Mimetics of the tri- and tetrasaccharide epitope of GQ1bα as myelin-associated glycoprotein (MAG) ligands

Article abstract of DOI:10.1016/j.bmc.2007.07.033

The synthesis of phenoxyphenyl, phenoxybenzyl, biphenyl, and phenyltriazole substituted sialic acid derivatives as mimics of the tri- and tetrasaccharide epitopes of GQ1bα is described. These synthetically easily available sialosides show comparable or ev

Full text of DOI:10.1016/j.bmc.2007.07.033

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7459–7469
Mimetics of the tri- and tetrasaccharide epitope of GQ1ba
as myelin-associated glycoprotein (MAG) ligands
Ganpan Gao,a Martin Smiesko,a Oliver Schwardt,a Heiko Ga¨thje,b Soerge Kelm,b
Angelo Vedania and Beat Ernsta,*
aInstitute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland
bDepartment of Physiological Biochemistry, University of Bremen, 28334 Bremen, Germany
Received 16 March 2007; accepted 6 July 2007
Available online 21 August 2007
Abstract—The synthesis of phenoxyphenyl, phenoxybenzyl, biphenyl, and phenyltriazole substituted sialic acid derivatives as mim-
ics of the tri- and tetrasaccharide epitopes of GQ1ba is described. These synthetically easily available sialosides show comparable or
even enhanced affinity to MAG compared with the natural tri- and tetrasaccharide epitopes and form a new class of potential MAG
antagonists.
ꢀ 2007 Elsevier Ltd. All rights reserved.
OH
1. Introduction
OH
To date, three inhibitor proteins of axonal regeneration
in the CNS, the myelin-associated glycoprotein
(MAG),1 together with Nogo2 and OMgp,3 have been
identified. They all bind to the Nogo receptor NgR,3,4
which interacts with p75NTR to transduce the inhibitory
signal across the membrane of a neuron. There, the
RhoA-ROCK cascade is activated, which finally leads
to growth cone collapse.5
α(2-6)-linked NeuNAc
OH
AcHN
HO
OH
CO2H
O
O
HO
HO
HO
O
O
AcHN
O
OH
O
O
OH
AcHN
OH
OH
HO
CO2H
α(2-3)-linked NeuNAc
In addition, MAG also binds to brain gangliosides, such
as GD1a, GT1b, and GQ1ba. The MAG-ganglioside
complex interacts with the same coreceptor p75NTR as
the NgR complexes to initiate the inhibitory cascade.6
Therefore, molecules that specifically block the binding
of gangliosides to MAG may enhance neurite outgrowth
and functional recovery after CNS injury. Structure–
activity relationship (SAR) studies of a number of glyco-
sphingolipids indicated that the branched tetrasaccha-
ride moiety Neu5Aca(2-3)Galb(1-3)[Neu5Aca(2-6)]
GalNAc (1) (Fig. 1) of GQ1ba, the ganglioside with
the highest affinity to MAG identified so far, represents
the minimal binding epitope.6a Further SAR studies
suggested that the a(2-3)-linked sialic acid on the termi-
1
Figure
1. Neu5Aca(2-3)Galb(1-3)[Neu5Aca(2-6)]GalNAc
(1),
tetrasaccharide epitope of GQ1ba; the disaccharide core Galb(1-
3)GalNAc is highlighted in box.
nal Gal moiety is the primary determinant for MAG
binding,7 while the additional a(2-6)-linked sialic acid
on the GalNAc moiety only moderately increases the
binding affinity.8 Compared to the contribution of these
two sialic acids, the disaccharide core Galb(1-3)GalNAc
(Fig. 1, in box) behaves more like a spacer to position
the two sialic acid moieties in the appropriate spatial ori-
entation needed for binding to MAG.6a,9
The tetrasaccharide 1 served as the starting point for
structural modifications. When Kelm et al.7c investi-
gated partial structures thereof, they found that Neu5A-
caBn (2) has an approximately tenfold higher affinity for
MAG than the corresponding a-methyl sialoside 3, sug-
Keywords: MAG; GQ1ba; Carbohydrate mimetics; Affinity; Suzuki
coupling.
*
Corresponding author. Tel.: +41 61 267 15 51/50; fax: +41 61 267 15
52; e-mail: beat.ernst@unibas.ch
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.033

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G. Gao et al. / Bioorg. Med. Chem. 15 (2007) 7459–7469
Table 1. Relative inhibitory potencies (rIPs) of sialosides
Structure
rIP
Structure
rIP
OH
OH
OH
AcHN
HO
OH
OH
OH
HO
HO
O
HO
CO2Na
O
O
O
HO
O
125
4
3024a
1
AcHN
OSE
O
O
O
OH
AcHN
OH
OH
HO
HO
HO
CO2Na
O
O
AcHN
OSE
O
O
O
OH
AcHN
OH
OH
HO
HO
CO2Na
HO
HO
AcHN
AcHN
27c
4a
5a
0.62
0.27
0.89
37c
4b
5b
0.07
n.a.
n.a.
O
O
OMe
CO2Na
OH
OH
OH
OH
HO
CO2Na
HO
HO
AcHN
CO2Na
AcHN
O
O
O
O
O
O
OH
OH
HO
HO
OH
OH
HO
HO
O
O
CO2Na
O
HO
HO
AcHN
CO2Na
AcHN
O
OH
OH
OH
OH
CO2Na
O
O
HO
AcHN
HO
CO2Na
O
6a
0.38
6b
n.a.
AcHN
O
O
O
OH
OH
HO
O
OH
OH
HO
HO
CO2Na
COONa
HO
CO2Na
N
HO
AcHN
O
N
AcHN
7
1.0
0.9
27
29
1.1
1.7
O
O
O
N
OH
OH
OH
OH
CO2Na
HO
HO
CO2Na
N
HO
N
N
HO
N
N
AcHN
O
O
28
N
AcHN
CO2Na
O
OH
OH
HO
OH
OH
CO2Na
CO2Na
The rIP of each sialoside was calculated by dividing the IC50 of the reference compound 30 by the IC50 of the compound of interest. This results in
rIPs above 1.0 for derivatives binding better than 30 and rIPs below 1.0 for compounds with a lower affinity than 30; n.a., not applicable, less than
50% inhibition at the highest concentration tested (20 mM); OSE = O(CH2)2SiMe3.
gesting an additional hydrophobic interaction of the
benzyl residue with the receptor site (see Table 1). To
further explore such hydrophobic interactions, and to
use a hydrophobic replacement of the Galb(1-3)GalNAc
core to position the carboxylic acid function of the a(2-
6)-linked sialic acid in the appropriate spatial position,
mimetics of epitope 1 containing aromatic and hetero
aromatic cores were synthesized.
areas.11 On the search for carbohydrate mimetics,
biphenyls were successfully employed to mimic the
Galb(1-4)GlcNAc core in selectin antagonists12 and the
Galb(1-3)GalNAc core in MAG antagonists.13 There-
fore, the sialic acid derivatives 4–7 (Fig. 2) were designed
with phenoxybenzyl, phenoxyphenyl, and biphenyl as
reducing end substituents. Whereas 4–6 were synthesized
to explore the hydrophobic contact suggested by the gain
in affinity found for Neu5AcaBn (2),7c 7 contains an extra
acetic acid attached to the biphenyl moiety to mimic the
a(2-6)-linked sialic acid of tetrasaccharide 1.
2. Results and discussion
Phenoxyphenyl and biphenyl scaffolds are privileged
structures frequently found in drugs.10 It is interesting
to note that biphenyl substructures are present in more
than 4% of the established drugs from various therapeutic
As shown in Scheme 1, mimetics 4 and 6 were success-
fully prepared by standard procedures. The glycosyla-
tion of 3-phenoxybenzyl alcohol (8) or 4-biphenyl
methanol (9) with the sialyl donor 1014 was promoted

G. Gao et al. / Bioorg. Med. Chem. 15 (2007) 7459–7469
7461
HO
AcHN
O
OR
OH
HO
CO2H
OH
O
O
R:
CO2Na
4
5
6
7
Figure 2. With the sialic acid derivatives 4-6, additional hydrophobic contacts are explored; 7 contains an additional acetic acid attached to the
biphenyl moiety to mimic the a(2-6)-linked sialic acid of the tetrasaccharide 1.
HO
AcO
OAc
R1O
OR1
O
AcHN
AcHN
8
O
O
SMe
O
O
a)
CO2R2
AcO
R1O
CO2Me
OAc
OR1
11αβ: R1: Ac; R2: Me
4αβ: R1: H; R2: Na
10 [14]
c)
HO
R1O
OR1
AcHN
9
O
O
b)
R1O
CO2R2
OR1
12αβ: R1: Ac; R2: Me
6αβ: R1: H; R2: Na
c)
HO
AcO
OAc
R1O
OR1
O
13
AcHN
AcHN
O
O
O
CO2Me
d)
AcO
R1O
OH
CO2R2
OAc
OR1
O
14 [19]
15αβ: R1: Ac; R2: Me
5αβ: R1: H; R2: Na
c)
˚
˚
Scheme 1. Reagents and conditions: (a) NIS-TfOH/TMSOTf, MS 3 A, MeCN, ꢀ30 ꢁC, 16 h (81%, a:b = 2.5:1); (b) NIS-TfOH/TMSOTf, MS 3 A,
MeCN, ꢀ30 ꢁC, 16 h (88%, a:b = 3:1); (c) NaOMe, MeOH, rt, 4 h; aq NaOH, rt, 16 h (4a 60%, 4b 63%, 5a 81%, 5b 95%, 6a 60%, 6b 57%); (d) Ph3P,
DEAD, MeCN, 0 ꢁC, 3 h (15 75%, a:b = 1.7:1).18
16
by NIS-TfOH15 or TMSOTf
in MeCN at ꢀ30 ꢁC.
graphic separation of the anomers followed by hydroly-
sis, the two diastereomers 5a and 5b were obtained.
Excellent yields (11 81%; 12 89%) and good stereoselec-
tivities (11a:11b = 2.5:1; 12a:12b = 3:1) were achieved.
After chromatographic separation of the anomers,
´
deprotection under Zemplen conditions followed by
hydrolysis in aq NaOH afforded the target compounds
For the synthesis of mimetic 7, a Suzuki coupling reaction
was applied (Scheme 2). Arylboronic ester 16 was ob-
tained by esterification of 4-bromophenylacetic acid (17)
in the presence of CAN in MeOH, followed by palla-
dium-catalyzed Miyaura boronation.20 The slow borona-
tion reaction was decisively accelerated employing
microwave radiation (300 W for 1.5 h) leading to the
boronic ester 16 in 50% yield. Because the subsequent Su-
zuki coupling reaction was extremely slow, an elevated
reaction temperature was applied, leading to severe
decomposition and only traces of the desired product
19. The reaction between 16 and sialyl derivative 1813
(Scheme 2) was substantially accelerated by microwave
conditions, leading to the desired product 19, however,
in only 8% yield, along with 3% of 7 (deprotected 19)
and recovered starting material 18 (25%).
4 and 6 in approx. 60% yields.
However, under identical conditions the sialylation of
4-phenoxyphenol (13) was not successful. Using
DMTST17 as promoter in MeCN at 0 ꢁC afforded only
10% of 15 with a reversed stereoselectivity (a:b = 1:2).
With NIS-TfOH as promotor, 15 was not formed at all.
This failure is most probably due to the low nucleophilic-
ity of phenol 13 compared to the benzyl alcohols 8 and 9.
As previously described,18 phenol 13 can be sialidated
with hemiketal 1419 in the presence of Ph3P and DEAD
in MeCN at 0 ꢁC, affording an anomeric mixture of aryl
sialoside 15 in good yield (Scheme 1). After chromato-

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G. Gao et al. / Bioorg. Med. Chem. 15 (2007) 7459–7469
HO2C
Br
17
a)
AcO
OAc
AcHN
MeO2C
O
O
O
O
Br
b)
B
+
AcO
CO2Me
OAc
18 [13]
16
AcO
OAc
HO
CO2Me
CO2Na
AcHN
AcHN
O
O
O
O
+
OH
OH
AcO
HO
CO2Me
CO2Na
OAc
19
7
c)
Scheme 2. Reagents and conditions: (a) CAN, MeOH, rt, 16 h (90%); bis(pinacolato)diboron, PdCl2 (dppf), dppf, KOAc, dioxane, 120 ꢁC
(microwave 300 W), 1.5 h (50%); (b) PdCl2 (dppf), dppf, BHT, K3PO4, dioxane, 170 ꢁC (microwave 300 W), 2.25 h (19 8%; 7 3%); (c) NaOMe,
MeOH, rt, 4 h; aq NaOH, rt, 16 h (87%).
As a consequence of the low yielding Suzuki coupling
reaction, we strived for a suitable replacement of biphe-
nyl-based mimetics. Because triazoles recently attracted
broad attention in medicinal chemistry,21 the corre-
sponding phenyltriazoles were considered as convenient
substitutes. In silico investigations supported this ap-
proach, since a detailed conformational comparison of
the mimetics 7 and 27 with parent compound 1 clearly
showed that their low energy conformations are super-
imposable (Fig. 3). The encircled area indicates that
the carboxylic acid substituents in 7 and 27 adopt a sim-
ilar spatial orientation as the carboxylic acid of the a(2-
6)-linked sialic acid in tetrasaccharide 1. Phenyl-1,2,3-
triazoles are accessible by copper-(I)-catalyzed cycload-
ditions, which recently have drawn broad attention as
so-called click chemistry.21 This modified Huisgen 1,3-
dipolar cycloaddition exhibits a number of convincing
synthetic advantages such as mild reaction conditions,
high yields, and adequate regioselectivity. In addition,
triazoles are more than just passive linkers, because they
are able to interact with biological targets through
hydrogen bonding and dipole interactions.21
The phase transfer reaction with donor 2022 and com-
mercially available 3-hydroxyphenylacetylene (21)
afforded sialyl derivative 22 in 60% yield (Scheme 3).
Deprotection by transesterification with NaOMe in
MeOH followed by hydrolysis in aq NaOH afforded
23 in 84% yield. The subsequent copper-(I)-catalyzed
triazole formations with ethyl azidoacetate (24), 4-azi-
dobenzoic acid (25), and benzyl azide (26) were carried
out under standard conditions21 giving the 1,2,3-triaz-
oles 27–29 in approx. 90% yield. It should be noted
that the triazole formation starting from the acetate-
protected precursor 22 was only partially successful.
To force the reaction to completion, the amount of cat-
alyst (Cu(II)SO4Æ5H2O), from which Cu(I) is generated
in situ by sodium ascorbate, had to be increased as
much as tenfold. Under these conditions, the desired
product was accompanied by the formation of fully
and partially deprotected side products. A probable
explanation for the different outcome of the cycloaddi-
tion reaction is the formation of complexes of Cu(II)
with the acetyl protecting groups, thus facilitating their
cleavage.
For the inhibition assays, a recombinant protein consist-
ing 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 pro-
tein A-agarose.23 The relative inhibitory potency (rIP) of
the sialosides was determined in microtiter plates coated
with covalently attached sialic acids as binding target for
Fc-MAGd1-3. By complexing the Fc-part with alkaline
phosphatase-labeled anti-Fc antibodies and measuring
the initial velocity of fluorescein release from fluorescein
diphosphate, the amount of bound MAG could be
determined. The affinities were measured relative to
the reference compound 30,24 which has a rIP of 1
(Table 1). Compound 30 was selected as the reference
Figure 3. Superimposition of biphenyl and phenyltriazole sialic acid
derivatives (7 and 27) with the tetrasaccharide 1. Mimetic 7 is in blue
and mimetic 27 in green, while tetrasaccharide 1 is shown in colors
according to atom type. The encircled area indicates that the carboxyl
group of both synthetic mimics 7 and 27 can reach the region, where a
charged interaction of the a(2-6)-linked NeuNAc carboxyl group with
the binding site is expected.

G. Gao et al. / Bioorg. Med. Chem. 15 (2007) 7459–7469
7463
HO
AcO
AcO
OAc
AcHN
AcHN
21
O
b)
O
O
CO2Me
OAc
OAc
a)
c)
AcO
AcO
CO2Me
Cl
OAc
20 [22]
22
HO
HO
N
N
AcHN
N
AcHN
R
O
O
CO2Na
O
O
OH
OH
OH
OH
HO
HO
CO2Na
23
27-29
CO2Na
R:
CO2Na
27
28
29
Scheme 3. Reagents and conditions: (a) BnEt3N+Clꢀ, CHCl3, aq NaOH, reflux, 2 h (60%); (b) NaOMe, MeOH, rt, 2.5 h; aq NaOH, rt, 2.5 h (84%);
(c) ethyl azidoacetate (24), 4-azidobenzoic acid (25), or benzyl azide (26), sodium ascorbate (0.1 equiv), CuSO4Æ5H2O (0.01 equiv), tBuOH-H2O, 16 h
(27, 93%; 28, 87%; 29, 89%).
compound because it represents the minimal carbohy-
drate epitope required for binding to MAG as identified
by Schnaar et al. in their study on gangliosides.6a High
rIPs represent compounds with high affinity, whereas
low rIPs indicate low affinity compounds.
4. Experimental
4.1. General methods
NMR spectra were recorded on a Bruker Avance DMX-
500 (500 MHz) spectrometer. Assignment of 1H and 13
C
Results from the bioassay confirmed that the disaccha-
ride Galb(1-3)GalNAc in the reference compounds 1
and 30 can be replaced by a-linked aromatic aglycons
like benzyloxyphenyl (4a), phenoxyphenyl (5a) or biphe-
nyl (6a). The introduction of an additional carboxylate
to mimic the acid function of the a(2-6)-linked sialic acid
(6a ! 7) led to a substantial increase of the affinity to
MAG. As expected,7c the b-sialosides (4b–6b) showed
no binding affinities, even at the highest concentration
tested (20 mM). In addition, phenyltriazole proved to
be a valuable replacement of the biphenyl linker, as
demonstrated by similar rIPs for 7 and 27. Moreover,
the unexpected high affinity of mimetic 29 (rIP 1.7) com-
pared with 28 (rIP 0.9) implies that instead of the anio-
nic charge, which was supposed to be the key
contribution of the a(2-6)-linked sialic acid, a hydropho-
bic contact in this area of MAG might also contribute to
binding.
NMR spectra was achieved using 2D methods (COSY,
HSQC). Chemical shifts are expressed in ppm using
residual CHCl3 as references. Optical rotations were
measured using a Perkin-Elmer Polarimeter 241. ESI-
MS analyses were carried out using a Waters Micromass
ZQ Detector system. The spectra were recorded in posi-
tive ESI mode. HR-MS was measured on a Bruker
micrOTOF detector in positive mode. Microwave as-
sisted synthesis was done using a CEM Explorer appara-
tus. Reactions were monitored by TLC using glass
plates coated with silica gel 60 F254 (Merck) and visual-
ized by using UV light and/or by charring with a molyb-
date solution (0.02 M solution of ammonium cerium
sulfate dihydrate and ammonium molybdate tetrahy-
drate in aqueous 10% H2SO4). Column chromatography
was performed on silica gel (Fluka, 40–60 mesh). Meth-
anol (MeOH) was dried by refluxing with sodium meth-
oxide and distilled immediately before use. Dioxane was
dried by refluxing with sodium and distilled immediately
before use. Acetonitrile (MeCN) and dichloromethane
(DCM) were dried by filtration over Al2O3 (Fluka, type
3. Conclusion
˚
5016 A basic). Molecular sieves (3 A) were activated in
vacuo at 500 ꢁC for 2 h immediately before use.
Benzyloxyphenyl, phenoxyphenyl, biphenyl and phenyl-
triazole based mimetics (4–7, 27–29) were designed and
synthesized. Their affinities to MAG are comparable
to the natural carbohydrate ligands 1 and 30. Because
of the pronounced structural simplification combined
with the expected improvement of their pharmacoki-
netic properties,26 the presented sialosides are the first
members of a new generation of MAG antagonists.
Especially, the facile, efficient and versatile synthetic
accessibility of the phenyltriazole derivatives will allow
to further elucidate the structure–affinity relationship
of this class of antagonists.
4.2. General procedure A: Sialylation of alcohols with
methyl 2-thiosialoside 10
To a solution of 1014 (1.0 equiv) and alcohol (1.5 equiv)
in MeCN was added activated powdered molecular
˚
sieves 3 A. The reaction mixture was stirred at rt under
argon for 5 h, then cooled to ꢀ30 ꢁC. After addition of
NIS (2.0 equiv) and TfOH or TMSOTf (0.2 equiv), stir-
ring was continued for 16 h at ꢀ30 ꢁC. Then the mixture
was diluted with DCM (20 ml) and filtered through a

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G. Gao et al. / Bioorg. Med. Chem. 15 (2007) 7459–7469
pad of Celite. The Celite was washed with DCM
(3· 10 ml), and the combined filtrates were washed with
20% aq Na2S2O3 (30 ml), 5% aq KHCO3 (2· 20 ml), and
H2O (20 ml), dried over Na2SO4, filtered and concen-
trated under reduced pressure. The residue was purified
by silica gel chromatography to afford a- and b-sialo-
sides as anomeric mixtures.
NaOMe/MeOH (0.5 ml), followed by H2O (0.5 ml). 4b
(12.0 mg, 63%) was obtained as a white solid after
purification.
1
[a]D +7.8 (c 0.62, H2O); H NMR (500 MHz, D2O): d
1.39 (dd, J = 11.6, 13.0 Hz, 1H, H-3a), 1.75 (s, 3H,
NHCOCH3), 2.10 (dd, J = 4.9, 13.1 Hz, 1H, H-3b),
3.25 (d, J = 9.6 Hz, 1H, H-6), 3.38 (dd, J = 5.5,
12.0 Hz, 1H, H-9a), 3.53 (dd, J = 2.7, 12.0 Hz, 1H, H-
9b), 3.59–3.63 (m, 3H, H-5, H-7, H-8), 3.75 (m, 1H,
H-4), 3.98, 4.28 (A, B of AB, J = 10.4 Hz, 2H, ArCH2),
6.73–6.75, 6.79–6.81, 6.86, 6.91–6.97, 7.11–7.16 (m, 9H,
C6H4, C6H5); 13C NMR (125 MHz, D2O): d 22.3
(NHCOCH3), 40.8 (C-3), 52.7 (C-5), 64.1 (C-9), 65.1
(ArCH2), 67.7 (C-4), 68.9 (C-7), 70.6 (C-6), 71.0 (C-8),
100.8 (C-2), 119.1, 119.5, 119.6, 124.6, 124.6, 130.8,
130.9, 140.1, 157.4, 157.6 (12C, C6H4, C6H5), 175.8
4.3. General procedure B: Cleavage of O-acetyl groups
and methyl esters
To a solution of the sialoside in MeOH was added
freshly prepared 1 M NaOMe/MeOH. The mixture
was stirred at rt for 5 h under argon, then H2O was
added and stirring was continued for 16 h. The solution
was neutralized with 0.5% HCl and concentrated. The
residue was purified by RP chromatography (5% gradi-
ent MeOH in H2O), ion-exchange chromatography
(Dowex 50X8, Na+ type), and P2 size exclusion chroma-
tography to afford the target molecule after a final
lyophilization from H2O.
(2C,
2 CO); HR-MS Calcd for C24H29NNaO10
[M+H+]: 514.1689; Found: 514.1691.
4.7. Sodium (40-phenoxyphenyl 5-acetamido-3,5-dideoxy-
D-glycero-a-D-galacto-2-nonulopyranosid)onate (5a)
4.4. General procedure C: Formation of 1,2,3-triazoles
According to General procedure B, 15a18 (55.9 mg,
84.7 lmol) in MeOH (4.5 ml) was treated with 1 M
NaOMe/MeOH (0.5 ml), followed by H2O (0.5 ml). 5a
(34.1 mg, 81%) was obtained as a white solid after
purification.
To a suspension of 3-ethinylphenyl sialoside 23 (1.0
equiv) and azide (1.0 equiv) in tert-BuOH/H2O (1:1)
was added sodium ascorbate (0.1 equiv, as freshly pre-
pared 1 M aqueous solution), followed by CuSO4Æ5H2O
(0.01 equiv, as aqueous solution). The heterogeneous
mixture was stirred vigorously at rt for 16 h and then
lyophilized. The resulting solid was purified by RP chro-
matography (5% gradient MeOH in H2O) to afford the
target molecule after a final lyophilization from H2O.
1
[a]D +29.4 (c 0.85, H2O); H NMR (500 MHz, D2O): d
1.73 (t, J = 11.9 Hz, 1H, H-3a), 1.85 (s, 3H, NHC-
OCH3), 2.73 (dd, J = 3.6, 12.2 Hz, 1H, H-3b), 3.41–
3.45 (m, 2H, H-7, H-9a), 3.56 (m, 1H, H-4), 3.61–3.74
(m, 4H, H-5, H-6, H-8, H-9b), 6.78–6.80, 6.84–6.85,
6.97–6.99, 7.19–7.22 (m, 9H, C6H4, C6H5); 13C NMR
(125 MHz, D2O): d 22.2 (NHCOCH3), 40.8 (C-3), 51.7
(C-5), 62.2 (C-9), 68.3 (2C, C-4, C-7), 72.1 (C-8), 73.4
(C-6), 103.0 (C-2), 118.5, 119.8, 123.5, 123.7, 130.2,
149.7, 153.3, 157.2 (12C, C6H4, C6H5), 172.6, 175.2 (2
CO); HR-MS Calcd for C23H26NNa2O10 [M+Na+]:
522.1352; Found: 522.1351.
4.5. Sodium (30-phenoxybenzyl 5-acetamido-3,5-dideoxy-
D-glycero-a-D-galacto-2-nonulopyranosid)onate (4a)
According to general procedure B, 11a (66.0 mg,
98.0 lmol) in MeOH (4.5 ml) was treated with 1 M
NaOMe/MeOH (0.5 ml), followed by H2O (0.5 ml). 4a
(30.0 mg, 60%) was obtained as a white solid after
purification.
4.8. Sodium (40-phenoxyphenyl 5-acetamido-3,5-dideoxy-
D-glycero-b-D-galacto-2-nonulopyranosid)onate (5b)
1
[a]D ꢀ27.3 (c 0.75, H2O); H NMR (500 MHz, D2O) d
1.58 (t, J = 12.1 Hz, 1H, H-3a), 1.94 (s, 3H, NHC-
OCH3), 2.66 (dd, J = 4.4, 12.3 Hz, 1H, H-3b), 3.50 (m,
1H, H-7), 3.52 (dd, J = 5.9, 12.1 Hz, 1H, H-9a), 3.58
(m, 1H, H-4), 3.62 (m, 1H, H-6), 3.66 (m, 1H, H-8),
3.70–3.80 (m, 2H, H-5, H-9b), 4.39, 4.62 (A, B of AB,
J = 11.2 Hz, 2H, ArCH2), 6.91–6.92, 6.97–6.98, 7.09–
7.16, 7.26–7.34 (m, 9H, C6H4, C6H5); 13C NMR
(125 MHz, D2O): d 22.4 (NHCOCH3), 40.8 (C-3), 52.2
(C-5), 62.8 (C-9), 66.9 (ArCH2), 68.5 (C-7), 68.6 (C-4),
72.0 (C-8), 73.1 (C-6), 101.3 (C-2), 118.8, 119.1, 119.3,
124.1, 124.3, 130.5, 130.6, 139.8, 157.0, 157.2 (12C,
C6H4, C6H5), 173.8, 175.4 (2 CO); HR-MS Calcd for
C24H29NNaO10 [M+H+]: 514.1689; Found: 514.1687.
According to general procedure B, 15b18 (14.8 mg,
22.4 lmol) in MeOH (4.5 ml) was treated with 1 M
NaOMe/MeOH (0.5 ml), followed by H2O (0.5 ml). 5b
(10.6 mg, 95%) was obtained as a white solid after
purification.
1
[a]D ꢀ45.5 (c 0.73, H2O); H NMR (500 MHz, D2O): d
1.65 (dd, J = 11.5, 13.0 Hz, 1H, H-3a), 1.89 (s, 3H,
NHCOCH3), 2.41 (dd, J = 4.9, 13.1 Hz, 1H, H-3b),
3.31 (d, J = 9.3 Hz, 1H, H-7), 3.44 (dd, J = 5.5,
11.8 Hz, 1H, H-9a), 3.55 (m, 1H, H-8), 3.59 (dd,
J = 2.6, 11.8 Hz, 1H, H-9b), 3.63 (d, J = 10.5 Hz, 1H,
H-6), 3.85 (t, J = 10.3 Hz, 1H, H-5), 4.12 (ddd, J = 4.9,
10.3, 11.2 Hz, 1H, H-4), 6.85, 6.89, 6.92–6.94, 6.99–
7.02, 7.23–7.26 (m, 9H, C6H4, C6H5); 13C NMR
(125 MHz, D2O): d 22.5 (NHCOCH3), 41.0 (C-3), 52.2
(C-5), 63.5 (C-9), 67.2 (C-4), 68.7 (C-7), 72.6 (C-8),
71.5 (C-6), 100.9 (C-2), 118.3, 118.4, 120.7, 122.9,
4.6. Sodium (30-phenoxybenzyl 5-acetamido-3,5-dideoxy-
D-glycero-b-D-galacto-2-nonulopyranosid)onate (4b)
According to general procedure B, 11b (25.0 mg,
37.1 lmol) in MeOH (4.5 ml) was treated with 1 M

G. Gao et al. / Bioorg. Med. Chem. 15 (2007) 7459–7469
7465
123.7, 125.7, 126.2, 130.4, 151.0, 151.1, 157.9 (12C,
C6H4, C6H5), 175.1, 175.2 (2 CO); HR-MS Calcd for
C23H26NNa2O10 [M+Na+]: 522.1352; Found: 522.1356.
11a (66.0 mg, 58%) and 11b (26.5 mg, 23%) as white
foams.
1
Compound 11a: [a]D +2.5 (c 1.26, CHCl3); H NMR
4.9. Sodium (40-biphenylmethyl 5-acetamido-3,5-dideoxy-
(500 MHz, CDCl3): d 1.82 (s, 3H, NHCOCH3), 1.95
(m, 1H, H-3a), 1.96, 1.97, 2.06, 2.08 (4s, 12H, 4
OCOCH3), 2.57 (dd, J = 4.6, 12.8 Hz, 1H, H-3b), 3.62
(s, 3H, OCH3), 4.01 (q, J = 10.8 Hz, 1H, H-5), 4.03
(m, 1H, H-9a), 4.07 (dd, J = 2.1, 10.7 Hz, 1H, H-6),
4.22 (dd, J = 2.7, 12.5 Hz, 1H, H-9b), 4.32, 4.73 (A, B
of AB, J = 12.2 Hz, 2H, ArCH2), 4.81 (ddd, J = 4.6,
10.0, 12.3 Hz, 1H, H-4), 5.12 (d, J = 10.8 Hz, 1H,
NH), 5.27 (dd, J = 2.1, 8.6 Hz, 1H, H-7), 5.37 (ddd,
J = 2.7, 5.5, 8.4 Hz, 1H, H-8), 6.82–7.28 (m, 9H, C6H4,
C6H5); 13C NMR (125 MHz, CDCl3): d 20.7, 20.8,
20.8, 21.1 (4 OCOCH3), 23.2 (NHCOCH3), 38.7 (C-3),
49.4 (C-5), 52.6 (OCH3), 62.3 (C-9), 66.5 (ArCH2),
67.2 (C-7), 68.4 (C-4), 69.0 (C-8), 72.5 (C-6), 98.5 (C-
2), 118.1, 118.3, 118.8, 122.6, 123.2, 129.5, 129.7,
139.3, 157.1, 157.2 (12C, C6H4, C6H5), 168.3, 170.0,
170.1, 170.2, 170.6, 171.0 (6 CO); ESI-MS Calcd for
C33H39NNaO14 [M+Na+]: 696.2; Found: 696.2.
D-glycero-a-D-galacto-2-nonulopyranosid)onate (6a)
According to general procedure B, 12a (51.3 mg,
78.0 lmol) in MeOH (4.5 ml) was treated with 1 M
NaOMe/MeOH (0.5 ml), followed by H2O (0.5 ml). 6a
(23.0 mg, 60%) was obtained as a white solid after
purification.
1
[a]D ꢀ22.4 (c 0.74, H2O); H NMR (500 MHz, D2O): d
1.42 (t, J = 12.1 Hz, 1H, H-3a), 1.74 (s, 3H, NHC-
OCH3), 2.50 (dd, J = 4.6, 12.4 Hz, 1H, H-3b), 3.30
(dd, J = 1.8, 9.4 Hz, 1H, H-7), 3.32 (dd, J = 5.8,
12.1 Hz, 1H, H-9a), 3.58 (m, 3H, H-4, H-6, H-8), 3.52
(dd, J = 2.3, 12.0 Hz, 1H, H-9b), 3.54 (t, J = 10.1 Hz,
1H, H-5), 4.27, 4.47 (A, B of AB, J = 11.3 Hz, 2H,
ArCH2), 7.11–7.14, 7.17–7.25, 7.36–7.40 (m, 9H, C6H4,
C6H5); 13C NMR (125 MHz, D2O):
d
22.4
(NHCOCH3), 40.9 (C-3), 52.3 (C-5), 62.8 (C-9), 67.2
(ArCH2), 68.5 (C-7), 68.7 (C-4), 72.0 (C-8), 73.1 (C-6),
101.5 (C-2), 127.3, 127.4, 128.1, 129.5, 129.7, 136.9,
140.6, 140.6 (12C, C6H4, C6H5), 174.0, 175.1 (2 CO);
HR-MS Calcd for C24H29NNaO10 [M+H+]: 498.1740;
Found: 498.1739.
1
Compound 11b: [a]D ꢀ8.1 (c 0.82, CHCl3); H NMR
(500 MHz, CDCl3): d 1.81 (s, 3H, NHCOCH3), 1.86
(m, 1H, H-3a), 1.89, 1.94, 1.95, 2.08 (4s, 12H, 4
OCOCH3), 2.47 (dd, J = 5.0, 13.0 Hz, 1H, H-3b), 3.67
(s, 3H, OCH3), 3.90 (dd, J = 2.2, 10.5 Hz, 1H, H-6),
4.03 (dd, J = 7.6, 12.4 Hz, 1H, H-9a), 4.08 (q,
J = 10.4 Hz, 1H, H-5), 4.41, 4.46 (A, B of AB,
J = 12.1 Hz, 2H, ArCH2), 4.74 (dd, J = 2.5, 12.4 Hz,
1H, H-9b), 5.17 (m, 1H, H-8), 5.21 (m, 1H, H-4), 5.26
(d, J = 10.2 Hz, 1H, NH), 5.32 (dd, J = 2.2, 4.3 Hz,
1H, H-7), 6.85–7.30 (m, 9H, C6H4, C6H5); 13C NMR
(125 MHz, CDCl3): d 20.8, 20.9 (4C, 4 OCOCH3),
23.1 (NHCOCH3), 38.2 (C-3), 48.3 (C-5), 51.7
(OCH3), 62.3 (C-9), 65.3 (ArCH2), 68.2 (C-7), 68.9 (C-
4), 71.7, 71.9 (C-6, C-8), 98.5 (C-2), 117.6, 118.1,
119.1, 122.0, 122.4, 129.8, 129.9, 138.7, 157.0, 157.5
(12C, C6H4, C6H5), 170.3, 170.6, 171.0 (6C, 6 CO);
ESI-MS Calcd for C33H39NNaO14 [M+Na+]: 696.2;
Found: 696.1.
4.10. Sodium (40-biphenylmethyl 5-acetamido-3,5-dide-
oxy-D-glycero-b-D-galacto-2-nonulopyranosid)onate (6b)
According to general procedure B, 12b (24.5 mg,
37.3 lmol) in MeOH (4.5 ml) was treated with 1 M
NaOMe/MeOH (0.5 ml), followed by H2O (0.5 ml). 6b
(10.5 mg, 57%) was obtained as a white solid after
purification.
1
[a]D +21.2 (c 0.70, H2O); H NMR (500 MHz, D2O): d
1.53 (t, J = 12.3 Hz, 1H, H-3a), 1.87 (s, 3H, NHC-
OCH3), 2.24 (dd, J = 4.9, 13.2 Hz, 1H, H-3b), 3.41 (d,
J = 9.7 Hz, 1H, H-7), 3.51 (dd, J = 5.6, 12.0 Hz, 1H,
H-9a), 3.69 (dd, J = 2.3, 12.0 Hz, 1H, H-9b), 3.73–3.80
(m, 2H, H-5, H-8), 3.83 (d, J = 10.5 Hz, 1H, H-6),
4.12. Methyl (40-biphenylmethyl 5-acetamido-4,7,8,9-tet-
ra-O-acetyl-3,5-dideoxy-D-glycero-a and b-D-galacto-2-
nonulopyranosid)onate (12a) and (12b)
3.90 (m, 1H, H-4), 4.15, 4.47 (A,
B of AB,
J = 10.2 Hz, 2H, ArCH2), 7.24–7.27, 7.33–7.39, 7.52–
7.53 (m, 9H, C6H4, C6H5); 13C NMR (125 MHz,
D2O): d 22.5 (NHCOCH3), 40.3 (C-3), 52.4 (C-5), 64.0
(C-9), 65.0 (ArCH2), 67.4 (C-4), 68.6 (C-7), 70.4 (C-8),
70.7 (C-6), 100.5 (C-2), 127.3, 127.5, 128.2, 129.5,
129.8, 136.7, 140.6, 140.7 (12C, C6H4, C6H5), 175.2,
175.6 (2 CO); HR-MS Calcd for C24H29NNaO10
[M+H+]: 498.1740; Found: 498.1740.
According to general procedure A, 10 (94.4 mg,
0.181 mmol) and 9 (50.0 mg, 0.271 mmol) in MeCN
(6 ml) were treated with NIS (81.4 mg, 0.362 mmol)
and TMSOTf (6.5 lL, 36.2 lmol). Silica gel chromatog-
raphy (toluene/AcOEt 2:1) afforded 12a (75.0 mg, 63%)
and 12b (24.5 mg, 21%) as white foams.
1
4.11. Methyl (30-phenoxybenzyl 5-acetamido-4,7,8,9-tet-
ra-O-acetyl-3,5-dideoxy-D-glycero-a and b-D-galacto-2-
nonulopyranosid)onate (11a) and (11b)
Compound 12a: [a]D +5.5 (c 0.87, CHCl3); H NMR
(500 MHz, CDCl3): d 1.82, 1.96, 1.98, 2.08, 2.11 (5s,
15H, NHCOCH3, 4 OCOCH3), 1.99 (m, 1H, H-3a),
2.61 (dd, J = 4.6, 12.8 Hz, 1H, H-3b), 3.63 (s, 3H,
OCH3), 4.01–4.07 (m, 2H, H-5, H-9a), 4.09 (dd,
J = 2.1, 12.7 Hz, 1H, H-6), 4.28 (dd, J = 2.7, 12.4 Hz,
1H, H-9b), 4.40, 4.79 (A, B of AB, J = 12.0 Hz, 1H,
ArCH2), 4.82 (ddd, J = 4.6, 9.9, 12.3 Hz, 1H, H-4),
5.20 (d, J = 9.7 Hz, 1H, NH), 5.29 (dd, J = 2.0, 8.5 Hz,
According to general procedure A, 10 (86.8 mg,
0.166 mmol) and 8 (43.4 lL, 0.250 mmol) in MeCN
(6 ml) were treated with NIS (75.0 mg, 0.332 mmol)
and TfOH (3.0 lL, 33.2 lmol). Silica gel chromatogra-
phy (1% gradient of MeOH in DCM) afforded

7466
G. Gao et al. / Bioorg. Med. Chem. 15 (2007) 7459–7469
1H, H-7), 5.37 (ddd, J = 2.7, 5.6, 8.4 Hz, 1H, H-8), 7.26–
7.29, 7.33–7.38, 7.49–7.52 (m, 9H, C6H4, C6H5); 13C
NMR (125 MHz, CDCl3): d 20.8, 20.8, 20.9, 21.1, 23.2
(NHCOCH3, 4 OCOCH3), 38.1 (C-3), 49.4 (C-5), 52.7
(OCH3), 62.4 (C-9), 66.6 (ArCH2), 67.3 (C-7), 68.5 (C-
8), 68.1 (C-4), 72.5 (C-6), 98.5 (C-2), 127.0, 127.0,
127.3, 128.3, 128.7, 129.0, 136.1, 140.7, 140.8 (12C,
C6H4, C6H5), 168.4, 170.1, 170.2, 170.3, 170.7, 171.0 (6
CO); ESI-MS Calcd for C33H39NNaO13 [M+Na+]:
680.2; Found: 680.3.
4.14. Methyl [40-(methyl acetate-2-yl)-biphenyl-3-yl 5-
acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-
a-D-galacto-2-nonulopyranosid]onate (19) and sodium [40-
(sodium acetate-2-yl)-biphenyl-3-yl 5-acetamido-3,5-dideoxy-
D-glycero-a-D-galacto-2-nonulopyranosid]onate (7)
A microwave tube was charged with 1813 (149 mg,
0.230 mmol), BHT (35.5 mg, 0.160 mmol), dry K3PO4
(146.5 mg, 0.690 mmol), PdCl2(dppf) (5.60 mg,
6.90 lmol) and dppf (3.80 mg, 6.90 lmol). The tube
was closed, evacuated through a needle at 60 ꢁC for
15 min, and flushed with argon. Dioxane (5.0 ml) and
16 (70.0 mg, 0.254 mmol) were added under vigorous
stirring. The mixture was then degassed in ultrasonic
bath for 20 min, and heated by microwave irradiation
to 170 ꢁC for 2.25 h. The solvent was evaporated in va-
cuo, the residue dissolved in DCM (50 ml) and washed
with H2O (2· 50 ml). The organic layer was dried
(Na2SO4) and concentrated under reduced pressure.
The residue was purified by silica gel chromatography
(PE/AcOEt 3:1–1:6) to afford 19 (12.5 mg, 8%) as white
foam. The aqueous layer was concentrated in vacuo to
give a brown solid, which was stirred with cold MeOH
(10 ml). After filtration and concentration under re-
duced pressure, the residue was subsequently purified
by silica gel chromatography (MeOH/H2O 10:1), RP
chromatography (5% gradient MeOH in H2O), ion-ex-
change chromatography (Dowex 50X8, Na+ type), and
P2 size exclusion chromatography to afford 7 (4.00 mg,
3%) as a white solid after a final lyophilization from
H2O.
1
Compound 12b: [a]D ꢀ15.5 (c 0.80, CHCl3); H NMR
(500 MHz, CDCl3): d 1.88 (dd, J = 11.6, 13.0 Hz, 1H,
H-3a), 1.81, 1.91, 1.95, 1.98, 2.09 (5s, 15H, NHCOCH3,
4 OCOCH3), 2.51 (dd, J = 5.0, 13.0 Hz, 1H, H-3b), 3.68
(s, 3H, OCH3), 3.96 (dd, J = 2.2, 10.5 Hz, 1H, H-6), 4.06
(dd, J = 7.8, 12.4 Hz, 1H, H-9a), 4.08 (q, J = 10.3 Hz,
1H, H-5), 4.48, 4.53 (A, B of AB, J = 11.9 Hz, 2H,
ArCH2), 4.80 (dd, J = 2.5, 12.4 Hz, 1H, H-9b), 5.21–
5.28 (m, 2H, H-4, H-8), 5.32 (d, J = 10.2 Hz, 1H,
NH), 5.35 (dd, J = 2.2, 4.1 Hz, 1H, H-7), 7.27–7.54
(m, 9H, C6H4, C6H5); 13C NMR (125 MHz, CDCl3): d
20.8, 20.8, 20.9, 20.9, 23.1 (4 OCOCH3, NHCOCH3),
37.4 (C-3), 49.4 (C-5), 52.7 (OCH3), 60.4 (C-9), 65.6
(ArCH2), 68.4 (C-7), 69.0 (C-8), 71.9 (2C, C-4, C-6),
98.5 (C-2), 127.1, 127.3, 127.4, 128.0, 128.8, 135.6,
140.7, 140.9 (12C, C6H4, C6H5), 167.4, 170.2, 170.3,
170.6, 170.8, 171.0 (6 CO); ESI-MS Calcd for
C33H39NNaO13 [M+Na+]: 680.2; Found: 680.2.
4.13. Methyl 40-(4,4,5,5-tetramethyl-1,2,3-dioxaborolan-
2-yl)-phenylacetate (16)
According to general procedure B, 19 was treated with
NaOMe/MeOH, followed by H2O to give 7 in 87% yield.
To a solution of 4-bromophenylacetic acid (17) (700 mg,
3.25 mmol) in MeOH (20 ml) was added CAN (2.68 g,
4.88 mmol) at rt. The mixture was stirred for 16 h and
evaporated under reduced pressure. The residue was di-
luted with DCM (50 ml), washed with H2O (2· 30 ml),
and dried (Na2SO4). After filtration and concentration
methyl 4-bromophenylacetate (660 mg, 90%) was ob-
tained as colorless oil, which was used without
purification.
1
Compound 19: [a]D +11.4 (c 0.67, CHCl3); H NMR
(500 MHz, CDCl3): d 1.90 (s, 3H, NHCOCH3), 2.01,
2.02, 2.03, 2.11 (4s, 12H, 4 OCOCH3), 2.20 (t,
J = 12.6 Hz, 1H, H-3a), 2.69 (dd, J = 4.7, 13.0 Hz, 1H,
H-3b), 3.66 (s, 2H, ArCH2), 3.70, 3.71 (2s, 6H, 2
OCH3), 4.06 (q, J = 10.4 Hz, 1H, H-5), 4.18 (dd,
J = 5.5, 12.5 Hz, 1H, H-9a), 4.33–4.36 (m, 2H, H-6, H-
9b), 4.99 (ddd, J = 4.7, 10.4, 12.1 Hz, 1H, H-4), 5.24
(d, J = 10.1 Hz, 1H, NH), 5.34 (dd, J = 1.9, 7.7 Hz,
1H, H-7), 5.40 (m, 1H, H-8), 7.08–7.10, 7.29–7.30,
7.33–7.35, 7.54–7.55 (m, 8H, 2 C6H4); 13C NMR
A microwave tube was charged with methyl 4-bromoph-
enylacetate (150 mg, 655 lmol), potassium acetate
(193 mg, 1.96 mmol), bis(picanolato)diborane (200 mg,
786 lmol), PdCl2(dppf) (16.0 mg, 19.7 lmol), and dppf
(10.9 mg, 19.7 lmol). The tube was closed, evacuated
through a needle, and flushed with argon. Dioxane
(5 ml) was added with vigorous stirring. The solvent
was degassed in an ultrasonic bath for 15 min and
flushed with argon for another 5 min. The tube was
heated by microwave irradiation to 120 ꢁC for 1.5 h.
The solvent was evaporated in vacuo and the residue
dissolved in DCM (50 ml), washed with water
(2· 50 ml), dried (Na2SO4), and concentrated. The resi-
due was purified by silica gel chromatography (1% gra-
dient of AcOEt in toluene) to afford 16 (91.6 mg, 50%)
as white foam.
(125 MHz, CDCl3):
d 20.6, 20.8, 20.9, 21.0 (4
OCOCH3), 23.2 (NHCOCH3), 37.6 (C-3), 40.8
(ArCH2), 49.5 (C-5), 52.1 (OCH3), 62.0 (C-9), 67.5 (C-
7), 68.9 (C-4), 69.5 (C-8), 73.4 (C-6), 100.1 (C-2),
119.2, 119.6, 123.0, 127.3, 129.7, 129.7, 133.4, 139.3,
142.0, 153.8 (12C, 2 C6H4), 160.1. 170.1, 170.1, 170.3,
170.6, 171.0 (6 CO); ESI-MS Calcd for C35H41NNaO15
[M+Na+]: 738.2; Found: 738.2.
Compound 7: [a]D +12.5 (c 0.26, H2O); 1H NMR
(500 MHz, D2O): d 1.96 (t, J = 12.2 Hz, 1H, H-3a),
2.04 (s, 3H, NHCOCH3), 2.86 (dd, J = 4.7, 12.5 Hz,
1H, H-3b), 3.41–3.45 (m, 2H, H-7, H-9a), 3.57 (s, 2H,
ArCH2), 3.60–3.65 (m, 2H, H-7, H-9a), 3.75 (ddd,
J = 4.7, 9.2, 12.0 Hz, 1H, H-4), 3.80 (dd, J = 2.4,
12.1 Hz, 1H, H-9b), 3.87–3.94 (m, 3H, H-5, H-6, H-8),
7.12–7.14, 7.38, 7.40–7.50, 7.63 (m, 8H, 2 C6H4); 13C
1H NMR (500 MHz, CDCl3): d 1.33 (s, 12H, 4 CH3),
3.64 (s, 2H, CH2Ph), 3.68 (s, 3H, OCH3), 7.29, 7.77
(AA0, BB0 of AA0BB0, J = 8.0 Hz, 4H, C6H4).

G. Gao et al. / Bioorg. Med. Chem. 15 (2007) 7459–7469
7467
NMR (125 MHz, D2O): d 22.3 (NHCOCH3), 41.3 (C-3),
44.4 (ArCH2), 52.1 (C-5), 62.9 (C-9), 68.4 (2C, C-4, C-
7), 72.3 (C-8), 73.7 (C-6), 103.1 (C-2), 120.1, 120.5,
123.2, 127.3, 130.1, 130.2, 138.3, 141.9, 153.3, 154.6
(12C, 2 C6H4); HR-MS Calcd for C25H28NNa2O11
[M+H+]: 564.1458; Found: 564.1456.
172.8, 175.5 (2 CO); ESI-MS Calcd for C19H23NNaO9
[M+H+]: 432.1; Found: 432.1.
4.17. Sodium {30-[1-(sodium acetate-2-yl)-1,2,3-triazol-4-
yl]-phenyl 5-acetamido-3,5-dideoxy-D-glycero-a-D-galac-
to-2-nonulopyranosid}onate (27)
4.15. Methyl (30-ethinylphenyl 5-acetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyr-
anoside)onate (22)
According to general procedure C, 23 (10 mg,
23.2 lmol) and ethyl azidoacetate (24) (2.4 lL,
23.2 lmol) in tert-BuOH-H2O (0.4 ml, 1:1) were treated
with sodium ascorbate (2.3 lmol, 2.3 lL of a 1 M aque-
ous solution) and CuSO4Æ5H2O (0.06 mg, 0.232 lmol, in
0.8 lL of H2O) to afford the ethyl ester of 27, which was
then treated with 0.1 M aq NaOH to give 27 (12.0 mg,
93%) after purification.
To a solution of methyl 2-deoxy-2-chloro-4,7,8,9-
tetra-O-acetyl-N-acetyl-neuraminidate(20)22
(295 mg,
0.579 mmol) in CHCl3 (10 ml) was added 3-ethinylphe-
nol (21) (0.300 ml, 2.89 mmol) dissolved in 0.2 M aq
NaOH (10 ml) at rt, followed by benzyltriethylammoni-
um chloride (290 mg, 1.27 mmol). The reaction mixture
was stirred vigorously at 100 ꢁC for 2 h, then diluted
with DCM (30 ml) and washed with 0.1 M NaOH
(2· 20 ml) and water (2· 20 ml). The organic layer was
dried (Na2SO4) and concentrated in vacuo. The residue
was purified by silica gel chromatography (0.5% gradi-
ent of MeOH in DCM) to afford 22 (205 mg, 60%) as
white foam.
1
[a]D +13.8 (c 0.75, H2O); H NMR (500 MHz, D2O): d
1.96 (m, 1H, H-3a), 2.03 (s, 3H, NHCOCH3), 2.92 (m,
1H, H-3b), 3.60–3.62 (m, 2H, H-7, H-9a), 3.75 (m, 1H,
H-4), 3.82 (m, 1H, H-9b), 3.89–3.93 (m, 3H, H-5, H-6,
H-8), 5.08 (s, 2H, CH2CO), 7.16 (d, J = 8.0 Hz, 1H,
C6H4), 7.43 (t, J = 7.8 Hz, 1H, C6H4), 7.60–7.61 (m,
2H, C6H4), 8.28 (s, 1H, C2HN3); 13C NMR (125 MHz,
D2O): d 22.5 (NHCOCH3), 41.4 (C-3), 52.2 (C-5), 63.1
(C-9), 68.6, 68.7 (C-4, C-7), 72.4 (C-8), 73.9 (C-6),
119.2, 121.9, 122.2, 130.6, 147.5, 150.1, 154.8 (8C,
C6H4, C2HN3), 175.5 (3C, 3 CO); HR-MS Calcd for
C21H24N4Na3O11 [M+Na+]: 577.1135; Found: 577.1144.
[a]D +13.1 (c 0.90, CHCl3); 1H NMR (500 MHz,
CDCl3): d 1.91 (s, 3H, NHCOCH3), 2.04, 2.05, 2.13,
2.14 (4s, 12H, 4 OCOCH3), 2.21 (m, 1H, H-3a), 2.64
(dd, J = 4.7, 13.0 Hz, 1H, H-3b), 3.07 (s, 1H, C„CH),
3.70 (s, 3H, OCH3), 4.08 (q, J = 10.4 Hz, 1H, H-5),
4.19 (dd, J = 4.9, 12.4 Hz, 1H, H-9a), 4.32–4.37 (m,
2H, H-6, H-9b), 4.97 (ddd, J = 4.7, 10.5, 11.9 Hz, 1H,
H-4), 5.34–5.38 (m, 2H, H-7, H-8), 5.40 (d,
J = 10.2 Hz, 1H, NH), 7.11–7.28 (m, 4H, C6H4); 13C
NMR (125 MHz, CDCl3): d 20.6, 20.6, 20.7, 20.8 (4
OCOCH3), 23.0 (NHCOCH3), 37.4 (C-3), 49.2 (C-5),
52.9 (OCH3), 61.9 (C-9), 67.2 (C-7), 68.6 (C-4), 69.2
(C-8), 73.2 (C-6), 82.8 (C„CH), 99.9 (C-2), 121.0,
122.93, 124.1, 127.9, 129.2, 153.10 (7C, C6H4, C„CH),
167.7, 169.9, 169.9, 170.1, 170.4, 170.7 (6 CO); Anal.
Calcd for C28H33NO13: C, 56.85; H, 5.62; N, 2.37.
Found: C, 56.24; H, 5.52; N, 2.37.
4.18. Sodium {30-[1-(sodium benzoate-4-yl)-1,2,3-triazol-
4-yl]-phenyl 5-acetamido-3,5-dideoxy-D-glycero-a-D-
galacto-2-nonulopyranosid}onate (28)
According to general procedure C, 23 (8.0 mg,
19.0 lmol) and 4-azidobenzoic acid (25) (3.0 mg,
19.0 lmol) in tert-BuOH-H2O (0.4 ml, 1:1) were treated
with sodium ascorbate (1.9 lmol, 1.9 lL of a 1 M aque-
ous solution) and CuSO4Æ5H2O (0.05 mg, 0.190 lmol, in
0.7 lL of H2O) to afford 28 (10.2 mg, 87%) as a white
solid.
1
[a]D +13.9 (c 1.16, H2O); H NMR (500 MHz, D2O): d
1.96 (m, 1H, H-3a), 2.04 (s, 3H, NHCOCH3), 2.92 (dd,
J = 4.6, 12.5 Hz, 1H, H-3b), 3.60–3.64 (m, 2H, H-7, H-
9a), 3.74–3.81 (m, 2H, H-4, H-9b), 3.89–3.97 (m, 3H, H-
5, H-6, H-8), 7.12–7.14, 7.37–7.40, 7.55–7.58, 7.59, 7.80–
7.81, 8.00–8.02, 8.71 (m, 9H, 2 C6H4, C2HN3); 13C
NMR (125 MHz, D2O): d 23.4 (NHCOCH3), 41.3 (C-
3), 52.1 (C-5), 62.9 (C-9), 68.5, 68.6 (C-4, C-7), 72.3
(C-8), 73.7 (C-6), 103.1 (C-2), 119.0, 120.7, 122.0,
122.0, 130.5, 130.5, 130.7, 138.3, 147.8, 154.6 (14C, 2
C6H4, C2HN3), 172.9, 175.4 (3C, 3 CO); HR-MS Calcd
for C26H27N4Na2O11 [M+H+]: 617.1472; Found:
617.1475.
4.16. Sodium (30-ethinylphenyl 5-acetamido-3,5-dideoxy-
D-glycero-a-D-galacto-2-nonulopyranoside)onate (23)
According to general procedure B, 22 (45.0 mg,
76.1 lmol) in MeOH (2.0 ml) was treated with 1 M
NaOMe/MeOH (0.2 ml), followed by H2O (0.5 ml). 23
(27.5 mg, 84%) was obtained as a white solid after
purification.
1
[a]D +24.3 (c 0.91, H2O); H NMR (500 MHz, D2O): d
1.92 (t, J = 12.2 Hz, 1H, H-3a), 2.03 (s, 3H, NHC-
OCH3), 2.88 (dd, J = 4.7, 12.5 Hz, 1H, H-3b), 3.49 (s,
1H, C„CH), 3.60 (dd, J = 1.5, 9.2 Hz, 1H, H-7), 3.63
(dd, J = 6.3, 12.2 Hz, 1H, H-9a), 3.74 (ddd, J = 4.7,
9.6, 11.8 Hz, 1H, H-4), 3.84–3.93 (m, 4H, H-5, H-6,
H-8, H-9b), 7.18–7.22, 7.31–7.32 (m, 4H, C6H4); 13C
NMR (125 MHz, D2O): d 22.4 (NHCOCH3), 41.1 (C-
3), 52.1 (C-5), 63.0 (C-9), 68.4, 68.5 (C-4, C-7), 72.3
(C-8), 73.7 (C-6), 78.7 (C„CH), 103.1 (C-2), 122.6
(C„CH), 122.9, 125.5, 128.6, 129.9, 153.9 (6C, C6H4),
4.19. Sodium [30-(1-benzyl-1,2,3-triazol-4-yl)-phenyl 5-
acetamido-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulo-
pyranosid]onate (29)
According to general procedure C, 23 (10.0 mg,
23.2 lmol) and benzyl azide (26) (2.9 lL, 23.2 lmol) in
tert-BuOH-H2O (0.4 ml, 1:1) were treated with sodium
ascorbate (2.3 lmol, 2.3 lL of a 1 M aqueous solution)

7468
G. Gao et al. / Bioorg. Med. Chem. 15 (2007) 7459–7469
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and CuSO4Æ5H2O (0.06 mg, 0.232 lmol, in 0.8 lL of
H2O) to afford 29 (11.7 mg, 89%) as a white solid.
1
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[a]D +15.6 (c 0.58, H2O); H NMR (500 MHz, D2O): d
1.95 (t, J = 12.2 Hz, 1H, H-3a), 2.03 (s, 3H, NHC-
OCH3), 2.91 (dd, J = 4.7, 12.5 Hz, 1H, H-3b), 3.53
(dd, J = 5.8, 12.0 Hz, 1H, H-9a), 3.61 (dd, J = 1.6,
9.2 Hz, 1H, H-7), 3.67 (dd, J = 2.4, 12.0 Hz, 1H, H-
9b), 3.75 (ddd, J = 4.7, 9.5, 11.9 Hz, 1H, H-4), 3.82
(ddd, J = 2.4, 5.8, 8.7 Hz, 1H, H-8), 3.87 (dd, J = 1.7,
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(s, 2H, PhCH2), 7.11–7.13, 7.32–7.42, 7.48–7.51 (m,
9H, C6H4, C6H5), 8.21 (s, 1H, C2HN3); 13C NMR
(125 MHz, D2O): d 22.2, (NHCOCH3), 41.0 (C-3),
51.9 (C-5), 54.1 (PhCH2), 62.6 (C-9), 68.2, 68.3 (C-4,
C-7), 72.0 (C-8), 73.5 (C-6), 102.9 (C-2), 118.9, 121.7,
121.7, 122.5, 128.2, 128.9, 129.3, 130.3, 130.6, 135.0,
147.3, 154.5 (14C, C6H4, C6H5, C2HN3), 172.7, 175.2
(2 CO); HR-MS Calcd for C26H30N4NaO9 [M+H+]:
565.1910; Found: 565.1915.
7. (a) Collins, B. C.; Kiso, M.; Hasegawa, A.; Tropak, M.
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4.20. Molecular modeling
The low-energy 3D-structures of tetrasaccharide 1 and
the synthetic mimetics 7 and 27 were identified in the
MacroModel 6.5 modeling environment.27 Conforma-
tion analysis and geometry optimization were performed
using the GBSA solvation model and the AMBER force
field for carbohydrates published by Still,28 augmented
with parameters derived by Kolb and Ernst.29 A flexible
alignment algorithm implemented in MacroModel 6.5
was used to superimpose synthetic mimics 7 and 27 to
tetrasaccharide 1 represented in its global-minimum
structure. The best possible overlap of the mimics with
the a(2-3)-linked NeuNAc moiety and the carboxyl
group of a(2-6)-linked NeuNAc of 1 was achieved by
varying torsion angles of five rotatable bonds of the lin-
ker between the saccharide subunit and the terminal car-
boxyl group (Fig. 3). The encircled area indicates that
the carboxyl groups of both synthetic mimetics 7 and
27 populate the same region in the 3D-space, where a
charged interaction of the carboxyl group of the a(2-
6)-linked NeuNAc moiety of 1 with MAG is expected.
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Acknowledgements
13. Schwizer, D.; Ga¨thje, H.; Kelm, S.; Porro, M.; Schwardt,
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We thank the Volkswagen Foundation (center project
grant ‘Conformational Control of the Biomolecular
Functions’) for financial support.
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