Examination of the biological role of the α(2→6)-linked sialic acid in gangliosides binding to the Myelin-Associated Glycoprotein (MAG)

Article abstract of DOI:10.1021/jm801058n

The tetrasaccharide 1, a substructure of ganglioside GQ1bα, shows a remarkable affinity for the myelin- associated glycoprotein (MAG) and was therefore selected as starting point for a lead optimization program. In our search for structurally simplified a

Full text of DOI:10.1021/jm801058n

J. Med. Chem. 2009, 52, 989–1004
989
Examination of the Biological Role of the r(2f6)-Linked Sialic Acid in Gangliosides Binding to
the Myelin-Associated Glycoprotein (MAG)
Oliver Schwardt,† Heiko Ga¨thje,‡ Angelo Vedani,† Stefanie Mesch,† Gan-Pan Gao,† Morena Spreafico,† Johannes von Orelli,†
Sørge Kelm,‡ and Beat Ernst*,†
Institute of Molecular Pharmacy, Pharmacenter, UniVersity of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland, Institute for
Physiological Biochemistry, UniVersity Bremen, D-28334 Bremen, Germany
ReceiVed August 25, 2008
The tetrasaccharide 1, a substructure of ganglioside GQ1bR, shows a remarkable affinity for the myelin-
associated glycoprotein (MAG) and was therefore selected as starting point for a lead optimization program.
In our search for structurally simplified and pharmacokinetically improved mimics of 1, modifications of
the core disaccharide, the R(2f3)- and the R(2f6)-linked sialic acid were synthesized. Biphenylmethyl
and (S)-lactate were identified as suitable replacements for the R(2f6)-linked sialic acid. Combined with
a core modification and the earlier found aryl amide substituent in the 9-position of the R(2f3)-linked
sialic acid, high affinity MAG antagonists were identified. All mimics were tested in a competitive target-
based binding assay, providing relative inhibitory potencies (rIP). Compared to the reference tetrasaccharide
1, the rIPs of the most potent antagonists 59 and 60 are enhanced nearly 400-fold. Their KDs determined in
surface plasmon resonance experiments are in the low micromolar range. These results are in semiquantitative
agreement with molecular modeling studies. This new class of glycomimetics will allow to validate the role
of MAG in the axon regeneration process.
Introduction
Unlike the peripheral nervous system (PNS), the injured adult
mammalian central nervous system (CNS) inherently lacks the
capacity for axon regeneration.1 Although neurite outgrowth is
in principle possible,2 it is actively blocked by inhibitor proteins
expressed on residual myelin and on astrocytes recruited to the
site of injury.3 To date, three major inhibitor proteins have been
identified: Nogo A,4 oligodendrocyte myelin glycoprotein
(OMpg),a5 and myelin-associated glycoprotein (MAG).6 These
three proteins all bind to the Nogo receptor (NgR)7 located on
the neuron surface. The complex then formed by the activated
NgR with the neurotrophin receptor p75NTR permits the trans-
duction of the inhibitory signal into the cytosol of the neuron.8
There, the RhoA-ROCK9 cascade is activated, which finally
leads to growth cone collapse (Figure 1).
It was shown that the RhoA-ROCK inhibitory cascade can
also be triggered by the interaction of a complex formed by
MAG and brain gangliosides10 with p75NTR 11 MAG (siglec-4)
.
belongs to the siglec family12 of sialic acid-binding immuno-
globulin-like lectins. The biological role of MAG as an inhibitor
of axonal regrowth after injury was broadly investigated.13
Schnaar10 reported that a limited set of structurally related
Figure 1. MAG, Nogo66, and oligodendrocyte myelin glycoprotein
(OMgp) all bind to the Nogo-66 receptor (NgR). The inhibitory signal
is transduced into the cytosol of the neuron via the coreceptor p75NTR
.
MAG also binds to GT1b with p75NTR as coreceptor and thereby
transduces the inhibitory signal into the cytosol. Intracellularly, the small
GTPase RhoA is activated, which leads to a collapse of the growth
cone (adapted from Filbin et al.3b).
* To whom correspondence should be addressed. Phone: 0041 267 15
51. Fax: 0041 267 15 52. E-mail: beat.ernst@unibas.ch.
†
Institute of Molecular Pharmacy, Pharmacenter, University of Basel.
Institute for Physiological Biochemistry, University Bremen.
‡
gangliosides like GT1b and GQ1bR (Figure 2), known to be
expressed on myelinated neurons in vivo, are functional ligands
for MAG. These gangliosides have been synthesized in prepara-
tive amounts,14 and were therefore available for a structure-
affinity relationship (SAR) study. MAG was found to bind
preferentially to Neu5AcR(2f3)-Galꢀ(1f3)-GalNAc and Neu5-
AcR(2f3)-Galꢀ(1f3)-[Neu5AcR(2f6)]-GalNAc, the terminal
trisaccharide and tetrasaccharide of the brain gangliosides GT1b
and GQ1bR, respectively.10,15,16 The SAR profile was then
further refined by numerous synthetic contributions based on
ganglioside fragments17 and neuraminic acid derivatives.18
a Abbreviations: CHO, Chinese hamster ovary; DCE, 1,2-dichloroethane;
DCM, dichloromethane; DDQ, 2,3-dichloro-5,6-dicyanobenzoquinone;
DMAP, 4-dimethylamino-pyridine; DME, 1,2-dimethoxyethane; DMF, N,N-
dimethylformamide; DMP, 2,2-dimethoxypropane; DMTST, dimethyl(m-
ethylthio)-sulfonium triflate; Gal, galactose; GalNAc, N-acetylgalactosamine;
IgG, immunoglobulin G; KD, dissociation constant; MAG, myelin-associated
glycoprotein; MPM, p-methoxybenzyl; MS, molecular sieves; Neu5Ac,
N-acetylneuraminic acid; NgR, Nogo receptor; NIS, N-iodosuccinimide;
NMR, nuclear magnetic resonance; OMgp, oligodendrocyte myelin glyco-
protein; OSE, 2-(trimethylsilyl)ethoxy; pyr, pyridine; rIP, relative inhibitory
potency; SAR, structure-affinity relationship; STD, saturation transfer
difference; TCA, trichloroacetyl; Tf, trifluoromethanesulfonyl; THF, tet-
rahydrofurane; Ts, p-toluylsulfonyl.
10.1021/jm801058n CCC: $40.75
2009 American Chemical Society
Published on Web 01/28/2009

990 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Schwardt et al.
Figure 2. The brain gangliosides GT1b and GQ1bR.
Figure 3. Relative inhibitory potencies (rIPs) of oligosaccharides 1-4.22,23 rIPs were calculated by dividing the IC50 of the reference compound
1 by the IC50 of the compound of interest. This results in rIPs above 1.00 for compounds that inhibit better than the reference compound and lower
than 1.00 for substances inhibiting weaker. OSE ) 2-(trimethylsilyl)ethoxy.
Although the exact biological role of the MAG/ganglioside
interaction has not been resolved yet, in some systems, axon
regeneration inhibited by MAG could be completely reversed
by sialidase treatment, suggesting that MAG indeed uses
sialidated glycans as its major axonal ligands.19 The excellent
correlation of the degree of neurite outgrowth with the binding
affinities of the tested oligosaccharides further contributes to
the validation of MAG as a therapeutic target.20 Overall, these
findings suggest that potent glycan inhibitors of MAG could
be valuable therapeutics to enhance axon regeneration.
A comparison of the R-series ganglioside GQ1bR with the
closely related GT1b (Figure 2) shows that an additional sialic
acid linked R(2f6) to GalNAc enhances binding affinity to
MAG 10-fold.15b,17b A detailed SAR study revealed that the
hydroxyl groups of the glycerol side chain of the R(2f6)-linked
sialic acid are not required for binding.17b When the R(2f6)-
Neu5Ac was substituted by a sulfate group, MAG-mediated
adhesion was found to be retained, indicating that an important
contribution of this moiety originates from the acid func-
tion.17b-d,21 Finally, gangliosides or therefrom deduced glycans,
which are missing the terminal R(2f3)-linked Neu5Ac, showed
no or only weak interactions with MAG, indicating the
outstanding relevance of this moiety for binding.15b,17a,b,21
The binding properties of the tri- and tetrasaccharides 1-422,23
(Figure 3) toward a soluble MAG-Fc chimera have been
investigated using a hapten inhibition assay.24 The affinities were
measured relative to the reference compound 1, which has a
relative inhibitory potency (rIP) of 1. Interestingly, the replace-
ment of the GalNAc moiety present in the natural epitope 1 by
a Gal residue (f2) had no significant influence on the rIP. A
similar trend was observed for the binding affinities of the
trisaccharides 3 and 4. As expected,15b,17b the tetrasaccharides
1 and 2 showed an improved affinity (approximately 4-fold)
compared to the corresponding trisaccharides 3 and 4.
Results and Discussion
In this study, we present the synthesis and biological
evaluation of mimics of tetrasaccharide 1. For that purpose, aryl
amide modifications in the 9-position of the R(2f3)-linked
Neu5Ac were combined with the replacement of the R(2f6)-
linked Neu5Ac with various noncarbohydrate substitutents.
Modification of the r(2f3)-Linked Neu5Ac. Initially, the
previously identified beneficial effect of aryl amide modifications
in the 9-position of methyl and benzyl sialosides18b,d was applied
to trisaccharide 4 (f 16, Scheme 1).
For the synthesis of 16, the known galactose building block
525 was transformed by standard procedures into the p-
methoxybenzyl-protected acceptor 7. The galactosylation of 7
with donor 826 in the presence of dimethyl(methylthio)-
sulfonium triflate (DMTST)27 yielded disaccharide 9 in 76%.
After deprotection applying Zemple´n conditions (f10), selective
6-O-benzoylation (f11) was accomplished with benzoyl cya-
nide at -40 °C in 67% yield. Sialidation of 11 with donor 1228
using DMTST as promoter gave 13 in 63% with good
stereoselectivity (R:ꢀ ) 3:1). After acetylation, the azido
derivative 14 was converted into benzamide 15 using modified
Staudinger conditions.18d,29 Finally, deprotection yielded test
compound 16 in 74% yield ready for biological testing in the
hapten inhibition assay (Table 1).
Replacement of the r(2f6)-Linked Neu5Ac by Lactates.
In a saturation transfer difference (STD) NMR study, the binding
contribution of the R(2f6)-linked sialic acid moiety has been
determined.30 In the complex of MAG with 1, only one sizable

R(2f6)-Linked Sialic Acid in Gangliosides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 991
Scheme 1a
a (a) p-methoxybenzyl chloride, NaH, DMF, 0°C, 3 h, 61%. (b) 80% aq AcOH, 90°C, 1.5 h, 50%. (c) DMTST,27 DCM, 0 °C, 16 h, 76%. (d) NaOMe/
MeOH, rt, 2 h, 85%. (e) BzCN, MeCN/NEt3 (4:1), -45°C, 1 h, 67%. (f) DMTST,27 MeCN, 0 °C, 16 h, 47% (R-isomer), 16% (ꢀ). (g) Ac2O, pyr, DMAP,
rt, 16 h, 87%. (h) BzCl, Ph3P, DCE, rt, 16 h, 57%. (i) i. DDQ, DCM/H2O, rt, 3 h; ii. NaOMe/MeOH, rt, 5 h, then aq NaOH, rt, 3 h, 74%.
Scheme 2a
a (a) i. Bu2SnO, benzene, 80°C, 3 h; ii. 18, 19 or 20, CsF, DME, rt, 1 d; 21: 83%; 22: 61%; 23: 83%. (b) Bu3SnH, cat. AIBN, benzene, 80°C, 3 h; 24:
72%; 25: 86%; 26: 87%. (c) NaOMe/MeOH, rt, 1 d, then aq NaOH, rt, 7 h; 27: 68%; 28: 87%; 29: 60%.

992 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Schwardt et al.
Scheme 3a
a (a) H2 (1 bar), 10% Pd-C, MeOH, 3 h, 97%. (b) NaOMe/MeOH, rt, 3 h, 99%. (c) PhCH(OMe)2, cat. p-TsOH, MeCN, rt, 18 h, 84%. (d) DMTST,27
MS 3 Å, DCM, 0°C, 2 d, 79%. (e) 80% aq AcOH, 60°C, 3 h, 89%. (f) NaOMe/MeOH, rt, 18 h, then aq NaOH, rt, 6 h, 73%. (g) i. Bu2SnO, benzene, 80
°C, 3 h; ii. 18, 19, or 20, CsF, DME, rt, 1 d; 38: 83%; 39: 72%; 40: 85%. (h) NaOMe/MeOH, rt, 1 d, then aq NaOH, rt, 7 h; 41: 81%; 42: 76%; 43: 96%.
STD effect arising from its N-acetyl group could be detected,
thus confirming the reported moderate contribution of this
moiety to the binding to MAG.18c In addition, from affinity
studies with a MAG mutant as well as from docking studies to
a homology model of MAG, a salt bridge formed by the
carboxylate of the R(2f6)-linked Neu5Ac and Lys67 was
postulated.22 To mimic this salt bridge, the 6-OH of GalNAc
was alkylated with lactic acid derivatives.
The synthesis of the derivatives 27-29 (Scheme 2) started
from trisaccharide 17.22 The regioselective alkylation of the 6-O-
position of the GalNAc-moiety was achieved in a two-step
procedure:31 (i) activation of the 4,6-diol 17 with dibutyltin oxide
to yield a 4,6-O-dibutylstannylene acetal intermediate and (ii)
in situ treatment under basic conditions with the enantiomerically
pure lactic acid triflates (R)-18, (R)-19, or (S)-20.32 Complete
inversion at the lactate stereocenter afforded exclusively (S)-
configured 21 and 22 and (R)-configured 23. The N-trichloro-
acetyl group was then reduced with tributyltin hydride,33
yielding the acetates 24-26. Whereas the 3-phenyllactate
derivative 22 exclusively formed the desired product 25, the
lactates 24 and 26 were both obtained as inseparable 2:1-
mixtures with an unidentified byproduct (presumably an orthoa-
mide). However, the pure mimics 28-29 could chromatographi-
cally be isolated after the final hydrolytic deprotection (Table
1).
Replacement of GalNAc by lyxo-Hexitol. STD NMR
investigations30 revealed only a marginal contribution of the
N-acetate of the GalNAc unit to binding. This observation
suggested the use of the corresponding 1,2-dideoxy-galactose
building block leading to less polar derivatives (Scheme 3).
Catalytic hydrogenation of the commercially available galactal
derivative 30 afforded peracetylated 1,2-dideoxygalactose 31.
Transesterification with sodium methoxide gave 32, which
was transformed into the corresponding benzylidene 33. The
DMTST27-promoted coupling of 33 with glycosyl donor 3434

R(2f6)-Linked Sialic Acid in Gangliosides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 993
Scheme 4a
a (a) Me3N·BH3 (4 eq), AlCl3 (6 eq), H2O (2 eq), THF, rt, 4 h, 74%. (b) NaOMe/MeOH, rt, 17 h, then aq NaOH, rt, 7 h, 68%. (c) i. biphenyl-4-
carbaldehyde, cat. p-TsOH, MeCN, 70 °C, 4 h; ii. Me3N·BH3 (4 eq), AlCl3 (6 eq), H2O (2 eq), THF, rt, 3 h, 51%. (d) NaOMe/MeOH, rt, 16 h, then aq
NaOH, rt, 7 h, 70%.
yielded 35 in 79%. Deprotection (f36), followed by
saponification finally gave trisaccharide 37.
enhances the affinity,18d p-Cl-benzamide 60 was synthesized
as well (Scheme 5).
The lactic acid derivatives 41-43 were obtained by the
previously applied strategy. Activation of diol 36 with dibutyltin
oxide and subsequent treatment with the lactic acid triflates (R)-
18, (R)-19, or (S)-20 in the presence of cesium fluoride yielded
the lactic acid derivatives 38-40 with complete inversion at
the lactate stereocenter. After deprotection, the test compounds
41-43 were isolated in good yields ready for evaluation in the
hapten inhibition assay (Table 1).
Replacement of the r(2f6)-Linked Neu5Ac by Benzyl
Ethers. To explore the already mentioned lipophilic contact of
the N-acetate of the R(2f6)-linked Neu5Ac predicted by STD
NMR,30 the benzyl ether derivatives 45 and 47 were synthesized
(Scheme 4). Starting from 35, reductive opening of the
benzylidene acetal with trimethylaminoborane and aluminum
chloride35 (f 44, 74%) followed by saponification gave the
6-O-benzyl derivative 45 in 68% yield. For the synthesis of the
biphenylmethyl analogue 47, diol 36 was first treated with
biphenyl-4-carbaldehyde in the presence of cat. amounts of
p-TsOH to yield the corresponding acetal intermediate, which
was subsequently reduced to ether 46 (51% yield from 36). Final
deprotection afforded test compound 47 (Table 1).
Starting from acceptor 33, disaccharide 48 was obtained in
87% by galactosylation with donor 8 using NIS/TfOH as
promoter. After removal of the benzylidene protection, diol 49
was reacted with biphenyl-4-carbaldehyde and the acetal
intermediate reduced to ether 50. Subsequent debenzoylation
gave 51 in 82% yield. Acetonide formation (f52), peracety-
lation (f53), and cleavage of the acetal protection afforded 54.
The coupling of 54 and 12 was promoted by NIS/TfOH, yielding
almost exclusively the R-isomer of 55. After acetylation (f
56), the azido group was converted into the benzamides 57 and
58 using modified Staudinger conditions. Finally, deprotection
yielded the test compounds 59 and 60 (Table 1).
Biological Evaluation
Hapten Inhibition Assay. For the biological evaluation of
the synthesized MAG antagonists, the previously reported
inhibition assay24 was applied. Thus, a recombinant protein
consisting of the three N-terminal domains of MAG and the Fc
part of human IgG (Fc-MAGd1-3) was produced by expression
in CHO cells and affinity purification on protein A-agarose.36
The relative inhibitory potencies (rIP) of the test compounds
were determined in microtiter plates coated with covalently
Combined Modifications of the r(2f3)- and the
r(2f6)-Neu5Ac. In the last series of compounds, the two most
successful modifications, namely the introduction of a benza-
mide at the 9-position of the R(2f3)-linked Neu5Ac (see
compound 16, Table 1) and the replacement of the R(2f6)-
linked Neu5Ac by a biphenylmethyl substituent (see compound
47, Table 1) were combined in antagonist 59. Because an
additional p-chloro substituent on the benzamide moiety further
attached sialic acids as competitive ligands for Fc-MAGd1-3
.
By complexing the Fc-part with alkaline phosphatase-labeled
anti-Fc antibodies and measuring the initial velocity of fluo-
rescein release from fluorescein diphosphate, the amount of
bound MAG could be determined. At least three independent
titrations were performed for each compound. To ensure

994 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Schwardt et al.
comparability with earlier results,22 the affinities were measured
relative to the reference compound 122 (Table 1, entry 1).
The biological evaluation of trisaccharide 16 (Table 1, entry
3) revealed an approximately 60-fold improvement in affinity.18b,d
However, the replacement of the R(2f6)-linked sialic acid
moiety in the natural epitope 1 (entry 1) by lactate residues led
to a relevant decrease in affinity for both stereoisomers 27 and
29 (entries 4 and 5). The additional introduction of a lipophilic
phenyl group at C-3 of the (S)-lactic acid moiety (compound
28, entry 6) did not show a measurable effect.
Table 1. Relative Inhibitory Potencies (rIPs) of Oligosaccharide
Mimicsa
In a second approach, the contribution of the N-acetate of
the GalNAc moiety was explored. Its replacement by 1,2-
dideoxy-Gal in trisaccharide 37 (entry 7) raised the affinity close
to the one of the parent tetrasaccharide 1 (rIP 0.76 vs 1.0). When
the similar modification was applied to the pseudo-tetrasaccha-
rides 27-29, improved affinities for the lactic acid derivatives
41 (entry 8) and 43 (entry 9), but not for the phenyllactic acid
derivative 42 (entry 10), were obtained.
For further investigating the lipophilic contact emanating from
the R(2f6)-linked sialic acid moiety,30 benzyl ether 45 (entry
11) and biphenylmethyl ether 47 (entry 12) were synthesized.
Whereas 45 (entry 11) turned out to be less active than the parent
tetrasaccharide 1 (rIP 0.59 vs 1.0), 47 (entry 12) proved to be
superior (rIP 1.25).
In a last step, the affinity enhancing modifications at the site
of the R(2f3)- as well as at the R(2f6)-linked sialic acid were
combined in one molecule leading to mimic 59 (entry 13), which
binding affinity is improved by more than 300-fold compared
to the parent tetrasaccharide 1 (entry 1). Similar to previous
findings,18d the p-chloro substituent on the benzamide in 60
(entry 14) led to a further significant enhancement of the affinity
(rIP: 377 vs 314), making oligosaccharide mimic 60 one of the
most active synthetic MAG ligands described to date.
Surface Plasmon Resonance. The interaction between MAG
and the MAG-antagonists 16, 59, and 60 has been analyzed by
a surface plasmon resonance based biosensor (Biacore) at 25
°C.37 Figure 4 shows the sensorgram for 59, which was used
for the kinetics analysis of the binding process. The curve was
globally fitted to a Langmuir 1:1 binding model employing
standard Biacore software.
Association (kon) and dissociation rate constants (koff) as well
as affinities (KD) were determined (Table 2). The KDs correspond
well with the results obtained in the hapten inhibition assay24
(Table 1). Whereas for trisaccharide 16 a KD of 32 µM was
measured, the introduction of the biphenylmethyl moiety in 59
or 60 improved affinities to the low micromolar level. The
beneficial effect18d of an additional p-chloro substituent on the
benzamide as realized in 60 could also be confirmed.
By resolution of the affinities into the individual association
and dissociation rate constants, it was possible to reveal the
binding kinetics of the three antagonists 16, 59, and 60. The
increase in lipophilicity originating from the introduction of
the biphenylmethyl moiety (59 and 60) led to a substantial
improvement of the association rate constants by a factor of 20
and 30, respectively. Because the three investigated antagonists
show rather similar koffs (0.3-1.05 s-1), the improved affinity
of 59 and 60 results predominantly from enhanced association
rate constants.
a The rIP of each substance was calculated by dividing the IC50 of the
reference compound 122 by the IC50 of the compound of interest. This results
in rIPs above 1.00 for derivatives binding better than 1 and rIPs below
1.00 for compounds with a lower affinity than 1. The previously used
reference compound 422,23 with a 3.3-fold lower affinity, reflecting the
contribution of the R(2f6)-linked sialic acid moiety, is also included in
Table 1 (entry 2).
Molecular Modeling
The homology model for the ligand-binding domain of MAG
(mouse; UniProt ) P20917) was based on the three-dimensional
structure of sialoadhesin (mouse, SIGLEC-1, PDB code )

R(2f6)-Linked Sialic Acid in Gangliosides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 995
Scheme 5a
a (a) NIS/TfOH, DCM, -20 °C, 87%. (b) 80% AcOH, 60°C, 83%. (c) i. biphenyl-4-carbaldehyde, cat. p-TsOH, MeCN; ii. Me3N·BH3 (4 eq), AlCl3 (6
eq), THF, rt, 55%. (d) NaOMe/MeOH, 82%. (e) DMP, p-TsOH, MeCN, 78%. (f) Ac2O, pyr, 94%. (g) 80% AcOH, 80°C, 81%. (h) NIS/TfOH, MeCN,
-40°C, 61%. (i) Ac2O, pyr, 80%. (j) BzCl or p-Cl-BzCl, PPh3, DCE, rt, 16 h; 57: 65%; 58: 75%. (k) NaOMe/MeOH, rt, 3 h, then aq NaOH, rt, 16 h; 59:
79%; 60: 75%.
Figure 4. Biacore sensorgram (A) and fitting curve (B) of compound 59 binding to MAG (for the immobilization see Experimental Section) for
which a kinetics analysis was performed. The Sensorgram was fitted to a Langmuir 1:1 binding model utilizing standard Biacore software in the
global fitting mode. Eight concentrations for 59 of a 2-fold dilution series starting from 50 µM (top) were used. The global fit yielded the following
parameters: KD ) 5.00 × 10-6 M, RUmax ) 19.4, koff ) 1.05 s-1, kon ) 2.1 × 105 M-1s-1. From koff, a half-life of 0.66 s is calculated.
1QFP, 2.8 Å resolution) and generated using the Fugue38 and
Orchestrar39,40 concepts as implemented in Sybyl 7.341 following
a standard protocol. The identity in primary sequence between
the two species is 29.9%. After generation, the homology model
was subjected to a full refinement in aqueous solution using
the AMBER 7.0 software package.42
optimized in aqueous solution by means of the AMBER* force
field.44 Atomic partial charges (MNDO/ESP) were then gener-
ated using MOPAC.45 The ligands were first manually docked
to the binding pocket of the MAG model using the salt-bridge
to Arg118 and the hydrogen bonds to Asn125, Gln126, Thr127,
and Tyr128 as anchor points. Next, the protein-ligand complex
was minimized in aqueous solution and then subjected to a
molecular dynamics equilibration protocol (10 ps at 100 K,
The three-dimensional structures of compounds 1, 59, and
60 were generated using the MacroModel software43 and

996 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Schwardt et al.
Table 2. Dissociation Constants KD from Surface Plasmon Resonance
Experiments (Equilibrium Analysis) Performed at 298 K with
Fc-MAGd1-3 Immobilized on a CM5 Chip
at 293 K during 100 ps (at 0.2 ps intervals). The lowest-energy
binding modes of tetrasaccharide 1 and mimic 60 are compared
in Figure 5.
ligand
Both compounds, 1 and 60, form an important salt bridge
(carboxylate of the R(2f3)-linked Neu5Ac with Arg118), a
relevant hydrogen bridge (backbone carbonyl of Phe 129 with
9-OH and 9-NH, respectively), and a crucial hydrophobic
contact (between the acetamido group of the R(2f3)-linked
Neu5Ac and the side chains of Trp22 and Tyr124). Whereas 1
establishes a second salt bridge between the carboxylate of the
R(2f6)-linked Neu5Ac and Lys67, prominent interactions with
the two hydrophobic pockets near the surface of MAG are
16
61.5
59
314
60
377
rIP
KD, µM
kon, M-1 s-1
koff, s-1
t1/2, s
32.6
5.00
2.83
1.1 × 104
2.2 × 105
3.1 × 105
0.3
1.05
0.8
2.3
0.67
0.87
heating to 310 K during 50 ps, 10 ps at 310 K, cooling during
10 ps to 293 K; 1 ps ) 10-12 s), followed by structure sampling
Figure 5. A homology model of MAG, based on a 3D-structure of sialoadhesin (siglec-1),46 complexed with tetrasaccharide 1 (left) and glycomimetic
60 (right). The images have been generated using the VMD software.47 In both compounds, the carboxylate of the R(2f3)-linked Neu5Ac forms
a salt bridge with Arg118 and hydrophobic interactions are established between its acetamido group and the side chains of Trp22 and Tyr124,
which are part of a small hydrophobic pocket. Both compounds form an important hydrogen bridge with the backbone carbonyl of Phe129; in 1,
with OH in the 9-position of the R(2f3)-linked Neu5Ac and in 60, with the corresponding amide NH. Tetrasaccharide 1 establishes a second salt
bridge between the carboxylate of the R(2f6)-linked Neu5Ac and Lys67. In contrast, antagonist 60 seeks for additional hydrophobic interactions:
(i) the p-chloro benzamide substituent and the side chains of Tyr127 and Glu131 defining a secondary hydrophobic pocket, and (ii) the biphenylmethyl
moiety and the side chains of Tyr60, Tyr69, and Tyr116 lining the main hydrophobic pocket.
Table 3. Semiquantitative Estimation of the Binding Affinitiesa
ligand
KD (exp.)
rIP
Eligand-receptor
Edesolvation
T∆S
Einternal strain
Ebinding
KD (calcd)
1
59
60
>1.0 × 10-3
5.0 × 10-6
2.8 × 10-6
1.0
314
377
-21.4
-23.2
-23.2
+10.0
+6.9
+6.8
+6.3
+4.3
+4.3
+3.6
+4.8
+3.5
-1.5
-7.2
-8.6
7.6 × 10-2
4.3 × 10-6
0.4 × 10-6
a All energies are given in kcal/mol, KD in M. The desolvation energies reflect the partial desolvation of the ligands at the protein surface. Proteinsligand
and internal energies were calculated using the AMBER force field44 as implemented in MacroModel43 and appropriately scaled.
Figure 6. Comparison (in stereo) of the bound conformation and the nearest low-energy conformation in aqueous solution of tetrasaccharide 1 (left) and
the glycomimetic 60 (right). The bound conformation is colored by atoms, the low-energy conformation in aqueous solution is depicted in blue.

R(2f6)-Linked Sialic Acid in Gangliosides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 997
formed by 60: Glu131 and Tyr127 are homing the p-chloro
benzamide substituent and the side chains of Trp59, Tyr60,
Tyr69, and Tyr116 lining the main hydrophobic pocket accom-
modate the biphenylmethyl moiety.
The introduction of the biphenylmethyl substituent in the
6-position of the lyxo-hexitol moiety caused a 5- to 6-fold
improvement of affinity. Interestingly, the major contribution
results from an enhanced association rate. Thus, the kinetic
analysis for 16, 59, and 60 (Figure 4) reveals that kon is changing
from 104 to 3 × 105 M-1 s-1 and koff is rather stable (0.3-1
s-1). For all three antagonists, the corresponding half-lives are
in the range of approximately 1 s. In general, high efficacy of
a ligand in vivo correlates with fast association and slow
dissociation rates.50 The association rate kon is concentration
dependent, therefore slow association rates can be compensated
by high intracellular concentrations. Slow dissociation rates koff,
however, must be achieved by optimizing the contact of ligand
and target. It was shown for HIV-1 protease inhibitors that it is
beneficial to reduce the dissociation rate to a point where the
compound acts close to an irreversible binder (koff 10-4s10-3
s-1), resulting in half-lives ranging from minutes to hours.50
Taking this into account, the structure of the ligands presented
in this study have to be further optimized in order to achieve
more druglike properties.
To assess the feasibility of the bound conformation (as
identified by molecular mechanics and molecular dynamics
simulations), we conducted a conformational search of com-
pounds 1, 59, and 60 in aqueous solution and identified the
lowest conformer featuring a similar folding as the bound state.
For the tetrasaccharide 1, the closest identified conformation is
located only 1.9 kcal/mol above the global minimum; for the
glycomimetics 59 and 60, the corresponding energies are 2.8
and 1.3 kcal/mol, respectively. This suggests that all three
ligands can adopt the bound conformation at very moderate
costs. Comparison of the calculated binding energies with the
experimental binding affinity (Table 3) support the binding
hypothesis identified in silico: the low binding affinity of
tetrasaccharide 1 can be explained by its high desolvation energy
combined with entropic costs; in contrast hereto, the glycomi-
metics 59 and 60 display a higher protein-ligand interaction
combined with lower entropic and desolvation energies which
explains their higher binding affinity (Table 3).
With docking studies of the most active ligand 60 to a
homology model of MAG, the beneficial contribution of the
two lipophilic substituents could be rationalized (Figure 5). Both,
the p-chloro group at the 9-position of R(2f3)-linked sialic acid
and the biphenylmethyl substitutent in the 6-position of the lyxo-
hexitol contribute to binding by hydrophobic contacts. Because
both hydrophobic pockets, especially the one formed by Trp59,
Tyr60, Tyr69, and Tyr116, are quite spacious, improved affinity
may be realized by further modifications of these substitutents.
Conclusions
To investigate the biological role of the sialic acid moi-
eties in the lead tetrasaccharide Neu5AcR(2f3)Galꢀ(1f3)-
[Neu5AcR(2f6)]GalNAc (1), a number of oligosaccharide
mimics were prepared. On the basis of STD NMR data30 and
previous results,18b,d,22 the terminal R(2f3)-linked Neu5Ac was
modified by benzamides at the nonreducing end, the
Galꢀ(1f3)GalNAc core was simplified, and the R(2f6)-linked
sialic acid was replaced by hydrophilic lactate residues or
lipophilic benzyl ethers. The biological evaluation of the mimics
was performed in a competitive target-based assay and by
surface plasmon resonance.
According to docking studies to a homology model of MAG,
the R(2f6)-linked sialic acid moiety is forming a salt bridge
with Lys67. When Lys67 was mutated, a substantial loss in the
affinity of tetrasaccharide 1 resulted.22 The loss in affinity caused
by the replacement of the sialic acid moiety by lactic acid (f27
and 29, entries 4 and 5, Table 1) or phenyllactic acid (f28,
entry 6, Table 1) is probably the result of an insufficient
preorganization of the carboxylate in the bioactive conformation.
Similar modifications for ligands of E-selectin48 and cholera
toxin49 were successful and rationalized by preorganization of
the bound conformation.
The replacement of the GalNAc moiety in trisaccharide 4 by
lyxo-hexitol resulted in a modestly improved affinity (f37, entry
7). On the basis of this new scaffold, the (R)- and (S)-lactic
acid derivatives 41 and 43 (entries 8 and 9) showed affinities
in the range of the parent tetrasaccharide 1. Interestingly, both
stereoisomers, the (S)- and the (R)-lactic acid derivatives 41
and 43, show comparable affinities.
Because STD-NMR investigations indicated a lipophilic
contact for the N-acetate of the R(2f6)-linked sialic acid in
1,30 lipophilic substitutents in the 6-position of the lyxo-hexitol
moiety in 37 were successfully explored (f45, entry 11 and
47, entry 12).
Another important issue to be addressed is the metabolic
stability of the presented oligosaccharide mimics. In general,
the substrate specificity of mammalian sialidases is determined
by the linkage type of the terminal sialic acid residue (2f3,
2f6, or 2f8) and does not depend on the structure of the
underlying oligosaccharide.51 Therefore, it cannot be excluded
that the presented mimics are metabolically cleaved by siali-
dases. Nevertheless, the new class of MAG blockers presented
in this study constitute an important step toward the development
of potent oligosaccharide mimics, although pharmacokinetic
aspects have to be further optimized.
Experimental Section
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). Chemical shifts are expressed in ppm using residual
CHCl3, CHD2OD, and HDO as references. Optical rotations were
measured using Perkin-Elmer polarimeters 241 and 341. Electron
spray ionization mass spectra (ESI-MS) were obtained on a Waters
micromass ZQ. The LC/HRMS analysis were carried out using a
Agilent 1100 LC equipped with a photodiode array detector and a
Micromass QTOF I equipped with a 4 GHz digital-time converter.
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
performed on silica gel (Fluka, 40-60 mesh). Methanol was dried
by refluxing with sodium methoxide and distilled immediately
before use. Pyridine was freshly distilled under argon over CaH2.
Dichloromethane (DCM), dichloroethane (DCE), 1,2-dimethoxy-
ethane (DME), acetonitrile (MeCN), toluene, and benzene were
dried by filtration over Al2O3 (Fluka, type 5016 A basic). Molecular
sieves (3 and 4 Å) were activated in vacuo at 500 °C for 2 h
immediately before use.
Finally, concomitant modifications of the reducing and
nonreducing sugars, each with the best so far identified
substituent, proved the additivity of the beneficial effects. Thus,
both 59 and 60 (entries 13 and 14) showed a more than 300-
fold enhancement in MAG-binding affinity.
The dissociation constants KD obtained by Biacore experi-
ments (Table 2) support the results of the inhibition assay.24

998 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Schwardt et al.
2-(Trimethylsilyl)ethyl (Methyl 5-Acetamido-4,7,8-tri-O-
acetyl-9-azido-3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonulopy-
ranosynate)-(2f3)-(6-O-benzoyl-ꢀ-D-galactopyranosyl)-(1f3)-
2,6-di-O-(4-methoxybenzyl)-ꢀ-D-galactopyranoside (13). A mixture
of 11 (77.0 mg, 97.8 µmol), donor 1228 (98.0 mg, 196 µmol), and
activated powdered MS 3 Å (1.00 g) was stirred in MeCN (5 mL)
at rt for 6 h under argon. Then DMTST (202 mg, 0.783 mmol)
was added at 0 °C. The reaction mixture was stirred at 0 °C for
16 h, diluted with DCM (5 mL), and filtered through a pad of celite.
The celite was washed with DCM (3 × 5 mL), and the combined
filtrates were washed with saturated aq NaHCO3 (2 × 10 mL) and
H2O (10 mL). The organic layer was dried (Na2SO4), filtered, and
concentrated under reduced pressure. The residue was purified by
silica gel chromatography (0.25% gradient MeOH in DCM) to
afford 13 (90.5 mg, 47%) as a white foam (ꢀ-isomer: 30.0 mg,
16%).
20.8, 20.9, 21.2, 23.08 (7C, 7 COCH3), 36.9 (Sia-C3), 49.0 (Sia-
C5), 51.2 (Sia-C9), 53.0, 55.2 (3C, 3 OCH3), 61.2 (Gal′-C6), 67.2
(Gal′-C4), 67.7 (2C, Sia-C7, OCH2), 68.6, 69.1 (Gal-C4, Sia-C8),
69.2 (Gal-C6), 69.5 (Sia-C4), 70.2 (Gal′-C2), 70.4 (Gal′-C5), 71.5
(Gal′-C3), 72.2 (Sia-C6), 72.9 (Gal-C5), 73.2, 74.7 (2 C6H4CH2),
78.3 (Gal-C2), 80.2 (Gal-C3), 96.7 (Sia-C2), 101.0 (Gal′-C1), 103.0
(Gal-C1), 113.5, 113.7, 128.2, 129.2, 129.3, 129.6, 129.7, 133.0,
130.1, 131.1, 159.0, 159.2 (18C, C6H5, 2 C6H4), 165.6, 167.9, 169.7,
170.2, 170.2, 170.4 (9C, 9 CO).
2-(Trimethylsilyl)ethyl (Methyl 5-Acetamido-4,7,8-tri-O-
acetyl-9-benzamido-3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonu-
lopyranosynate)-(2f3)-(2,4-di-O-acetyl-6-O-benzoyl-ꢀ-D-galac-
topyranosyl)-(1f3)-4-O-acetyl-2,6-di-O-(4-methoxybenzyl)-ꢀ-D-
galactopyranoside (15). Compound 14 (79.3 mg, 57.9 µmol) and
benzoylchloride (27.0 µL, 0.232 mmol) were dissolved in DCE (5
mL) and stirred at rt under argon. Ph3P (33.0 mg, 0.127 mmol) in
DCE (1 mL) was added after 5 min. The reaction was stirred at rt
for 16 h and then diluted with DCM (10 mL) and washed with
saturated aq NaHCO3 (2 × 10 mL) and H2O (10 mL). The organic
layer was dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by silica gel chromatography (0.25% gradient
MeOH in DCM) to afford 15 (47.5 mg, 57%) as a colorless foam.
[R]D +2.3, (c 0.90, CHCl3). 1H NMR (500 MHz, CDCl3): δ 0.00
(s, 9H, SiMe3), 0.98-1.07 (m, 2H, SiCH2), 1.70 (t, J ) 12.4 Hz,
1H, Sia-H3a), 1.84, 1.95, 2.00, 2.08, 2.09, 2.10 (7s, 21H, 7 COCH3),
2.58 (dd, J ) 4.6, 12.7 Hz, 1H, Sia-H3b), 2.82 (dt, J ) 3.9, 15.0
Hz, 1H, Sia-H9a), 3.42-3.49 (m, 2H, Gal-H6), 3.53 (dd, J 2.6,
10.8 Hz, 1H, Sia-H6), 3.59-3.63 (m, 3H, Gal-H2, Gal-H5, OCH2-
Ha), 3.71, 3.77, 3.78 (3s, 9H, 3 OCH3), 4.00-4.05 (m, 2H, Gal′-
H5, OCH2-Hb), 4.11 (q, J ) 10.4 Hz, 1H, Sia-H5), 4.18 (dd, J )
7.6, 11.0 Hz, 1H, Gal′-H6a), 4.31-4.38 (m, 2H, Gal′-H6b, Sia-
H9b), 4.36 (d, J ) 7.8 Hz, 1H, Gal-H1), 4.39, 4.42 (A, B of AB,
J ) 11.2 Hz, 2H, C6H4CH2), 4.66, 4.76 (A, B of AB, J ) 10.3 Hz,
2H, C6H4CH2), 4.67 (m, 1H, Gal′-H3), 4.85 (ddd, J ) 4.6, 10.5,
12.2 Hz, 1H, Sia-H4), 4.91 (d, J ) 7.9 Hz, 1H, Gal′-H1), 5.07 (d,
J ) 2.7 Hz, 1H, Gal′-H4), 5.10 (dd, J ) 7.9, 10.1 Hz, 1H, Gal′-
H2), 5.13 (d, J ) 10.4 Hz, 1H, NH), 5.20 (dd, J ) 2.6, 9.2 Hz,
1H, Sia-H7), 5.34 (dt, J ) 3.2, 9.2 Hz, 1H, Sia-H8), 5.39 (d, J )
3.6 Hz, 1H, Gal-H4), 6.82-6.88, 7.23-7.26, 7.31-7.34, 7.39-7.55,
7.77-7.79, 8.01-8.03 (m, 18H, 2 C6H5, 2 C6H4). 13C NMR (125
MHz, CDCl3): δ -1.5 (SiMe3), 18.5 (SiCH2), 20.7, 20.9, 20.9, 21.3,
23.1 (7 COCH3), 37.5 (Sia-C3), 38.3 (Sia-C9), 48.2 (Sia-C5), 53.0,
55.2, 55.2 (3 OCH3), 61.3 (Gal′-C6), 67.3 (Gal′-C4), 67.7 (OCH2),
67.9 (Sia-C7), 68.5 (Sia-C8), 69.3 (Gal-C6), 69.3 (Sia-C4), 69.8
(Gal-C4), 70.3, 70.4 (Gal′-C2, Gal′-C5), 71.7 (Gal′-C3), 72.1 (Sia-
C6), 73.0 (Gal-C5), 73.2, 74.7 (2 C6H4CH2), 78.3 (Gal-C2), 80.1
(Gal-C3), 96.7 (Sia-C2), 101.0 (Gal′-C1), 103.0 (Gal-C1), 113.6,
113.7, 113.8, 126.9, 128.2, 128.4, 128.5, 128.6, 129.3, 129.4, 129.5,
129.7, 129.8, 130.1, 130.1, 131.5, 131.9, 133.0, 134.2, 159.1, 159.2
(24C, 2 C6H5, 2 C6H4), 165.6, 165.7, 167.1, 167.7, 169.6, 170.1,
170.3, 170.7, 170.9, 171.7 (10 CO). Anal. Calcd for C71H90N2O28Si:
C, 58.91; H, 6.27; N, 1.94. Found: C, 58.31; H, 6.24; N, 1.67.
[R]D -3.3, (c 0.91, CHCl3). 1H NMR (500 MHz, CDCl3): δ 0.01
(s, 9H, SiMe3), 1.01-1.04 (m, 2H, SiCH2), 1.90, 2.04, 2.10, 2.13
(4s, 12H, 4 COCH3), 2.11 (m, 1H, Sia-H3a), 2.69 (dd, J ) 4.6,
13.1 Hz, 1H, Sia-H3b), 3.11 (dd, J ) 5.4, 13.6 Hz, 1H, Sia-H9a),
3.44 (dd, J ) 2.6, 13.6 Hz, 1H, Sia-H9b), 3.49-3.59 (m, 3H, Gal-
H5, Gal-H6a, OCH2-Ha), 3.61-3.71 (m, 4H, Gal-H2, Gal-H3, Gal-
H6b, Gal′-H4), 3.75-3.81 (m, 1H, Gal′-H2), 3.77, 3.78, 3.80 (3s,
9H, 3 OCH3), 3.84 (m, 1H, Gal′-H5), 4.00 (d, J ) 3.2 Hz, 1H,
Gal-H4), 4.01-4.13 (m, 4H, Gal′-H3, Sia-H5, Sia-H6, OCH2-Hb),
4.32 (d, J ) 7.5 Hz, 1H, Gal-H1), 4.39, 4.43 (A, B of AB, J )
11.5 Hz, 2H, C6H4CH2), 4.50 (dd, J ) 7.6, 11.6 Hz, 1H, Gal′-
H6a), 4.60 (d, J ) 7.8 Hz, 1H, Gal′-H1), 4.61 (m, 1H, Gal′-H6b),
2
4.75, 4.78 (A, B of AB, J ) 10.7 Hz, 2H, C6H4CH2), 4.94 (ddd,
J ) 4.6, 10.0, 12.0 Hz, 1H, Sia-H4), 5.24 (d, J ) 9.5 Hz, 1H,
NH), 5.28-5.34 (m, 2H, Sia-H7, Sia-H8), 6.82-6.86, 7.21-7.23,
7.34-7.36, 7.39-7.42, 7.53-7.56, 8.01-8.03 (m, 13H, C6H5, 2
C6H4). 13C NMR (125 MHz, CDCl3): δ -1.5 (SiMe3), 18.4 (SiCH2),
20.7, 20.8, 21.1, 22.1 (4 COCH3), 37.4 (Sia-C3), 49.4 (Sia-C5),
51.0 (Sia-C9), 53.2, 55.2, 55.2 (3 OCH3), 63.2 (Gal′-C6), 67.1
(OCH2), 67.6, 67.6 (Gal′-C4, Sia-C7), 68.5 (Sia-C4), 68.6 (Gal-
C4), 68.9 (Sia-C8), 69.3 (Gal′-C2), 69.5 (Gal-C6), 72.1 (Gal′-C5),
72.8 (Sia-C6), 73.1 (C6H4CH2), 73.2 (Gal-C5), 74.5 (C6H4CH2),
76.1 (Gal′-C3), 77.5 (Gal-C2), 84.0 (Gal-C3), 97.5 (Sia-C2), 102.8
(Gal-C1), 103.8 (Gal′-C1), 113.6, 113.7, 128.4, 129.2, 129.6, 129.6,
129.8, 130.3, 133.3, 159.1 (18C, C6H5, 2 C6H4), 166.2, 168.4, 170.0,
170.1, 170.3, 170.9 (6 CO). Anal. Calcd for C58H78N4O24Si: C,
56.03; H, 6.32; N, 4.51. Found: C, 55.81; H, 6.32; N, 4.35.
2-(Trimethylsilyl)ethyl (Methyl 5-Acetamido-4,7,8-tri-O-
acetyl-9-azido-3,5-dideoxy-D-glycero-r-D-galacto-2-nonulopyra-
nosynate)-(2f3)-(2,4-di-O-acetyl-6-O-benzoyl-ꢀ-D-galactopyr-
anosyl)-(1f3)-4-O-acetyl-2,6-di-O-(p-methoxybenzyl)-ꢀ-D-
galactopyranoside (14). To a stirred solution of 13 (87.0 mg, 70.0
µmol) in pyridine (0.4 mL) at 0 °C was added Ac2O (0.2 mL). The
mixture was stirred at rt for 16 h under argon and then quenched
by addition of MeOH (1 mL). The solution was concentrated and
coevaporated with toluene (3 × 5 mL). The residue was purified
by silica gel chromatography (10% gradient EtOAc in toluene) to
afford 14 (83.0 mg, 87%) as a colorless foam.
2-(Trimethylsilyl)ethyl (Sodium 5-Acetamido-9-benzamido-
3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonulopyranosynate)-
(2f3)-ꢀ-D-galactopyranosyl-(1f3)-ꢀ-D-galactopyranoside (16).
To a stirred solution of 15 (23.0 mg, 15.9 µmol) in DCM (1.8 mL)
were added DDQ (10.8 mg, 47.7 µmol) and H2O (0.1 mL). The
mixture was stirred at rt for 3 h, and then the precipitate was filtered
off and washed with DCM (2 × 5 mL). The combined filtrates
were washed with H2O (2 × 5 mL). The organic layer was dried
(Na2SO4), filtered, and concentrated under reduced pressure. The
residue was purified by silica gel chromatography (0.5% gradient
MeOH in DCM) to afford a white foam (16.2 mg), which was
dissolved in MeOH (1.8 mL) and treated with freshly prepared 1
M NaOMe/MeOH (0.2 mL). The mixture was stirred at rt for 5 h
under argon, and then H2O (0.5 mL) was added and stirring
continued for 3 h. The solution was neutralized with 0.5% HCl,
concentrated, and the residue purified by reversed-phase chroma-
tography (RP-18, 5% gradient MeOH in H2O), Dowex ion-exchange
chromatography (Na+ type), and P2 size exclusion chromatography
[R]D -8.6, (c 0.66, CHCl3). 1H NMR (500 MHz, CDCl3): δ 0.00
(s, 9H, SiMe3), 0.99-1.04 (m, 2H, SiCH2), 1.67 (t, J ) 12.5 Hz,
1H, Sia-H3a), 1.84, 1.92, 2.00, 2.02, 2.09, 2.11, 2.15 (7s, 21H, 7
COCH3), 2.58 (dd, J ) 4.6, 12.7 Hz, 1H, Sia-H3b), 3.09 (dd, J )
5.8, 13.6 Hz, 1H, Sia-H9a), 3.44-3.65 (m, 7H, Gal-H2, Gal-H5,
Gal-H6, Sia-H6, Sia-H9b, OCH2-Ha), 3.73, 3.78, 3.79 (3s, 9H, 3
OCH3), 3.86 (m, 1H, Gal-H3), 3.98-4.05 (m, 3H, Gal′-H5, Sia-
H5, OCH2-Hb), 4.16 (dd, J ) 7.9, 11.0 Hz, 1H, Gal′-H6a), 4.36
(d, J ) 7.8 Hz, 1H, Gal-H1), 4.38-4.44 (m, 3H, Gal′-H6b,
C6H4CH2), 4.54 (dd, J ) 3.3, 10.2 Hz, 1H, Gal′-H3), 4.65, 4.76
(A, B of AB, J ) 10.6 Hz, 2H, C6H4CH2), 4.83 (d, J ) 8.0 Hz,
1H, Gal′-H1), 4.85 (m, 1H, Sia-H4), 5.05 (d, J ) 3.3 Hz, 1H, Gal′-
H4), 5.07 (d, J ) 10.4 Hz, 1H, NH), 5.11 (dd, J ) 8.0, 10.2 Hz,
1H, Gal′-H2), 5.33 (dd, J ) 2.6, 8.7 Hz, 1H, Sia-H7), 5.40-5.43
(m, 2H, Gal-H4, Sia-H8), 6.84-6.87, 7.23-7.25, 7.32-7.33,
7.40-7.43, 7.53-7.56, 8.02-8.04 (m, 13H, C6H5, 2 C6H4). 13C
NMR (125 MHz, CDCl3): δ -1.6 (SiMe3), 18.4 (SiCH2), 20.7, 20.7,

R(2f6)-Linked Sialic Acid in Gangliosides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 999
to afford 16 as a white solid (10.1 mg, 74%) after a final
lyophilization from water.
C6), 65.6 (Hex-C1), 68.0 (2C, Hex-C4, Gal-C4), 69.7 (Gal-C2),
71.3 (Gal-C3), 71.8 (Gal-C5), 77.9 (Hex-C5), 78.7 (Hex-C3), 100.3
(Gal-C1), 128.3, 128.5, 128.6, 128.7, 128.8, 129.1, 129.2, 129.6,
129.7, 129.7, 130.0, 133.4, 133.4, 133.5, 133.7 (24C, 4 C6H5), 165.3,
165.5, 165.6, 166.0 (4 CO). ESI-MS calcd for C40H38NaO13 [M +
Na]+, 749.22; found, 749.28.
[R]D +2.2, (c 0.67, H2O). 1H NMR (500 MHz, D2O): δ 0.02 (s,
9H, SiMe3), 0.96 (td, J ) 5.2, 12.9 Hz, SiCH2-Ha), 1.05 (td, J )
5.5, 12.9 Hz, SiCH2-Hb), 1.80 (t, J ) 12.1 Hz, Sia-H3a), 2.00 (s,
3H, COCH3), 2.76 (dd, J ) 4.6, 12.2 Hz, 1H, Sia-H3b), 3.55-3.66
(m, 6H, Gal-H2, Gal-H5, Gal′-H2, Gal′-H5, Sia-H7, Sia-H9a),
3.67-3.78 (m, 9H, Gal-H3, Gal-H6, Gal′-H6, Sia-H4, Sia-H6, Sia-
H9b, OCH2-Ha), 3.87 (t, J ) 10.2 Hz, 1H, Sia-H5), 3.94 (d, J )
3.1 Hz, 1H, Gal′-H4), 3.99-4.07 (m, 2H, Sia-H8, OCH2-Hb), 4.11
(dd, J ) 3.1, 9.8 Hz, 1H, Gal′-H3), 4.14 (d, J ) 3.3 Hz, 1H, Gal-
H4), 4.37 (d, J ) 8.0 Hz, 1H, Gal-H1), 4.64 (d, J ) 7.8 Hz, 1H,
Gal′-H1), 7.53-7.56, 7.61-7.64, 7.78-7.80 (m, 5H, C6H5). 13C
NMR (125 MHz, D2O): δ -2.2 (SiMe3), 17.9 (SiCH2), 22.4
(COCH3), 40.1 (Sia-C3), 43.1 (Sia-C9), 52.0 (Sia-C5), 61.2, 61.3
(Gal-C6, Gal′-C6), 67.8 (Gal′-C4), 68.6 (OCH2), 68.8 (Gal-C4),
69.8 (Sia-C4), 70.1, 70.1 (3C, Gal-C2, Gal′-C2, Sia-C7), 70.7 (Sia-
C8), 75.0 (2C, Gal-C5, Gal′-C5), 75.3 (Sia-C6), 76.1 (Gal′-C3),
83.1 (Gal-C3), 100.2 (Sia-C2), 102.1 (Gal-C1), 104.5 (Gal′-C1),
127.5, 129.2, 132.5, 134.1 (6C, C6H5), 171.6, 174.1 (2 CO). HRMS
Calcd for C35H55N2O19Si [M - Na]-: 835.3168; Found: 835.3164.
(2,3,4,6-Tetra-O-benzoyl-ꢀ-D-galactopyranosyl)-(1f3)-1,5-an-
hydro-4,6-O-benzylidene-2-deoxy-D-lyxo-hexitol (48). A stirred
suspension of 33 (500 mg, 2.11 mmol), donor 826 (3.20 g, 2.54
mmol), and activated powdered molecular sieves 4 Å (1.00 g) in
DCM (20 mL) was cooled to -20 °C under argon. Then NIS (950
mg, 4.22 mmol) was added and stirring continued for 2 h at -20
°C. After the addition of TfOH (24.2 µL, 0.275 mmol), stirring
was continued for 1 h at -20 °C. The reaction was quenched with
NEt3 (2.5 mL) and filtered through a pad of celite. The celite was
washed with DCM (3 × 10 mL), and the combined filtrates were
successively washed with 20% aq Na2S2O3 (2 × 15 mL) and H2O
(10 mL). The organic layer was dried (Na2SO4), filtered, and
concentrated under reduced pressure. The residue was purified by
silica gel chromatography (petrol ether/EtOAc 2:1 to 1:1) to afford
48 (1.50 g, 87%) as a colorless foam.
(2,3,4,6-Tetra-O-benzoyl-ꢀ-D-galactopyranosyl)-(1f3)-1,5-an-
hydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (50).
To a solution of 49 (700 mg, 0.963 mmol) and biphenyl-4-
carbaldehyde (351 mg, 1.93 mmol) in MeCN (25 mL) was added
p-toluene sulfonic acid monohydrate (18.0 mg, 0.096 mmol) at rt.
The mixture was stirred for 23 h at 70 °C before NEt3 (1 mL) was
added. The solvents were removed and the residue was purified by
silica gel chromatography (petrol ether/EtOAc/NEt3, 85:15:1 to 25:
75:1) to give the acetal intermediate (563 mg). The acetal was
dissolved in THF (15 mL) and subsequently treated with Me3N·BH3
(185 mg, 2.53 mmol) and anhyd AlCl3 (505 mg, 3.79 mmol) with
stirring under argon. After 24 h, the reaction was quenched with
H2O (5 mL) followed by 0.1 M aq HCl (5 mL) and the solution
extracted with DCM (3 × 50 mL). The combined organic phases
were washed with saturated aq NaHCO3 (30 mL), dried (Na2SO4),
filtered, and concentrated under reduced pressure. The residue was
purified by silica gel chromatography (petrol ether/EtOAc/NEt3,
66:33:1 to 25:75:1) to afford 50 (475 mg, 55%) as a colorless foam.
1
[R]D +69.5 (c 1.00, CHCl3). H NMR (500 MHz, CDCl3): δ
1.52 (m, 1H, Hex-H2a), 2.00 (m, 1H, Hex-H2b), 2.46 (d, J ) 1.2
Hz, 1H, OH), 3.30 (m, 1H, Hex-H1a), 3.42 (m, 1H, Hex-H5), 3.47
(dd, J ) 4.1, 9.9 Hz, 1H, Hex-H6a), 3.63 (dd, J ) 7.4, 9.8 Hz, 1H,
Hex-H6b), 3.76 (m, 1H, Hex-H3), 3.91-3.94 (m, 2H, Hex-H1b,
Hex-H4), 4.28 (m, 1H, Gal-H5), 4.39 (dd, J ) 6.0, 11.4 Hz, 1H,
Gal-H6a), 4.44, 4.51 (A, B of AB, J ) 12.1 Hz, 2H, BiphCH2),
4.57 (dd, J ) 7.1, 11.4 Hz, 1H, Gal-H6b), 4.90 (d, J ) 7.9 Hz,
1H, Gal-H1), 5.55 (dd, J ) 3.5, 10.4 Hz, 1H, Gal-H3), 5.72 (dd, J
) 8.0, 10.3 Hz, 1H, Gal-H2), 5.92 (d, J ) 3.4 Hz, 1H, Gal-H4),
7.16-7.19, 7.25-7.38, 7.42-7.51, 7.55-7.58, 7.70-7.72, 7.85-7.87,
7.94-7.95, 8.02-8.04 (m, 29H, C6H4, 5 C6H5). 13C NMR (125
MHz, CDCl3): δ 26.0 (Hex-C2), 62.0 (Gal-C6), 65.6 (Hex-C1),
67.6, 67.9 (Hex-C4, Gal-C4), 69.7 (Gal-C2), 70.6 (Hex-C6), 71.4
(Gal-C3), 71.6 (Gal-C5), 73.3 (BiphCH2), 77.5 (Hex-C5), 78.4
(Hex-C3), 100.0 (Gal-C1), 127.1, 127.2, 127.2, 128.2, 128.3, 128.3,
128.5, 128.5, 128.6, 128.7, 128.7, 128.9, 129.1, 129.6, 129.8, 130.0,
133.4, 133.4, 133.7, 137.1 (36C, C6H4, 5 C6H5), 165.3, 165.5, 166.0
(4C, 4CO). ESI-MS calcd for C53H48NaO13 [M + Na]+, 915.30;
found, 915.34.
1
[R]D +92.9 (c 1.05, CH2Cl2). H NMR (500 MHz, CDCl3): δ
1.65 (m, 1H, Hex-H2a), 2.08 (m, 1H, Hex-H2b), 3.14 (m, 1H, Hex-
H5), 3.33 (ddd, J ) 1.7, 12.2, 13.8 Hz, 1H, Hex-H1a), 3.80 (m,
2H, Hex-H6), 3.83 (m, 1H, Hex-H3), 3.93 (m, 1H, Hex-H1b), 4.14
(dd, J ) 1.4, 1.8 Hz, 1H, Hex-H4), 4.28 (m, 1H, Gal-H5), 4.34
(m, 1H, Gal-H6a), 4.64 (dd, J ) 1.4, 11.3 Hz, 1H, Gal-H6b), 5.07
(d, J ) 7.9 Hz, 1H, Gal-H1), 5.43 (s, 1H, PhCH), 5.55 (dd, J )
3.5, 9.5 Hz, 1H, Gal-H3), 5.79 (dd, J ) 7.9, 9.7 Hz, 1H, Gal-H2),
5.92 (m, 1H, Gal-H4), 7.18-7.20, 7.25-7.38, 7.41-7.42, 7.72-7.73,
7.82-7.84, 7.95-7.97, 8.01-8.03 (m, 25H, 5 C6H5). 13C NMR
(125 MHz, CDCl3): δ 25.0 (Hex-C2), 61.3 (Gal-C6), 64.7 (Hex-
C1), 67.2 (Gal-C4), 68.7 (2C, Hex-C5, Gal-C2), 69.3 (Hex-C6),
70.5 (Gal-C5), 70.7 (Hex-C4), 74.1 (Gal-C3), 75.0 (Hex-C3), 98.4
(Gal-C1), 99.3 (PhCH), 125.0, 127.0, 127.3, 127.4, 127.5, 127.6,
127.7, 128.0, 128.4, 128.6, 128.8, 128.8, 129.1, 132.2, 132.3, 132.5,
132.7 (30C, 5 C6H5), 164.7, 165.0 (4C, 4 CO). ESI-MS calcd for
C47H42NaO13 [M + Na]+, 837.25; found, 837.33.
ꢀ-D-Galactopyranosyl-(1f3)-1,5-anhydro-2-deoxy-D-lyxo-hex-
itol (51). A solution of 50 (470 mg, 0.526 mmol) in MeOH (15
mL) was treated with freshly prepared 1 M NaOMe/MeOH (1.5
mL) and stirred at rt for 19 h under argon. Then the mixture was
neutralized with Dowex 50 × 8 ion-exchange resin (H+ type),
filtrated, and concentrated. The residue was purified by reversed-
phase chromatography (RP-18 column, 5% gradient MeOH in
water) to afford 51 (205 mg, 82%) as a colorless foam.
(2,3,4,6-Tetra-O-benzoyl-ꢀ-D-galactopyranosyl)-(1f3)-1,5-an-
hydro-2-deoxy-D-lyxo-hexitol (49). Compound 48 (948 mg, 1.16
mmol) was dissolved in 80% aq acetic acid (20 mL) and stirred
for 3 h at 60 °C. The solvent was removed in vacuo and the residue
coevaporated with toluene (2 × 10 mL). The remaining solid was
purified by chromatography on silica gel (DCM/MeOH, 20:1 to
10:1) to give 49 (705 mg, 83%) as a colorless foam.
1
[R]D +2.25 (c 0.80, MeOH). H NMR (500 MHz, CD3OD): δ
1.79 (dd, J ) 3.8, 12.3 Hz, 1H, Hex-H2a), 1.97 (m, 1H, Hex-H2b),
3.47 (m, 1H, Hex-H1a), 3.48 (dd, J ) 3.3, 9.7 Hz, 1H, Gal-H3),
3.52 (m, 1H, Gal-H5), 3.54 (dd, J ) 7.5, 9.7 Hz, 1H, Gal-H2),
3.57 (m, 1H, Hex-H5), 3.66 (dd, J ) 4.7, 10.2 Hz, 1H, Hex-H6a),
3.70 (dd, J ) 7.2, 10.1 Hz, 1H, Hex-H6b), 3.71 (dd, J ) 5.1, 11.4
Hz, 1H, Gal-H6a), 3.75 (dd, J ) 7.0, 11.4 Hz, 1H, Gal-H6b), 3.82
(d, J ) 3.0 Hz, 1H, Gal-H4), 3.92 (ddd, J ) 3.0, 4.7, 11.7 Hz, 1H,
Hex-H3), 3.98 (d, J ) 2.6 Hz, 1H, Hex-H4), 4.00 (m, 1H, Hex-
H1b), 4.40 (d, J ) 7.5 Hz, 1H, Gal-H1), 4.59, 4.62 (A, B of AB,
J ) 12.0 Hz, 2H, BiphCH2), 7.31-7.34, 7.41-7.44, 7.60-7.62
(m, 9H, C6H4, C6H5). 13C NMR (125 MHz, CD3OD): δ 27.0 (Hex-
C2), 62.6 (Gal-C6), 66.9 (Hex-C1), 69.4 (Hex-C4), 70.3 (Gal-C4),
71.6 (Hex-C6), 72.5 (Gal-C2), 74.1 (BiphCH2), 74.7 (Gal-C3), 76.7
(Gal-C5), 77.8 (Hex-C3), 79.0 (Hex-C5), 102.9 (Gal-C1), 127.9,
128.0, 128.3, 129.4, 129.9, 138.7 (12C, C6H4, C6H5). ESI-MS calcd
for C25H32NaO9 [M + Na]+, 499.19; found, 499.20.
[R]D +109 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3): δ 1.49
(m, 1H, Hex-H2a), 1.93 (m, 1H, Hex-H2b), 2.10, 2.58 (2s, 2H, 2
OH), 3.22 (m, 1H, Hex-H5), 3.28 (m, 1H, Hex-H1a), 3.45 (d, J )
11.4 Hz, 1H, Hex-H6a), 3.70-3.74 (m, 2H, Hex-H3, Hex-H6b),
3.91-3.94 (m, 2H, Hex-H1b, Hex-H4), 4.30 (m, 1H, Gal-H5), 4.43
(dd, J ) 5.3, 11.6 Hz, 1H, Gal-H6a), 4.57 (dd, J ) 7.5, 11.6 Hz,
1H, Gal-H6b), 4.89 (d, J ) 7.9 Hz, 1H, Gal-H1), 5.55 (dd, J )
3.4, 10.4 Hz, 1H, Gal-H3), 5.72 (dd, J ) 8.1, 10.2 Hz, 1H, Gal-
H2), 5.92 (m, 1H, Gal-H4), 7.17-7.20, 7.30-7.59, 7.70-7.72,
7.86-7.88, 7.95-7.97, 8.02-8.05 (m, 20H, 4 C6H5). 13C NMR
(125 MHz, CDCl3): δ 26.1 (Hex-C2), 62.2 (Gal-C6), 63.4 (Hex-

1000 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Schwardt et al.
(3,4-O-Isopropylidene-ꢀ-D-galactopyranosyl)-(1f3)-1,5-anhy-
dro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (52). To
a solution of 51 (195 mg, 0.409 mmol) and 2,2-dimethoxypropane
(75.2 µL, 0.614 mmol) in acetone (15 mL) was added p-toluene
sulfonic acid monohydrate (7.8 mg, 0.041 mmol). The mixture was
stirred for 3.5 h at 70 °C before NEt3 (0.5 mL) was added. The
solvents were removed in vacuo and the residue was purified by
silica gel chromatography (DCM/MeOH, 50:1 to 10:3) to give 52
(166 mg, 78%) and the 4,6-O-isopropylidene (33.3 mg, 16%) as
colorless foams.
1H, Gal-H6b), 4.48 (d, J ) 7.9 Hz, 1H, Gal-H1), 4.49, 4.60 (A, B
of AB, J ) 12.0 Hz, 2H, BiphCH2), 4.93 (dd, J ) 7.9, 9.8 Hz, 1H,
Gal-H2), 5.32 (d, J ) 2.9 Hz, 1H, Hex-H4), 7.33-7.36, 7.39-7.45,
7.55-7.59 (m, 9H, C6H5, C6H4). 13C NMR (125 MHz, CDCl3): δ
20.8, 20.9, 20.9 (3 COCH3), 27.1 (Hex-C2), 62.3 (Gal-C6), 65.8
(Hex-C1), 68.6 (Gal-C4), 69.0 (Hex-C4), 69.8 (Hex-C6), 72.1, 72.2
(Gal-C3, Gal-C5), 72.7 (Gal-C2), 73.3 (BiphCH2), 73.9 (Hex-C3),
77.0 (Hex-C5), 98.5 (Gal-C1), 127.1, 127.2, 127.3, 128.4, 128.7,
136.7, 140.7, 140.9 (12C, C6H4, C6H5), 170.6, 170.8, 171.1 (3 CO).
ESI-MS calcd for C31H38NaO12 [M + Na]+, 625.23; found, 625.22.
(Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-
D-glycero-r-D-galacto-2-nonulopyranosynate)-(2f3)-(2,6-di-O-
acetyl-ꢀ-D-galactopyranosyl)-(1f3)-4-O-acetyl-1,5-anhydro-6-
O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (55). A stirred
suspension of 54 (70.0 mg, 0.116 mmol), donor 1227 (146 mg, 0.290
mmol), and activated powdered molecular sieves 3 Å (1.50 g) in
MeCN (7 mL) was cooled to -40 °C under argon. Then NIS (131
mg, 0.580 mmol) was added and stirring continued for 1.5 h at
-40 °C. After the addition of TfOH (6.1 µL, 0.073 mmol), stirring
was continued for 19 h at -40 °C. The reaction was diluted with
DCM (15 mL) and filtered through a pad of celite. The celite was
washed with DCM (3 × 5 mL), and the combined filtrates were
successively washed with 20% aq Na2S2O3 (2 × 5 mL) and H2O
(10 mL). The organic layer was dried (Na2SO4), filtered, and
concentrated under reduced pressure. The residue was purified by
silica gel chromatography (0.33% gradient MeOH in DCM) to
afford the ꢀ-sialoside 55 (20.8 mg) as an inseparable mixture with
glycal and the pure R-sialoside 55 (75.4 mg, 61%) as a colorless
foam.
1
[R]D +20.8 (c 1.00, CHCl3). H NMR (500 MHz, CDCl3): δ
1.34, 1.52 (2s, 6H, C(CH3)2), 1.66 (s, 1H, OH), 1.72 (m, 1H, Hex-
H2a), 2.08 (m, 1H, Hex-H2b), 2.94 (s, 1H, OH), 3.46 (m, 1H, Hex-
H1a), 3.52 (m, 1H, Hex-H5), 3.60 (t, J ) 7.7 Hz, 1H, Gal-H2),
3.70 (dd, J ) 5.0, 10.0 Hz, 1H, Hex-H6a), 3.74 (dd, J ) 6.6,
10.0 Hz, 1H, Hex-H6b), 3.80 (m, 1H, Hex-H3), 3.82 (dd, J ) 3.8,
11.5 Hz, 1H, Gal-H6a), 3.87 (m, 1H, Gal-H5), 3.95 (dd, J ) 7.0,
11.4 Hz, 1H, Gal-H6b), 4.03 (s, 1H, Hex-H4), 4.07-4.11 (m, 2H,
Hex-H1b, Gal-H3), 4.16 (d, J ) 4.7 Hz, 1H, Gal-H4), 4.34 (d, J )
8.1 Hz, 1H, Gal-H1), 4.59, 4.63 (A, B of AB, J ) 12.1 Hz, 2H,
BiphCH2), 7.32-7.36, 7.40-7.45, 7.56-7.59 (m, 9H, C6H5, C6H4).
13C NMR (125 MHz, CDCl3): δ 26.2 (Hex-C2), 26.6, 28.3
(C(CH3)2), 62.5 (Gal-C6), 65.6 (Hex-C1), 68.1 (Hex-C4), 70.2
(Hex-C6), 73.0 (Gal-C2), 73.6 (BiphCH2), 73.7 (Gal-C4), 73.8 (Gal-
C5), 77.0 (Hex-C5), 78.1 (Hex-C3), 78.9 (Gal-C3), 100.8 (Gal-
C1), 105.9 (C(CH3)2), 127.2, 127.3, 127.5, 128.3, 128.7, 136.8,
140.5, 140.8 (12C, C6H4, C6H5). ESI-MS calcd for C28H36NaO9
[M + Na]+, 539.23; found, 539.25.
(2,6-Di-O-acetyl-3,4-O-isopropylidene-ꢀ-D-galactopyranosyl)-
(1f3)-4-O-acetyl-1,5-anhydro-6-O-(biphenyl-4-yl-methyl)-2-
deoxy-D-lyxo-hexitol (53). To a solution of 52 (161 mg, 0.312
mmol) in pyridine (5 mL) were subsequently added acetic anhydride
(442 µL, 4.68 mmol) and DMAP (10 mg) at 0 °C under argon.
The mixture was stirred for 21 h at rt and then concentrated in
vacuo. The residue was coevaporated with toluene (3 × 5 mL),
dissolved in DCM (20 mL), and washed with 0.1 N HCl (10 mL)
and saturated NaHCO3 (10 mL). The organic phase was dried
(Na2SO4), filtered, and concentrated in vacuo to give 53 (188 mg,
94%) as a colorless foam.
1
[R]D +1.63 (c 0.72, CHCl3). H NMR (500 MHz, CDCl3): δ
1.81 (m, 1H, Hex-H2a), 1.86-1.90 (m, 4H, Sia-H3a, COCH3), 1.95
(m, 1H, Hex-H2b), 2.03, 2.04, 2.06, 2.11, 2.15, 2.18 (6s, 18H, 6
COCH3), 2.46 (s, 1H, OH), 2.65 (dd, J ) 4.4, 12.7 Hz, 1H, Sia-
H3b), 3.22 (dd, J ) 6.7, 13.5 Hz, 1H, Sia-H9a), 3.42 (m, 1H, Gal-
H4), 3.48-3.52 (m, 4H, Hex-H1a, Hex-H6, Sia-H9b), 3.64-3.65
(m, 2H, Hex-H5, Gal-H5), 3.80 (s, 3H, OCH3), 3.90 (dd, J ) 3.6,
7.3 Hz, 1H, Hex-H3), 3.94 (dd, J ) 2.4, 10.8 Hz, 1H, Sia-H6),
4.06-4.12 (m, 2H, Hex-H1b, Sia-H5), 4.25-4.27 (m, 3H, Gal-
H3, Gal-H6), 4.50, 4.60 (A, B of AB, J ) 12.0 Hz, 2H, BiphCH2),
4.63 (d, J ) 7.9 Hz, 1H, Gal-H1), 4.79 (m, 1H, Sia-H4), 5.03 (dd,
J ) 8.1, 9.8 Hz, 1H, Gal-H2), 5.15 (d, J ) 10.2 Hz, 1H, NH), 5.31
(m, 1H, Hex-H4), 5.32 (dd, J ) 2.4, 8.8 Hz, 1H, Sia-H7), 5.48 (m,
1H, Sia-H8), 7.32-7.35, 7.39-7.44, 7.55-7.58 (m, 9H, C6H4,
C6H5). 13C NMR (125 MHz, CDCl3): δ 20.8, 21.2, 23.1 (7C, 7
COCH3), 27.1 (Hex-C2), 37.7 (Sia-C3), 49.0 (Sia-C5), 51.6 (Sia-
C9), 53.1 (OCH3), 62.9 (Gal-C6), 65.8 (Hex-C1), 67.3 (Gal-C4),
68.1 (Sia-C7), 68.7 (2C, Sia-C4, Sia-C8), 69.0 (Hex-C4), 69.1 (Gal-
C2), 70.0 (Hex-C6), 71.8 (Gal-C5), 72.6 (Sia-C6), 73.3 (Gal-C3),
73.5 (BiphCH2), 74.0 (Hex-C3), 77.1 (Hex-C5), 96.8 (Sia-C2), 98.5
(Gal-C1), 127.0, 127.1, 127.2, 128.3, 128.7, 136.8, 140.6, 140.9
(12C, C6H4, C6H5), 168.5, 169.3, 169.9, 170.3, 170.4, 170.6, 170.9
(8C, 8 CO). ESI-MS calcd for C49H62N4NaO22 [M + Na]+, 1081.38;
found, 1081.54.
1
[R]D +27.7 (c 1.01, CHCl3). H NMR (500 MHz, CDCl3): δ
1.32, 1.55 (2s, 6H, C(CH3)2), 1.76 (dd, J ) 3.6, 12.6 Hz, 1H, Hex-
H2a), 1.96 (m, 1H, Hex-H2b), 2.07 (s, 9H, 3 COCH3), 3.49-3.54
(m, 3H, Hex-H1a, Hex-H6), 3.65 (m, 1H, Hex-H5), 3.88 (m, 1H,
Hex-H3), 3.95 (dt, J ) 1.5, 6.1 Hz, 1H, Gal-H5), 4.10-4.16 (m,
3H, Hex-H1b, Gal-H3, Gal-H4), 4.32 (d, J ) 6.1 Hz, 2H, Gal-
H6), 4.43 (d, J ) 8.1 Hz, 1H, Gal-H1), 4.50, 4.60 (A, B of AB, J
) 11.9 Hz, 2H, BiphCH2), 4.93 (m, 1H, Gal-H2), 5.32 (d, J ) 3.0
Hz, 1H, Hex-H4), 7.32-7.36, 7.39-7.45, 7.55-7.59 (m, 9H, C6H5,
C6H4). 13C NMR (125 MHz, CDCl3): δ 20.8, 20.9, 20.9 (3 COCH3),
26.3 (C(CH3)2), 27.0 (Hex-C2), 27.6 (C(CH3)2), 63.2 (Gal-C6), 66.0
(Hex-C1), 68.7 (Hex-C4), 69.9 (Hex-C6), 70.9 (Gal-C5), 72.5 (Gal-
C2), 73.4 (Gal-C3), 73.4 (BiphCH2), 73.8 (Hex-C3), 76.7 (Gal-
C4), 77.3 (Hex-C5), 97.7 (Gal-C1), 110.8 (C(CH3)2), 127.1, 127.2,
127.2, 128.4, 128.7, 136.8, 140.7, 140.9 (12C, C6H4, C6H5), 169.3,
170.2, 170.8 (3 CO).ESI-MS calcd for C34H42NaO12 [M + Na]+,
665.26; found, 665.35.
(Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-
D-glycero-r-D-galacto-2-nonulopyranosynate)-(2f3)-(2,4,6-tri-
O-acetyl-ꢀ-D-galactopyranosyl)-(1f3)-4-O-acetyl-1,5-anhydro-
6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (56). To a
solution of 55 (108 mg, 0.102 mmol) in pyridine (3 mL) were
subsequently added acetic anhydride (96.0 µL, 1.02 mmol) and
DMAP (3 mg) at 0 °C under argon. The mixture was stirred for
17 h at rt, quenched by addition of MeOH (1 mL), and concentrated
in vacuo. The residue was coevaporated with toluene (3 × 5 mL)
and then purified by silica gel chromatography (0.25% gradient
MeOH in DCM) to afford 56 (89.5 mg, 80%) as a colorless foam.
(2,6-Di-O-acetyl-ꢀ-D-galactopyranosyl)-(1f3)-4-O-acetyl-1,5-
anhydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (54).
Compound 53 (180 mg, 0.280 mmol) was suspended in 80% aq
acetic acid (20 mL) and stirred for 2.5 h at 80 °C. The solvent was
removed in vacuo and the residue coevaporated with toluene (3 ×
5 mL). The remaining solid was purified by silica gel chromatog-
raphy (DCM/MeOH, 50:1 to 20:1) to give 54 (136 mg, 81%) as a
colorless foam.
1
1
[R]D +17.8 (c 1.00, CHCl3). H NMR (500 MHz, CDCl3): δ
[R]D +3.34 (c 1.00, CHCl3). H NMR (500 MHz, CDCl3): δ
1.76 (dd, J ) 3.8, 12.7 Hz, 1H, Hex-H2a), 1.95 (m, 1H, Hex-H2b),
2.07, 2.10 (2s, 9H, 3 COCH3), 3.03, 3.13 (2s, 2H, 2 OH), 3.47-3.53
(m, 3H, Hex-H1a, Hex-H6), 3.60 (dd, J ) 2.8, 9.8 Hz, 1H, Gal-
H3), 3.63-3.67 (m, 2H, Hex-H5, Gal-H5), 3.86 (d, J ) 3.1 Hz,
1H, Gal-H4), 3.91 (m, 1H, Hex-H3), 4.11 (m, 1H, Hex-H1b), 4.26
(dd, J ) 6.3, 11.5 Hz, 1H, Gal-H6a), 4.35 (dd, J ) 6.7, 11.5 Hz,
1.70 (t, J ) 12.4 Hz, 1H, Sia-H3a), 1.84 (m, 1H, Hex-H2a), 1.85
(s, 3H, COCH3), 1.97 (m, 1H, Hex-H2b), 2.00, 2.01, 2.06, 2.09,
2.10, 2.17, 2.21 (7s, 21H, 7 COCH3), 2.58 (dd, J ) 4.6, 12.7 Hz,
1H, Sia-H3b), 3.19 (dd, J ) 6.6, 13.5 Hz, 1H, Sia-H9a), 3.47-3.53
(m, 4H, Hex-H1a, Hex-H6, Sia-H9b), 3.63 (m, 1H, Hex-H5), 3.64
(dd, J ) 2.8, 10.7 Hz, 1H, Sia-H6), 3.82 (m, 1H, Gal-H5), 3.85 (s,

R(2f6)-Linked Sialic Acid in Gangliosides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1001
1
3H, OCH3), 3.89 (m, 1H, Hex-H3), 4.01 (m, 2H, Gal-H6), 4.08 (q,
J ) 10.5 Hz, 1H, Sia-H5), 4.09 (m, 1H, Hex-H1b), 4.48 (dd, J )
3.4, 10.2 Hz, 1H, Gal-H3), 4.50, 4.59 (A, B of AB, J ) 12.0 Hz,
2H, BiphCH2), 4.69 (d, J ) 7.9 Hz, 1H, Gal-H1), 4.85 (ddd, J )
4.6, 10.5, 12.1 Hz, 1H, Sia-H4), 4.89 (d, J ) 3.1 Hz, 1H, Gal-H4),
4.98 (dd, J ) 7.9, 10.2 Hz, 1H, Gal-H2), 5.18 (d, J ) 10.3 Hz,
1H, NH), 5.31 (d, J ) 3.1 Hz, 1H, Hex-H4), 5.33 (dd, J ) 2.7, 8.8
Hz, 1H, Sia-H7), 5.50 (ddd, J ) 3.3, 6.6, 8.7 Hz, 1H, Sia-H8),
7.32-7.35, 7.38-7.44, 7.54-7.58 (m, 9H, C6H4, C6H5). 13C NMR
(125 MHz, CDCl3): δ 20.6, 20.7, 20.7, 20.8, 20.8, 21.4, 23.1 (8C,
8 COCH3), 26.9 (Hex-C2), 37.4 (Sia-C3), 48.9 (Sia-C5), 51.6 (Sia-
C9), 53.2 (OCH3), 61.8 (Gal-C6), 65.8 (Hex-C1), 67.4 (Gal-C4),
68.0 (Sia-C7), 68.6 (Sia-C8), 68.8 (Hex-C4), 69.1 (Sia-C4), 69.5
(Gal-C2), 70.1 (Hex-C6), 70.7 (Gal-C5), 71.2 (Gal-C3), 72.2 (Sia-
C6), 73.3 (BiphCH2), 74.5 (Hex-C3), 77.2 (Hex-C5), 96.7 (Sia-
C2), 98.6 (Gal-C1), 127.1, 127.1, 127.2, 128.3, 128.7, 136.8, 140.6,
140.9 (12C, C6H4, C6H5), 167.9, 169.1, 169.8, 170.3, 170.4, 170.4,
170.7, 170.9 (9C, 9 CO). ESI-MS calcd for C51H64N4NaO23 [M +
Na]+, 1123.39; found, 1123.48.
[R]D +12.6 (c 1.00, CHCl3). H NMR (500 MHz, CDCl3): δ
1.73 (t, J ) 12.4 Hz, 1H, Sia-H3a), 1.80 (m, 1H, Hex-H2a), 1.86
(s, 3H, COCH3), 1.95 (m, 1H, Hex-H2b), 2.01, 2.03, 2.07, 2.09,
2.14, 2.18, 2.19 (7s, 21H, 7 COCH3), 2.58 (dd, J ) 4.6, 12.6 Hz,
1H, Sia-H3b), 2.87 (dt, J ) 3.6, 15.0 Hz, 1H, Sia-H9a), 3.47-3.52
(m, 3H, Hex-H1a, Hex-H6), 3.57 (dd, J ) 2.6, 10.8 Hz, 1H, Sia-
H6), 3.64 (m, 1H, Hex-H5), 3.82 (s, 3H, OCH3), 3.83 (m, 1H, Gal-
H5), 3.90 (m, 1H, Hex-H3), 4.02 (m, 2H, Gal-H6), 4.08 (m, 1H,
Hex-H1b), 4.16 (q, J ) 10.5 Hz, 1H, Sia-H5), 4.38 (ddd, J ) 2.9,
8.8, 15.1 Hz, 1H, Sia-H9b), 4.50, 4.59 (A, B of AB, J ) 12.0 Hz,
2H, BiphCH2), 4.52 (dd, J ) 3.5, 10.1 Hz, 1H, Gal-H3), 4.72 (d,
J ) 8.0 Hz, 1H, Gal-H1), 4.86 (ddd, J ) 4.6, 10.7, 12.0 Hz, 1H,
Sia-H4), 4.90 (d, J ) 3.0 Hz, 1H, Gal-H4), 4.98 (dd, J ) 8.0, 10.1
Hz, 1H, Gal-H2), 5.19 (d, J ) 10.5 Hz, 1H, NH), 5.21 (dd, J )
2.6, 9.7 Hz, 1H, Sia-H7), 5.31 (d, J ) 2.7 Hz, 1H, Hex-H4), 5.42
(dt, J ) 3.0, 9.7 Hz, 1H, Sia-H8), 6.91 (dd, J ) 4.0, 8.6 Hz, 1H,
NH), 7.32-7.35, 7.39-7.44, 7.55-7.58, 7.73-7.75 (m, 13H, 2
C6H4, C6H5). 13C NMR (125 MHz, CDCl3): δ 20.6, 20.7, 20.7, 20.8,
20.8, 21.1, 21.4, 23.1 (8 COCH3), 26.9 (Hex-C2), 37.4 (Sia-C3),
38.4 (Sia-C9), 49.2 (Sia-C5), 53.1 (OCH3), 61.8 (Gal-C6), 65.8
(Hex-C1), 67.5 (Gal-C4), 67.8 (Sia-C7), 68.3 (Sia-C8), 68.9 (Hex-
C4), 69.3 (Sia-C4), 69.6 (Gal-C2), 70.1 (Hex-C6), 70.6 (Gal-C5),
71.4 (Gal-C3), 72.0 (Sia-C6), 73.3 (BiphCH2), 74.3 (Hex-C3), 77.2
(Hex-C5), 96.7 (Sia-C2), 98.4 (Gal-C1), 127.1, 127.2, 127.2, 128.3,
128.7, 128.9, 132.5, 136.9, 140.6, 140.9 (18C, 2 C6H4, C6H5), 166.3,
169.2, 170.3, 170.4, 170.4, 170.9, 171.0 (10C, 10 CO). ESI-MS
calcd for C58H69ClN2NaO24 [M + Na]+, 1235.38; found, 1235.42.
(Sodium 5-Acetamido-9-benzamido-3,5,9-trideoxy-D-glycero-
r-D-galacto-2-nonulopyranosynate)-(2f3)-ꢀ-D-galactopyrano-
syl-(1f3)-1,5-anhydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-
lyxo-hexitol (59). To a solution of 57 (24.0 mg, 20.4 µmol) in
MeOH (3 mL) was added freshly prepared 1 M NaOMe/MeOH
(0.3 mL). The mixture was stirred at rt under argon for 6 h, and
then water (0.5 mL) was added and the mixture stirred for another
15 h at rt. The solution was neutralized with Dowex 50×8 ion-
exchange resin (H+ type), filtrated, and concentrated. The residue
was purified by reversed-phase chromatography (RP-18 column,
5% gradient MeOH in water), Dowex 50×8 ion-exchange chro-
matography (Na+ type), and P2 size exclusion chromatography to
afford 59 (14.3 mg, 79%) as a colorless solid after a final
lyophilization from water.
(Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-benzamido-3,5,9-
trideoxy-D-glycero-r-D-galacto-2-nonulopyranosynate)-(2f3)-
(2,4,6-tri-O-acetyl-ꢀ-D-galactopyranosyl)-(1f3)-4-O-acetyl-1,5-
anhydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (57).
Compound 56 (40.0 mg, 36.3 µmol) and benzoyl chloride (16.9
µL, 0.145 mmol) were dissolved in DCE (3 mL) and stirred at rt
under argon. Ph3P (20.8 mg, 79.9 µmol) in DCE (1 mL) was added
after 5 min. The reaction was stirred at rt for 18 h and then diluted
with DCM (10 mL) and washed with saturated aq NaHCO3 (5 mL)
and H2O (5 mL). The organic layer was dried (Na2SO4), filtered,
and concentrated in vacuo. The residue was purified by silica gel
chromatography (0.5% gradient MeOH in DCM) to afford 57 (27.9
mg, 65%) as a colorless foam.
1
[R]D +13.3 (c 0.73, CHCl3). H NMR (500 MHz, CDCl3): δ
1.72 (t, J ) 12.4 Hz, 1H, Sia-H3a), 1.83 (m, 1H, Hex-H2a), 1.86
(s, 3H, COCH3), 1.96 (m, 1H, Hex-H2b), 2.01, 2.06, 2.09, 2.15,
2.18, 2.19 (6s, 21H, 7 COCH3), 2.58 (dd, J ) 4.6, 12.7 Hz, 1H,
Sia-H3b), 2.90 (dt, J ) 3.8, 15.1 Hz, 1H, Sia-H9a), 3.44-3.51 (m,
3H, Hex-H1a, Hex-H6), 3.58 (dd, J ) 2.6, 10.8 Hz, 1H, Sia-H6),
3.61 (m, 1H, Hex-H5), 3.83 (s, 3H, OCH3), 3.84 (m, 1H, Gal-H5),
3.88 (m, 1H, Hex-H3), 4.01 (m, 2H, Gal-H6), 4.06 (m, 1H, Hex-
H1b), 4.15 (q, J ) 10.4 Hz, 1H, Sia-H5), 4.39 (ddd, J ) 2.8, 8.7,
15.1 Hz, 1H, Sia-H9b), 4.50, 4.59 (A, B of AB, J ) 12.0 Hz, 2H,
BiphCH2), 4.55 (dd, J ) 3.4, 10.2 Hz, 1H, Gal-H3), 4.72 (d, J )
7.9 Hz, 1H, Gal-H1), 4.86 (ddd, J ) 4.6, 10.5, 12.0 Hz, 1H, Sia-
H4), 4.90 (d, J ) 3.0 Hz, 1H, Gal-H4), 4.98 (dd, J ) 8.0, 10.1 Hz,
1H, Gal-H2), 5.20 (d, J ) 10.3 Hz, 1H, NH), 5.24 (dd, J ) 2.6,
9.4 Hz, 1H, Sia-H7), 5.30 (d, J ) 2.5 Hz, 1H, Hex-H4), 5.43 (dt,
J ) 3.2, 9.4 Hz, 1H, Sia-H8), 6.90 (dd, J ) 4.0, 8.5 Hz, 1H, NH),
7.32-7.34, 7.39-7.46, 7.50-7.58, 7.79-7.80 (m, 14H, C6H4, 2
C6H5). 13C NMR (125 MHz, CDCl3): δ 20.6, 20.7, 20.7, 20.8, 20.8,
21.1, 21.4, 23.1 (8 COCH3), 26.9 (Hex-C2), 37.4 (Sia-C3), 38.5
(Sia-C9), 49.1 (Sia-C5), 53.1 (OCH3), 61.8 (Gal-C6), 65.8 (Hex-
C1), 67.5 (Gal-C4), 67.9 (Sia-C7), 68.5 (Sia-C8), 68.9 (Hex-C4),
69.3 (Sia-C4), 69.6 (Gal-C2), 70.1 (Hex-C6), 70.6 (Gal-C5), 71.4
(Gal-C3), 72.0 (Sia-C6), 73.3 (BiphCH2), 74.3 (Hex-C3), 77.2 (Hex-
C5), 96.7 (Sia-C2), 98.4 (Gal-C1), 126.9, 127.1, 127.1, 127.2, 128.3,
128.6, 128.7, 134.2, 136.8, 140.6, 140.9 (18C, C6H4, 2 C6H5), 167.3,
167.8, 169.3, 170.3, 170.4, 170.4, 171.0, 171.7 (10C, 10 CO). ESI-
MS calcd for C58H70N2NaO24 [M + Na]+, 1201.42; found, 1201.76.
1
[R]D -4.56 (c 0.41, MeOH). H NMR (500 MHz, CD3OD): δ
1.73 (m, 1H, Hex-H2a), 1.75 (t, J ) 11.9 Hz, 1H, Sia-H3a), 1.92
(m, 1H, Hex-H2b), 1.98 (s, 3H, COCH3), 2.86 (dd, J ) 3.9, 12.1
Hz, 1H, Sia-H3b), 3.39-3.43 (m, 2H, Hex-H1a, Sia-H7), 3.49-3.53
(m, 3H, Hex-H5, Gal-H5, Sia-H9a), 3.58 (dd, J ) 7.8, 9.5 Hz, 1H,
Gal-H2), 3.62-3.76 (m, 7H, Hex-H6, Gal-H6, Sia-H4, Sia-H5, Sia-
H6), 3.78 (dd, J ) 2.9, 13.4 Hz, 1H, Sia-H9b), 3.88-3.96 (m, 4H,
Hex-H1b, Hex-H3, Hex-H4, Gal-H4), 4.00-4.05 (m, 2H, Gal-H3,
Sia-H8), 4.45 (d, J ) 7.7 Hz, 1H, Gal-H1), 4.57, 4.61 (A, B of
AB, J ) 12.1 Hz, 2H, BiphCH2), 7.31-7.34, 7.41-7.47, 7.51-7.54,
7.59-7.61, 7.83-7.84 (m, 14H, C6H4, 2 C6H5). 13C NMR (125
MHz, CD3OD): δ 22.6 (COCH3), 26.9 (Hex-C2), 42.0 (Sia-C3),
44.4 (Sia-C9), 53.8 (Sia-C5), 62.8 (Gal-C6), 66.9 (Hex-C1), 69.0
(Gal-C4), 69.3 (Hex-C4), 69.4 (Sia-C4), 70.8 (Gal-C2), 71.3 (Sia-
C8), 71.8 (Hex-C6), 72.1 (Sia-C7), 74.0 (BiphCH2), 74.7 (Sia-C6),
76.6 (Gal-C5), 77.6 (Gal-C3), 77.9 (Hex-C3), 79.0 (Hex-C5), 101.2
(Sia-C2), 102.7 (Gal-C1), 127.9, 128.0, 128.3, 128.3, 129.5, 129.6,
129.9, 132.6, 135.8, 138.6, 141.9, 142.1 (18C, C6H4, 2 C6H5), 170.3,
175.4 (3C, 3 CO). HRMS calcd for C43H54N2NaO17 [M + H]+,
893.3320; found, 893.3323.
(Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-(4-chlorobenzamido)-
3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonulopyranosynate)-
(2f3)-(2,4,6-tri-O-acetyl-ꢀ-D-galactopyranosyl)-(1f3)-4-O-acetyl-
1,5-anhydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-
hexitol (58). According to the procedure described for the synthesis
of 57, a solution of 56 (40.0 mg, 36.3 µmol) and p-chlorobenzoyl
chloride (18.6 µL, 0.145 mmol) in DCE (3 mL) was treated with
Ph3P (20.8 mg, 79.9 µmol) in DCE (1 mL) for 17 h. After workup,
the crude product was purified by silica gel chromatography (0.25%
gradient MeOH in DCM) to afford 58 (33.0 mg, 75%) as a colorless
foam.
(Sodium 5-Acetamido-9-(4-chlorobenzamido)-3,5,9-trideoxy-
D-glycero-r-D-galacto-2-nonulopyranosynate)-(2f3)-ꢀ-D-galac-
topyranosyl-(1f3)-1,5-anhydro-6-O-(biphenyl-4-yl-methyl)-2-
deoxy-D-lyxo-hexitol (60). According to the procedure described
for the synthesis of 59, a solution of 58 (19.0 mg, 15.7 µmol) in
MeOH (3 mL) was treated with 1 M NaOMe/MeOH (0.3 mL).
After 6 h, water (0.5 mL) was added and the mixture stirred for
another 16 h. After workup, the crude product was purified by
reversed-phase chromatography (RP-18 column, 5% gradient MeOH
in water), Dowex 50×8 ion-exchange chromatography (Na+ type),

1002 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Schwardt et al.
and P2 size exclusion chromatography to afford 60 (10.9 mg, 75%)
Supporting Information Available: Synthesis of compounds
6-11 and 21-47; HRMS data, HPLC traces, and 1H NMR spectra
for the target compounds 16, 27-29, 37, 41-43, 45, 47, 59, and
60. This material is available free of charge via the Internet at http://
pubs.acs.org.
as a colorless solid after a final lyophilization from water.
1
[R]D -4.94 (c 0.55, MeOH). H NMR (500 MHz, CD3OD): δ
1.72 (m, 1H, Hex-H2a), 1.88 (t, J ) 12.0 Hz, 1H, Sia-H3a), 1.94
(m, 1H, Hex-H2b), 1.97 (s, 3H, COCH3), 2.78 (dd, J ) 4.0, 12.5
Hz, 1H, Sia-H3b), 3.41-3.45 (m, 2H, Hex-H1a, Sia-H7), 3.51-3.56
(m, 3H, Hex-H5, Gal-H5, Sia-H9a), 3.60 (dd, J ) 7.9, 9.5 Hz, 1H,
Gal-H2), 3.64 (dd, J ) 4.7, 10.3 Hz, 1H, Gal-H6a), 3.66-3.74
(m, 4H, Hex-H6, Gal-H6b, Sia-H6), 3.76-3.84 (m, 3H, Sia-H4,
Sia-H5, Sia-H9b), 3.87 (ddd, J ) 2.9, 4.4, 11.6 Hz, 1H, Hex-H3),
3.93 (m, 1H, Gal-H4), 3.96-4.00 (m, 3H, Hex-H1b, Hex-H4, Sia-
H8), 4.07 (dd, J ) 2.6, 9.6 Hz, 1H, Gal-H3), 4.46 (d, J ) 7.7 Hz,
1H, Gal-H1), 4.57, 4.61 (A, B of AB, J ) 12.0 Hz, 2H, BiphCH2),
7.31-7.33, 7.41-7.44, 7.46-7.48, 7.59-7.61, 7.82-7.84 (m, 13H,
2 C6H4, C6H5). 13C NMR (125 MHz, CD3OD): δ 22.7 (COCH3),
27.0 (Hex-C2), 41.2 (Sia-C3), 44.8 (Sia-C9), 53.7 (Sia-C5), 62.5
(Gal-C6), 66.9 (Hex-C1), 68.9 (Sia-C4), 69.3 (Hex-C4), 69.4 (Gal-
C4), 70.7 (Gal-C2), 71.3 (Sia-C8), 71.7 (Hex-C6), 72.0 (Sia-C7),
74.0 (BiphCH2), 75.1 (Sia-C6), 76.4 (Gal-C5), 77.7 (Gal-C3), 78.1
(Hex-C3), 79.0 (Hex-C5), 100.8 (Sia-C2), 102.8 (Gal-C1), 127.9,
128.0, 128.3, 129.4, 129.7, 129.9, 130.1, 134.4, 138.7, 141.9, 142.2
(18C, 2 C6H4, C6H5), 169.4, 175.1 (3C, 3 CO). HRMS calcd for
C43H53ClN2NaO17 [M + H]+, 927.2930; found, 927.2926.
References
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Dergham, P.; Braun, P. E. Oligodendrocyte-Myelin Glycoprotein
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In Vitro Binding Assay. A recombinant protein consisting of
the N-terminal three domains of MAG and the Fc part of human
IgG (Fc-MAGd1-3) was produced by expression in CHO cells and
affinity purification on protein A-agarose as described.36 For the
hapten inhibition assay a modification of the previously described
assay17a was used. Instead of human erythrocytes as a target for
sialic acid-dependent binding of MAG, commercially available
microtiter plates with immobilized Neu5Ac (Lundonia, Lund,
Sweden) were used. In brief, to each well 10 µL of an oligosac-
charide solution was added followed by 20 µL of Fc-MAGd1-3
,
which had been precomplexed with an anti-Fc antibody labeled
with alkaline phosphatase. After an overnight incubation at 4 °C,
unbound Fc-MAG complexes were removed by washing and the
amount of bound Fc-MAG was determined via the alkaline
phosphatase with fluorescein diphosphate as substrate. For each
oligosaccharide, at least eight concentrations were used to determine
the concentration required for 50% inhibition (IC50). To compare
the results from different assays, compound 4 was included in each
test and used as a reference to calculate the relative inhibitory
potencies (rIP). At least three independent titrations were performed
for each compound.
Surface Plasmon Resonance Experiments (Biacore). Surface
plasmon resonance experiments were performed on a Biacore 3000
machine using CM5 chips. Goat antihuman IgG antibody (Fc
specific, Sigma) was immobilized following the standard Biacore
EDC/NHS immobilization procedure. For this, 2 µL of a Fc-
antibody solution of 2.1 mg/mL (in 50 mM phosphate buffer, pH
7.0) was added to 98 µL of 10 mM acetate buffer (pH 5.5). The
immobilization yielded between 4000 and 5000 resonance units.
A sample and a reference surface were prepared sequentially. For
capturing Fc-MAGd1-3 onto the sample surface, Fc-MAGd1-3 in
HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM
EDTA, 0.0005% surfactant P20; Biacore) was injected. Ligands
were dissolved in HBS-EP buffer. Eight ligand concentrations of a
2-fold dilution series starting from 50 µM were prepared. The
samples were injected in a randomized order. Five blank buffer
injections were performed before triplicate measurements and one
between each single experiment. The flow rate was 10 µL/min for
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Acknowledgment. We gratefully acknowledge the financial
support by the Swiss National Science Foundation (200020-
120628) and the Volkswagen Foundation (center project grant
“conformational control of biomolecular functions”).

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