solid. 1H NMR
(500 MHz, CD3OD): d 1.30, 1.48 (2s, 6H, C(CH3)2), 1.88 (dd, J = 2.0,
13.7 Hz, 1H, H-3a), 1.95 (s, 3H, NHAc), 2.62 (dd, J = 3.9, 13.7 Hz,
1H, H-3b), 4.02 (d, J = 3.8 Hz, 1H, H-4), 4.07 (t, J = 6.2 Hz, 2H, H-
9a, H-9b), 4.13 (dd, J = 2.6, 10.7 Hz, 1H, H-5), 4.20 (d, J = 6.9 Hz,
1H, H-7), 4.37 (d, J = 10.7 Hz, 1H, H-6), 4.43 (A of AB, J = 11.4 Hz,
1H, CH2Ar), 4.47 (tt, J = 4.5, 8.8 Hz, 1H, H-8), 4.90 (B of AB,
J = 11.4 Hz, 1H, CH2Ar), 7.04–7.13 (m, 1H, CHar), 7.13–7.22 (m,
2H, CHar), 7.44, 7.82 (AA0, BB0 of AA0BB0, J = 8.6 Hz, 4H, CHar); 13C
NMR (126 MHz, CD3OD): d 22.7 (NHAc), 25.8, 27.1 (C(CH3)2), 40.8
4.2.24. Methyl [(2,3-difluorobenzyl) 5-acetamido-9-(4-
chlorobenzamido)-3,5,9-trideoxy-4-methyl-D-glycero-a-D-talo-
nonulopyranosid]onate (18)
ZrCl4 (50.0 mg, 0.13 mmol, 4.0 equiv) was dried for 30 min at
30 °C under high vacuum. After addition of dry THF (1.7 mL), the
suspension was heated up to 50 °C for 20 min. Afterwards the col-
orless solution was cooled to ꢀ54 °C. MeLi (1 M in hexane, 0.5 mL)

1358
S. Mesch et al. / Carbohydrate Research 345 (2010) 1348–1359
(C-3), 41.9 (C-9), 50.1 (C-5), 52.7 (OMe), 60.5 (CH2Ar), 66.3 (C-4),
71.5 (C-7), 76.3 (C-6), 77.7 (C-8), 98.9 (C-2), 110.1 (C(CH3)2),
117.8, 125.4, 126.8, 128.3, 129.7, 130.1, 130.2, 134.3, 138.7 (12C,
C-Ar), 169.3, 171.4, 173.1 (3CO). ESI-MS calcd for C29H33Cl2F2N2O9
[M+Na]+: 649.18; found m/z 649.22.
10 mM sodium acetate, pH 5.0, to obtain a concentration of
30 g/mL. This solution was then injected over the activated sur-
face for 10 min at a flow rate of 10 L/min. Protein A densities
l
l
around 4’000 to 5’000 RU were achieved. Flow cells were blocked
with a 10-min injection of 1 M ethanolamine, pH 8.0. For captur-
ing, MAGd1-3-Fc solution (expressed and purified as described32
)
4.2.27. Methyl [(2,3-difluorobenzyl) 5-acetamido-9-(4-
was diluted to a 30–40
wards, MAGd1-3-Fc was injected at a flow rate of 1
10 min. Using HBS-EP, the surface was equilibrated overnight at
a flow rate of 5 L/min, achieving densities around 2000 RU. Ten-
l
g/mL concentration using HBS-EP. After-
chlorobenzamido)-3,5,9-trideoxy-
D
-glycero-
a-
D-talo-
lL/min for
nonulopyranosid]onate (21)
Compound 20 (20.0 mg, 30
lmol) was dissolved in 80% aq AcOH
l
(1.5 mL) and stirred for 3 h at 60 °C. Then, the reaction mixture was
cooled to rt and neutralized with 10% aq NaOH. After evaporation
of the solvents, the crude product was purified by chromatography
on silica gel (10% gradient of MeOH in DCM) to yield 21 (17 mg,
90%) as a colorless oil. 1H NMR (500 MHz, CD3OD): d 1.89–1.97
(m, 4H, H-3a, NHAc), 2.62 (dd, J = 3.4, 13.9 Hz, 1H, H-3b), 3.42
(dd, J = 5.1, 13.8 Hz, 1H, H-6), 3.52 (dd, J = 7.6, 13.8 Hz, 1H, H-9a),
3.70 (s, 3H, OMe), 3.78 (dd, J = 3.3, 13.9 Hz, 1H, H-9b), 4.01–4.11
(m, 3H, H-4, H-5, H-8), 4.35 (d, J = 11.4 Hz, 1H, H-7), 4.45, 4.76
(A, B of AB, J = 11.8 Hz, 2H, CH2Ar), 7.00–7.21 (m, 3H, CHar), 7.41,
7.78 (AA0, BB0 of AA0BB0, J = 8.6 Hz, 4H, CHar); 13C NMR (126 MHz,
CD3OD): d 22.7 (NHAc), 41.1 (C-3), 44.9 (C-9), 49.8 (C-5), 53.2
(OMe), 60.3 (CH2Ar), 67.0 (C-4), 71.3 (C-8), 71.8 (C-7), 72.5 (C-6),
99.0 (C-2), 117.9, 118.0, 125.5, 125.6, 126.7, 128.5, 128.6, 129.8,
130.2, 134.6, 138.8 (12C, C-Ar), 169.6, 172.0, 174.3 (3CO). ESI-MS
calcd for C26H29ClF2N2O9 [M+Na]+: 587.25; found m/z 587.25.
fold dilution series were freshly prepared in eluent buffer immedi-
ately before use (?14, 16a–c, 19, and 22). All binding experiments
were conducted at 25 °C at a flow rate of 20 lL/min. The samples
were injected over 1 min followed by 1 min dissociation. Each
sample was measured with a duplicate of one concentration, using
a randomized concentration order. Several buffer samples were in-
jected before the first concentration, and one blank between each
concentration, which was used for the double blank referencing
during data processing. Double referencing was applied to correct
for bulk effects and other systematic artifacts. Data processing and
equilibrium binding constant determinations were accomplished
with Scrubber (BioLogic Software, Version 1.1 g or 2.0c). Kinetic
data were simultaneously fit using Scrubber 2.0c. For the DMSO as-
say, DMSO (for molecular biology, >99.9%) was purchased from
Fluka. The stock solution of the test compounds (?16d–h) was
prepared in DMSO and was kept in glass vials to eliminate contam-
inations by, for example, softeners. The running buffer was 3%
4.2.28. Sodium [(2,3-difluorobenzyl) 5-acetamido-9-(4-
DMSO in HBS-EP. The surface was equilibrated at a flow of 5 lL/
chlorobenzamido)-3,5,9-trideoxy-
D
-glycero-
a-
D-talo-
min until the baseline was stable. In order to eliminate the influ-
nonulopyranosid]onate (22)
ence of DMSO on the signals, a calibration curve was done. There-
Compound 21 (17.0 mg, 30
l
mol) was dissolved in MeOH
fore, two solutions were prepared (A = 1 mL running buffer + 50
lL
(3 mL) and 10% aq NaOH (0.1 mL) was added. After 2 h the reaction
was neutralized with 7% aq HCl. The solvents were evaporated and
the residue was purified by chromatography on RP-8 (10% gradient
HBS-EP; B = 1 mL running buffer + 1 L DMSO). Solutions A and B
l
were mixed as indicated in Table 5 and used for calibration. DMSO
calibration solutions were injected after five blank injections and
before the sample solutions. The test compounds were diluted be-
fore measuring with HBS-EP to achieve a content of 3% DMSO. The
DMSO calibration was accomplished directly in ScrubberÓ (version
2.0c).
of MeOH in water) to yield 22 (2.0 mg, 34%). ½a D20
ꢀ41.6 (c 0.37,
ꢃ
MeOH); 1H NMR (500 MHz, D2O): d 1.85 (dd, J = 2.9, 14.2 Hz, 1H,
H-3a), 1.96 (s, 3H, NHAc), 2.62 (dd, J = 3.4, 14.2 Hz, 1H, H-3b),
3.44 (dd, J = 7.9, 14.2 Hz, 1H, H-9a), 3.49 (dd, J = 1.8, 8.9 Hz, 1H,
H-7), 3.67–3.79 (m, 2H, H-8, H-9b), 4.00 (dd, J = 2.8, 10.7 Hz, 1H,
H-5), 4.13 (q, J = 2.9 Hz, 1H, H-4), 4.37 (dd, J = 1.8, 10.7 Hz, 1H, H-
6), 4.53, 4.73 (A, B of AB, J = 11.7 Hz, 2H, CH2Ar), 6.97–7.25 (m,
3H, CHar), 7.49, 7.73 (AA0, BB0 of AA0BB0, J = 8.5 Hz, 4H, CHar); 13C
NMR (126 MHz, CD3OD) d 21.8 (NHAc), 39.2 (C-3), 42.6 (C-9),
48.2 (C-5), 60.1 (CH2Ar), 65.8 (C-4), 69.4 (C-6), 70.2, 70.3 (C-7, C-
8), 100.0 (C-2), 117.0, 125.9, 128.7, 128.8, 132.1, 137.5 (12C, C-
Ar), 170.0, 174.5 (3C, 3CO). HRMS calcd for C25H27ClF2N2O9
[M+Na]+: 595.1273; found m/z 595.1276.
4.4. Isothermal titration calorimetry
ITC experiments were performed using a VP-ITC instrument
from MicroCal, Inc. (Northampton, MA). The measurements were
performed at 25 °C. Injections of 5
from a computer controlled 300
l
L ligand solutions were added
l
L microsyringe at an interval of
5 min into the sample cell solution of MAGd1-3-Fc (cell volume
1.4512 mL) with stirring at 307 rpm. A control experiment was
performed, where the identical ligand solutions were injected into
buffer without protein. The enthalpogram showed negligible heat
development, resulting from dilution effects. The assay buffer
was HBS-E (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, pH 7.4).
The concentration of MAGd1-3-Fc was 48.4 lM, and 500 lM antag-
onist was injected. The experimental data were fitted to a theoret-
ical titration curve (one site binding model) using Origin version 7
4.3. Surface plasmon resonance (SPR) analysis
The SPR measurements were performed on a Biacore 3000 sur-
face plasmon resonance-based optical biosensor (Biacore AB, Swe-
den). Sensor chips (CM5), immobilization kits, maintenance
supply, and HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM
EDTA, 0.005% v/v surfactant P20) were purchased from Biacore
AB (HBS-EP ready-to-use; degassed and filtered). CM5 chips were
preconditioned prior to usage by injecting a series of conditioning
software (MicroCal), with
DH (enthalpy change in kcal/mol), KA
(association constant in Mꢀ1), and N (number of binding sites) as
adjustable parameters. The quantity c = KAꢁMt(0), where Mt(0) is
the initial macromolecule concentration, is of importance in titra-
solutions. A flow rate of 50
l
L/min was used and 2 ꢂ 20
lL of
50 mM NaOH, 10 mM HCl, 0.1% SDS, and 100 mM H3PO4 were in-
jected. The carboxy groups on the CM5 chip were activated for
10 min with a 1:1 mixture of 0.1 M N-hydroxysuccinimide (NHS)
and 0.1 M 3-(N,N-dimethylamino)propyl-N-ethylcarbodiimide
Table 5
Calibration solutions
(EDC) at a flow rate of 10 lL/min. Protein A (P6031) was purchased
Calibration
1
2
3
4
5
from Sigma. A sample and a reference surface were prepared
sequentially or in parallel. For immobilizing protein A, a stock solu-
tion (1 mg/mL in 50 mM phosphate buffer, pH 7.0) was diluted in
A (lL)
400
0
300
100
200
200
100
300
0
B (lL)
400

S. Mesch et al. / Carbohydrate Research 345 (2010) 1348–1359
1359
tion microcalorimetry.31 The experiment was performed with a c
value of 340. Thermodynamic parameters were calculated from
Eq. (1),
12. Yang, L. J. S.; Zeller, C. B.; Shaper, N. L.; Kiso; Hasegawa, A.; Shapiro, R. E.;
Schnaar, R. L. PNAS 1996, 93, 814–818.
13. Tang, S.; Shen, Y. J.; DeBellard, M. E.; Mukhopadhyay, G.; Salzer, J. L.; Crocker, P.
R.; Filbin, M. T. J. Cell Biol. 1997, 138, 1355–1366.
14. Vinson, M.; Strijbos, P. J. L. M.; Rowles, A.; Facci, L.; Moore, S. E.; Simmons, D. L.;
Walsh, F. S. J. Biol. Chem. 2001, 276, 20280–20285.
15. Yang, L. J. S.; Lorenzini, I.; Vajn, K.; Mountney, A.; Schramm, L. P.; Schnaar, R. L.
Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 11057–11062.
D
G ¼
D
H ꢀ T
G, DH, and
D
S ¼ RT ln KA ¼ ꢀRT ln KD
ð1Þ
where
D
D
S are the changes in free energy, enthalpy, and
entropy of binding, respectively, T is the absolute temperature, and
R = 1.98 cal/mol/K. For reasons of consistency the values were con-
verted to kJ (1 cal = 4.1868 J).
16. Kelm, S.; Brossmer, R.; Isecke, R.; Gross, H. J.; Strenge, K.; Schauer, R. Eur. J.
Biochem. 1998, 255, 663–672.
17. Shelke, S. V.; Gao, G. P.; Mesch, S.; Gäthje, H.; Kelm, S.; Schwardt, O.; Ernst, B.
Bioorg. Med. Chem. 2007, 15, 4951–4965.
18. Schwizer, D.; Gäthje, H.; Kelm, S.; Porro, M.; Schwardt, O.; Ernst, B. Bioorg. Med.
Chem. 2006, 14, 4944–4957.
4.5. HPLC
19. Schwardt, O.; Gaethje, H.; Vedani, A.; Mesch, S.; Gao, G.; Spreafico, M.; von
Orelli, J.; Kelm, S.; Ernst, B. J. Med. Chem. 2009, 52, 989–1004.
20. Ernst, B.; Magnani, J. L. Nat. Rev. Drug Disc. 2009, 8, 661–677.
21. Mesch, S.; Moser, D.; Strasser, D. S.; Kelm, A.; Cutting, B.; Rossato, G.; Vedani,
A.; Koliwer-Brandl, H.; Wittwer, M.; Rabbani, S.; Schwardt, O.; Kelm, S.; Ernst,
B. J. Med. Chem. 2010, 53, 1597–1615.
22. Swinney, D. J. PharmMed 2008, 22, 23–34.
23. Copeland, R. A.; Pompliano, D. L.; Meek, T. D. Nat. Rev. Drug Disc. 2006, 5, 730–
739.
24. Mehta, P.; Cummings, R. D.; McEver, R. P. J. Biol. Chem. 1998, 273, 32506–
32513.
25. Wild, M. K.; Huang, M. C.; Schulze-Horsel, U.; van der Merwe, P. A.; Vestweber,
D. J. Biol. Chem. 2001, 276, 31602–31612.
The concentration of MAGd1-3-Fc was determined via HPLC
against a standard curve of BSA at 210 nm using a Beckmann Gold
system, with UV detection (210 nm). The column used was Poros
R1/10 10
lm
(100 ꢂ 2 mm, Dr. Maisch HPLC Markensäulen,
po10.r1.s1002, Morvay Analytik GmbH). The running buffers were
A: H2O + 0.1% TFA and B: 90% MeCN + 0.09% TFA. All measurements
were performed at 75 °C, applying a gradient of 20–90% running
buffer B within 20 min at a flow rate of 0.2 mL/min.44
26. Herfurth, L.; Ernst, B.; Öhrlein, R.; Wagner, B.; Magnani, J. L.; Benie, A. J.; Peters,
T. J. Med. Chem. 2005, 48, 6879–6886.
Acknowledgments
27. Kati, W. M.; Montgomery, D.; Carrick, R.; Gubareva, L.; Maring, C.; McDaniel, K.;
Steffy, K.; Molla, A.; Hayden, F.; Kempf, D.; Kohlbrenner, W. Antimicrob. Agents
Chemother. 2002, 46, 1014–1021.
28. Markgren, P. O.; Hamalainen, M.; Danielson, U. H. Anal. Biochem. 2000, 279, 71–
78.
29. Markgren, P. O.; Lindgren, M. T.; Gertow, K.; Karlsson, R.; Hamalainen, M.;
Danielson, U. H. Anal. Biochem. 2001, 291, 207–218.
30. Bhunia, A.; Schwardt, O.; Gaethje, H.; Gao, G.; Kelm, S.; Benie, A. J.; Hricovini,
M.; Peters, T.; Ernst, B. ChemBioChem 2008, 9, 2941–2945.
31. Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L. N. Anal. Biochem. 1989, 179,
131–137.
We are greatly indebted to Dr. Francis Bitsch and Peggy Brunet
Lefeuvre, Novartis, for providing a HPLC method for protein con-
centration determination and for providing the VP-ITC instrument
as well as for advices regarding the experimental setup and analyz-
ing the data. In addition, we would like to thank the Swiss National
Science Foundation (project 200020-120628), the German Ministry
for Education and Research ((BMBF, project 031632A), and the
Tönjes-Vogt Foundation (project XXI) for their support of this
work.
32. Bock, N.; Kelm, S. Binding and Inhibition Assay for Siglecs. In Methods in
Molecular Biology; Brockhausen, I., Ed.; Humana Press: Totowa NJ, 2006; Vol.
347, pp 359–376.
33. Schwarz, F. P.; Ahmed, H.; Bianchet, M. A.; Amzel, L. M.; Vasta, G. R.
Biochemistry 1998, 37, 5867–5877.
References
34. Ladbury, J. E.; Klebe, G.; Feire, E. Nat. Rev. Drug Disc. 2010, 9, 23–27.
35. Regan, J.; Pargellis, C. A.; Cirillo, P. F.; Gilmore, T.; Hickey, E. R.; Peet, G. W.;
Proto, A.; Swinamer, A.; Moss, N. Bioorg. Med. Chem. Lett. 2003, 13, 3101–
3104.
1. Schwab, M. E.; Bandtlow, C. E. Nature 1994, 371, 658–659.
2. Cajal, R. Y. S. Degeneration and Regeneration of the Nervous System; Oxford Univ.
Press: London, 1928.
36. Hasegawa, A.; Ohki, H.; Nagahama, T.; Ishida, H.; Kiso, M. Carbohydr. Res. 1991,
212, 277–281.
37. Kitamura, M.; Isobe, M.; Ichikawa, Y.; Goto, T. J. Am. Chem. Soc. 1984, 106,
3252–3257.
3. Schwab, M. E.; Caroni, P. J. Neurosci. 1988, 8, 2381–2393.
4. Sandvig, A.; Berry, M.; Barrett, L. B.; Butt, A.; Logan, A. GLIA 2004, 46, 225–251.
5. Filbin, M. T. Nature Rev. Neurosci. 2003, 4, 703–713.
6. Quarles, R. H. Neurochem. Res. 2009, 34, 79–86.
38. Yoshida, M.; Ishida, H.; Kiso, M.; Hasegawa, A. Carbohydr. Res. 1996, 280, 331–
338.
7. Kelm, S.; Schauer, R.; Crocker, P. R. J. Glycoconj. 1996, 13, 913–926.
8. Crocker, P. R.; Kelm, S. The Siglec Family of I-type Lectins; Wiley-VCH Verlag
GmBH: Weinheim, Germany, 2000. pp 579–595.
9. Lauren, J.; Hu, F.; Chin, J.; Liao, J.; Airaksinen, M. S.; Strittmatter, S. M. J. Biol.
Chem. 2007, 282, 5715–5725.
10. Robak, L. A.; Venkatesh, K.; Lee, H.; Raiker, S. J.; Duan, Y.; Lee-Osbourne, J.;
Hofer, T.; Mage, R. G.; Rader, C.; Giger, R. J. J. Neurosci. 2009, 29, 5768–5783.
11. Kelm, S.; Pelz, A.; Schauer, R.; Filbin, M. T.; Tang, S.; de Bellard, M. E.; Schnaar,
R. L.; Mahoney, J. A.; Hartnell, A.; Bradfield, P. Curr. Biol. 1994, 4, 965–972.
39. Groves, D. R.; von Itzstein, M. J. Chem. Soc., Perkin Trans. 1 1996, 2817–2821.
40. Hartmann, M.; Christian, M.; Zbiral, E. Liebigs Ann. Chem. 1990, 1, 83–91.
41. Hartmann, M.; Zbiral, E. Liebigs Ann. Chem. 1991, 795–801.
42. Hartmann, M.; Zbiral, E. Monatsh. Chem. 1991, 122, 111–125.
43. Hartmann, M.; Zbiral, E. Monatsh. Chem. 1989, 120, 899–906.
44. Bitsch, F.; Aichholz, R.; Kallen, J.; Geisse, S.; Fournier, B.; Schlaeppi, J. M. Anal.
Biochem. 2003, 323, 139–149.