Design and synthesis of a multivalent homing device for targeting to murine CD22

Article abstract of DOI:10.1016/S0968-0896(00)00224-8

CD22 is a cell-surface glycoprotein uniquely located on mature B-cells and B-cell derived tumour cells. Current evidence suggests that binding of endogenous ligands to CD22 leads to modulation of B-cell activation by antigen. Incidentally, however, B-cell activation may derail, and lead to an undesired immune response, for example in cases of allergy, rheumatoid arthritis and Crohn's disease. In this situation, synthetic high-affinity ligands for CD22 may be of therapeutic value as inhibitors of B-cell activation. Recent studies have revealed that natural ligands for CD22 contain the trisaccharide NeuAcα-2,6-Lac as the basic binding motif. In addition, it has been demonstrated that binding to CD22 is strongly enhanced by multivalent presentation of the basic binding motif (cluster effect). In this paper, the stepwise development of a novel multivalent high-affinity ligand for CD22 is described. In the first stage, a series of monovalent NeuAcα-2,6-Glc(Y)X type binding motifs was prepared, and their affinity for murine CD22 was monitored, to obtain more insight into the effect of separate structure elements on ligand recognition. In the second stage, we prepared a trivalent cluster, based on the monovalent motif that displayed the highest affinity for CD22, NeuAcα-2,6-GlcNBzNO₂OMe (7). This cluster, TRIS(NeuAcα-2,6-GlcNBzNO₂)₃ (52), displayed a more than 58-fold higher affinity for CD22 than the reference structure NeuAcα-2,6-LacOMe (10). To our knowledge, the cluster 52 is one of the most potent antagonists for CD22 yet synthesised.

Full text of DOI:10.1016/S0968-0896(00)00224-8

Bioorganic & Medicinal Chemistry 9 (2001) 85±97
Design and Synthesis of a Multivalent Homing Device for
Targeting to Murine CD22
Leo A. J. M. Sliedregt,a,y Sabine M. W. van Rossenberg,a Reshma Autar,a
A. Rob P. M. Valentijn,b Gijs A. van der Marel,b Jacques H. van Boom,b
Christina Piperi,c P. Anton van der Merwe,c Johan Kuiper,a
Theo J.C. van Berkela and Erik A. L. Biessena,*
aLeiden/Amsterdam Center for Drug Research, Division of Biopharmaceutics, Sylvius Laboratories, Leiden University,
PO Box 9503, 2300 RA Leiden, The Netherlands
bLeiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
cSir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK
Received 9 February 2000; accepted 9 August 2000
AbstractÐCD22 is a cell-surface glycoprotein uniquely located on mature B-cells and B-cell derived tumour cells. Current evidence
suggests that binding of endogenous ligands to CD22 leads to modulation of B-cell activation by antigen. Incidentally, however, B-
cell activation may derail, and lead to an undesired immune response, for example in cases of allergy, rheumatoid arthritis and
Crohn's disease. In this situation, synthetic high-anity ligands for CD22 may be of therapeutic value as inhibitors of B-cell acti-
vation. Recent studies have revealed that natural ligands for CD22 contain the trisaccharide NeuAca-2,6-Lac as the basic binding
motif. In addition, it has been demonstrated that binding to CD22 is strongly enhanced by multivalent presentation of the basic
binding motif (cluster e€ect). In this paper, the stepwise development of a novel multivalent high-anity ligand for CD22 is
described. In the ®rst stage, a series of monovalent NeuAca-2,6-Glc(Y)X type binding motifs was prepared, and their anity for
murine CD22 was monitored, to obtain more insight into the e€ect of separate structure elements on ligand recognition. In the
second stage, we prepared a trivalent cluster, based on the monovalent motif that displayed the highest anity for CD22, NeuAca-
2,6-GlcNBzNO2OMe (7). This cluster, TRIS(NeuAca-2,6-GlcNBzNO2)3 (52), displayed a more than 58-fold higher anity for
CD22 than the reference structure NeuAca-2,6-LacOMe (10). To our knowledge, the cluster 52 is one of the most potent antago-
nists for CD22 yet synthesised. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
therapeutic value as inhibitors of B-cell activation. The
structural features that determine the anity of (syn-
thetic) ligands for human and/or murine CD22 have
been the subject of a number of studies.7 13 From these
studies, it appeared that CD22 binds a-2,6-sialylated
glycoproteins, whereas it does not recognise a-2,3-sialy-
lated glycoproteins.7,8 The basic monovalent binding
motif in natural ligands for CD22 was identi®ed as a-2,6-
sialyl-N-acetyllactosamine (Siaa-2,6-LacNAc).9 Unlike
in the case of the selectins,14 the presence of the Sia
moiety, and in particular the C7±C8±C9 glycerol side
arm and the axial anomeric carboxyl function, is
imperative for binding to CD22. The presence of a so-
called cluster e€ect for CD22 was also suggested, as
multiple sialylated structures were bound more avidly
than monovalent sialosides.9,10 While sharing the above
mentioned binding characteristics, human and murine
CD22 di€er in their preference for the substituent at the
C-5 position of Sia. Human CD22 binds equally well to
Sialoadhesins, or siglecs,1 are a group of cell adhesion
molecules within the immunoglobulin superfamily
(IgSF) that bind to glycans with terminal sialic acid (Sia)
residues. The siglec CD22 is a cell-surface glycoprotein
uniquely located on mature B-cells and B-cell derived
tumour cells.2,3 Binding of endogenous ligands to CD22
triggers a cascade of intracellular events, leading to B-cell
activation and proliferation.4 6 Incidentally, however, B-
cell activation may derail, and lead to an undesired
immune response, for example in cases of allergy, rheu-
matoid arthritis and Crohn's disease. In this situation,
synthetic high-anity ligands for CD22 may be of
*Corresponding author. Tel.: +31-71-5276040; fax: +31-71-5276032;
e-mail: biessen@lacdr.leidenuniv.nl
yCurrent address: Solvay Pharmaceuticals, PO Box 900, 1380 DA
Weesp, The Netherlands.
0968-0896/01/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00224-8

86
L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97
N-acetyl (NeuAc) and N-glycoloyl (NeuGc) sialic acid-
containing glycans, whereas murine CD22 prefers
NeuGc to NeuAc conjugates.12 In a recent report, Kelm
et al.13 investigated the binding of human and murine
CD22 to erythrocytes which presented di€erent N-
modi®ed sialyl glycans. Remarkably, replacement of the
respective native N-acyl group on the erythrocyte Sia
moiety by a halogenated acetyl, a formyl, or an ami-
noacetyl group did not a€ect binding by either human
or murine CD22. In contrast, in the same study,
binding to the siglec myelin associated glycoprotein
(MAG) was shown to improve by a factor 17 by sub-
stitution of the N-acetyl by an N-¯uoroacetyl group.
The most extensive QSAR study for CD22 was
reported by Powell et al.,10 revealing that the minimal
motif required for recognition by human CD22 is a
Figure 1. Possible variations in NeuAca-2,6-Glc(Y)X type ligands for
CD22.
Figure 3. Structures of sialoside donor 14 and acceptors 15±26.
Figure 2. Structures of monovalent ligands 1±13.

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97
87
Table 1. Structures, yields and a/b ratios of protected di- and trisaccharides prepared from donor 14 and acceptors 15±26
Acceptor
Product
X
Y
Yielda (a/b)b
15
16
17
18
27
28
29
30
OMe
N3
O(4-NO2)Ph
OMe
NHAc
NHAc
NHAc
N3
37% (5/1)
41% (4/1)
28% (6/1)
50% (5/1)
19
20
21
22
31
32
33
34
Ð
Ð
Ð
Ð
NHBz
NHOct
NH(2-Napht)
NH(4-NO2)Bz
42% (3/1)
30% (3/1)
25% (5/1)
44% (4/1)
23
24
35
36
OMe
N3
Ð
Ð
26% (2/1)
33% (3/1)
25
26
37
38
OMe
O(4-NO2)Ph
Ð
Ð
47% (6/1)
52% (4/1)
aa/b Mixture.
bAs gauged by 13C NMR analysis.
disaccharide NeuAca-2,6-Hex(NAc), Hex(NAc) being
Gal, GalNAc or GlcNAc. The most potent synthetic
binding motifs in this study displayed a threefold higher
anity for CD22 than the reference structure NeuAca-
2,6-Lac.
mido, benzoylamido, azido) of the glucosamine. In the
second stage, a trivalent cluster, based on the mono-
valent motif that displayed the highest anity for
murine CD22, was prepared and tested, to establish
whether the anity of a synthetic oligosaccharide could
be further enhanced by multivalent presentation.
In summary, from the literature it appears that (a) the
presence of a complete Sia ring structure is essential for
binding to CD22, (b) the group at the C-5 position of
the Sia moiety does not have a dramatic e€ect on bind-
ing, and (c) the anity for CD22 can be enhanced by
further optimisation of the (carbohydrate) structure
attached to the reducing end of the Sia moiety.
We were interested in developing a novel synthetic high-
anity ligand for CD22. The molecular structure of this
ligand should warrant maximal (selective) binding to
CD22, but also be easily accessible from a synthetic
point of view. These two requirements can be met by
preparing a trivalent glycoconjugate, based on a TRIS
dendritic core used by our group in earlier studies,15 and
a NeuAca-2,6-Hex(NAc) derived minimal motif as pro-
posed by Powell et al., in which (a) the terminal NeuAc
moieties are intact and unmodi®ed, and (b) glucosamine
serves as the basic skeleton for the underlying carbohy-
drate. In search of this high-anity ligand, a two-stage
approach was pursued. In the ®rst stage, a series of
monovalent NeuAca-2,6-Glc(Y)X type binding motifs
(Fig. 1) was prepared, and their anity for murine CD22
was monitored, to obtain insight into the e€ect of mod-
i®cations at both the C-1 position (X=methoxy, azido,
nitrophenoxy) and at the C-2 position (e.g., Y=aceta-
Scheme 1. Protecting group exchange of NeuAca-2,6-GalX type
disaccharides. Reagents: (i) NaOMe; (ii) Ac2O.

88
L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97
Results and Discussion
with the sialyl xanthate 1416 (Fig. 3) in combination
with N-iodosuccinimide/tri¯ic acid (NIS/TfOH)17,18 as
promoter system. The results of the glycosylation reac-
tions are depicted in Table 1.
For the ®rst phase of our study, nine NeuAca-2,6-
Glc(Y)X type ligands were prepared (1±9, see Fig. 2),
together with, as a frame of refererence, two NeuAca-2,6-
LacX (10 and 11) and two NeuAca-2,6-GalX (12 and
13) type ligands.
The a-isomers of the di- and trisaccharides 27±36 were
identi®ed by the characteristic 1H NMR chemical
shifts of the H-3eq [d(a-isomer)>d(b-isomer)] and H-4
(dÆ4.8 ppm) of the NeuAc moieties, and were separated
from the b-isomers by silica gel chromatography. For the
galactose derived disaccharides 37 and 38, separation of
The synthesis of the di- and trisaccharides 1±13 involved
sialylation of the partially protected acceptors 15±26
Table 2. Anity data of oligosaccharides 1±13a
Compound
Relative IC50
1
2
3
4
5
6
7
8
NeuAca-2,6-GlcNAcOMe
NeuAca-2,6-GlcN3OMe
NeuAca-2,6-GlcNH2OMe
NeuAca-2,6-GlcNHBzOMe
NeuAca-2,6-GlcNHOctOMe
NeuAca-2,6-GlcNHNaphtOMe
NeuAca-2,6-GlcNHBzNO2OMe
NeuAca-2,6-GlcNAcN3
NeuAca-2,6-GlcNAcOPhNO2
NeuAca-2,6-LacOMe
NeuAca-2,6-LacN3
NeuAca-2,6-GalOMe
NeuAca-2,6-GalOPhNO2
6.9
1.2
2.0
2.2
1.6
1.8
9.2
2.1
7.9
1.0
2.2
2.4
3.6
9
10
11
12
13
aThe relative IC50 was calculated by dividing the average IC50 found
for compound 10 by the average IC50 of the respective compound. A
relative IC50>1 therefore represents a better binding to CD22 than the
reference compound 10.
Scheme 2. Synthesis of 2-amino disaccharide 3. Reagents: (i) NaOMe;
(ii) NaOH; (iii) 1,3-propanedithiol.
Scheme 3. Synthesis of disaccharide ®t for coupling to TRIS dendritic core. Reagents: (i) HOCH2CH2OCH2CH2N3, TfOH; (ii) 1 M NaOH, re¯ux;
(iii) excess 4-NO2BzCl; (iv) NaOMe; (v) tBDMS-Cl; (vi) Ac2O; (vii) p-TsOH; (viii) NIS, TfOH; (ix) P(Ph)3, H2O.

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97
89
the respective a- and b-isomers could only be accom-
plished after replacing the benzoyl groups on the galac-
tose ring by acetyl groups (see Scheme 1). Having the
fully protected a-isomers of 27±36, 39 and 40 in hand,
two-step basic hydrolysis of the protecting groups then
gave the respective target ligands 1±2 and 4±13.
is slightly lower for murine CD22 than for human
CD22.12 The anity of 10 was de®ned as 1 and the IC50
values of all other ligands were expressed relative to that
of 10 (see Table 2).
From Table 2 it can be seen that, in line with the results
of Powell et al.,10 the glucosamine derived dimers 1±9 all
have a comparable or higher anity for CD22 than the
trisaccharide 10. Remarkably, substitution of an anome-
ric methoxy by an azido group appears to have a negative
e€ect (1 versus 8, respectively) at the disaccharide level,
but a bene®cial e€ect at the trisaccharide level (10 versus
11, respectively). An anomeric nitrophenoxy instead of
a methoxy group has a positive e€ect on binding, both
in the case of glucosamine- (9 versus 1, respectively) and
galactose-derived (13 versus 12, respectively) disac-
charides. Focusing on the C-2 position of the glucoside
in the NeuAca-2,6-Glc(Y)X template, substitution of
the acetamide in 1 by an azide or an amine impairs
binding to CD22 (2 and 3 versus 1, respectively). With
Finally, the 2-amino ligand 3 was prepared from the
deprotected 2-azido ligand 2 by mild reduction with 1,3-
propanedithiol19 (see Scheme 2).
The monovalent ligands 1±13 were tested on their a-
nity for CD22 in an in vitro competition assay based on
the binding of porcine erythrocytes to immobilised
murine CD22D1-3-Ig.20,21 The NeuAca-2,6-LacOMe
trisaccharide 10, which was selected as the reference
compound, displayed an average IC50 of 244 mM. This
compares well with the value reported by Powell et al.10
for binding of NeuAca-2,6-Lac to human CD22, taking
into account that the anity of NeuAc glycoconjugates
Scheme 4. Synthesis of the cluster based on 7. Reagents: (i) HBTU, HOBT; (ii) NaOMe; (iii) NaOH.

90
L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97
respect to 3, it should be noted that Kelm et al.13 also
reported that the presence of a free amine reduced the
anity for CD22, as well as for the other siglecs sia-
loadhesin (Sn) and MAG. By contrast, azido and amino
groups at the C-2 position of the glucose ring of sialyl-
LewisX were found22 to enforce the binding to E-selec-
tin by a factor of 4±6. Introduction of a more lipophilic
moiety at the C-2 position of a disaccharide by repla-
cing the N-acetyl in 1 by either an aromatic (benzoyl,
naphthoyl) or aliphatic (octanoyl) group negatively
a€ects binding to CD22 (4, 5 and 6 versus 1, respec-
tively). The latter result may look rather unexpected in
view of recent reports in which lipophilic groups confer
stronger binding in many di€erent ligand±receptor sys-
tems.23 29 On the other hand, addition of a single nitro
group to the aromatic benzoyl ring leads to a dramatic
increase in anity for CD22 (7 versus 1, 7 versus 4). The
nitrobenzoyl disaccharide 7 displayed an almost 10-fold
higher anity than the reference 10, establishing 7 as
the most potent ligand of all the monovalent carbohy-
drates tested in this study. The disaccharide 7 was
therefore selected as the basic motif for the multivalent
ligand to be prepared in the second phase of our study.
structure 10, a 58.1-fold improvement in binding was
observed. It cannot be excluded that the presence of the
lipophilic benzyloxycarbonyl group in 52 contributes to
the binding of the TRIS cluster. However, the results
found for the lipophilic monovalent ligands 19 and
21 suggest that the e€ect of a single phenyl ring on bind-
ing will not be very signi®cant. Therefore, we assume
that the strong gain in anity found for the multivalent
sialoside 52 is mainly due to the cluster e€ect.
Conclusion
We have prepared 13 sialoside oligosaccharides that
carry di€erent structural features on the hexose moieties
at the reducing terminus, and tested these oligosacchar-
ides on their ability to bind to CD22. The main result of
these tests is that the presence of 4-nitroaryl groups at
either the C-1 or C-2 position of the reducing hexose
enhances binding, while the presence of either lipophilic
or positively charged groups at C-2 does not. The most
potent disaccharide, the nitrobenzoyl derivative 7,
displays an almost 10-fold higher anity for CD22 than
the reference structure, NeuAca-2,6-LacOMe. Based on
disaccharide 7, a trivalent cluster (52) was prepared. This
trivalent sialoside 52 displayed a 6.3-fold higher anity
for CD22 than disaccharide 7, thereby providing strong
evidence that ligand valency is indeed an important
determinant for high-anity binding to CD22.9,10 To
our knowledge, the cluster 52 is one of the most potent
ligands for CD22 yet synthesised, and may ®nd appli-
cation as (ant)agonist or homing device for CD22-
expressing B-cells.
The synthesis of the multivalent ligand based on 7 started
with the introduction of a masked diethylene glycol
amino spacer moiety at the anomeric position of the
glucosamine ring. This spacer should allow smooth
attachment of the disaccharide to the TRIS dendritic
core, and warrant an optimal resemblance of the result-
ing cluster to the native multivalent ligand structure, in
terms of spacing of the terminal sialic acid residues.
To this end, the azide 42 was prepared by the addition
of 2-(2-azidoethoxy)ethanol to the known30 oxazoline
41 of glucosamine (see Scheme 3). Conversion into the
nitrobenzoyl derivative 45 proceeded via a sequence
of straightforward protection and deprotection steps.
Sialylation of 45 with the methyl thiosialoside 4631 and
subsequent Staudinger32 reduction of the azido group of
the dimer 47 a€orded the amine 48, ®t for coupling to
the TRIS dendritic core.
Experimental
General
All solvents were of analytical grade. Dry solvents were
stored over 4 A molecular sieves. Merck Kieselgel 60
F254 DC Alufolien was used for TLC analysis. Carbo-
hydrate compounds were visualised by charring with
sulfuric acid:ethanol (1:4, v/v). Compounds containing
NH functions were visualised by charring with a solution
of 4,40-tetramethyldiamino-diphenylmethane (TDM)33
after treatment of the TLC plates with chlorine, or
visualised by charring with a 0.3% solution of ninhy-
drine in acetic acid:n-butanol (3:100, v/v). Column
chromatography was performed with Kieselgel 60, 230±
400 mesh (Merck). Gel ®ltration was performed with
Sephadex LH-20 (Pharmacia). 1H NMR spectra
(200 MHz) and 13C{1H} NMR spectra (50.1 MHz) were
recorded with a Jeol JNM-FX200 spectrometer. 1H
NMR spectra (300 MHz) were recorded with a Bruker
WM-300 spectrometer. Matrix Assisted Laser Desorp-
tion Ionisation (MALDI) Time-of-Flight (TOF) mass
spectrometry34 was performed with a Perkin Elmer/
PerSeptive Biosystems Voyager-DE-RP MALDI-TOF
mass spectrometer equipped with delayed extraction.35
Exact masses were obtained by nano±ESI±TOF±MS on
a Micromass LCT with a resolution of 5000 FWHM
using pentaphenylalanine as reference mass. Murine
Condensation of TRIS-derived tricarboxylate 4915 with
48 (Scheme 4), under standard peptide coupling condi-
tions, furnished the protected cluster 50 in an excellent
yield. Finally, mild two-step removal of the protective
ester groups a€orded the desired sialoside 52.
It was found that the multivalent sialoside 52 displayed
a 6.2-fold higher anity for CD22 than 7 (see Table 3).
Compared to our initial NeuAca-2,6-LacOMe reference
Table 3. Anity data of oligosaccharides 10, 7 and the cluster 52a
Compound
Relative IC50
10
7
52
NeuAca-2,6-LacOMe
NeuAca-2,6-GlcNHBzNO2OMe
TRIS(NeuAca-2,6-GlcNHBzNO2)3
1.0
9.2
58.1
aThe relative IC50 was calculated by dividing the average IC50 found
for compound 10 by the average IC50 of the respective compound. A
relative IC50>1 therefore represents a better binding to CD22 than the
reference compound 10.

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97
91
CD22/IgG was puri®ed from a stable transfected CHO
cell line (kindly provided by Dr. P. Crocker, Dundee,
Scotland). Cells were grown in GMEM (First link UK)
containing 5% IgG-poor FCS (Gibco), 20 U pen/strep
and 400 mM MSX (Sigma). Production of CD22 was
induced by addition of 2 mM sodium butyrate (Sigma),
and medium was collected for 3 weeks. Murine CD22
was routineously puri®ed from the pooled media by
protein-A Sepharose chromatography (yieldÆ200 mg/
mL). Purity was routinely checked by PAGE analysis.
(CHarom benzoyl), 129.3±128.7 (Cq benzoyl), 99.5 (C-20),
88.9 (C-1), 73.6, 73.1, 72.5, 69.9. 68.6, 67.6, 67.3 (C-
3,4,5, C-40,60,70,80), 65.0, 63.2 (C-6, C-90), 53.7 (C-2),
52.7 (OCH3 ester), 48.7 (C-50), 37.2 (C-30), 22.9 (N-ace-
tyl), 20.9, 20.8, 20.6 (CH3 O-acetyl). b-Isomer d 98.0 (C-
2 ). H NMR (CDCl3): d 8.05±7.33 (m, 10H, CHarom),
6.52 (d, 1H, NH acetyl, JNH-2=9.8 Hz), 5.65 (dd, 1H,
H-3, J3 4=9.6 Hz), 5.57 (dd, 1H, H-4, J4 5=9.5 Hz),
0
1
5.37 (ddd, 1H, H-80, J8 9 a=2.7 Hz, J8
7.1 Hz), 5.23
0
0
0
0
9 b
(dd, 1H, H-70, J6 7 =1.2 Hz, J7 8 =9.5 Hz), 4.90 (d,
0
0
0
0
1H, H-1, J1 2=9.3 Hz), 4.72 (ddd, 1H, H-40, J4
0
0
=
5
10.0 Hz), 4.35 (dd, 1H, H-90a, J9 a 9 b=12.2 Hz), 4.30
(ddd, 1=H, H-2, J2 3=9.9 Hz), 4.19 (m, 3H, H-
0
0
Methyl 2-acetamido-3,4-di-O-benzoyl-2-deoxy-6-O-(methyl
4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-D-neuraminate-2-yl)-
ꢁ-D-glucopyranoside (27). A mixture of acceptor 15
(145 mg; 327 mmol), donor 14 (130 mg; 218 mmol)16 and
powdered molecular sieves (3 A) in acetonitrile (3 mL)
was stirred for 1 h under argon atmosphere. The tem-
perature of the mixture was lowered to 50 ꢀC, and a
solution of NIS (74 mg; 0.33 mmol) and TfOH (3 mL;
0.03 mmol) in acetonitrile (1 mL) was added. The mix-
ture was gradually warmed to room temperature over a
period of 3 h. The reaction was quenched by the addi-
tion of triethylamine (0.1 mL). The mixture was ®ltered
over Hy¯o Super Cel (Fluka), diluted with ethyl acetate
(50 mL), washed with aq Na2S2O3 (1 M; 50 mL), aq
NaHCO3 (1 M; 50 mL) and water (50 mL), dried (MgSO4),
concentrated, and applied to a silica gel column. Elution
was performed with DCM:methanol (1:0!96:4, v/v).
The fractions containing the product were pooled, con-
centrated, and applied to a Sephadex LH-20 column.
The disaccharide 27 was obtained as a/b mixture. Yield:
73 mg (80 mmol, 37%, a/b 5:1). The a-isomer was iso-
lated from the latter mixture by silica gel chromato-
graphy, with toluene:ethanol (1:0!8:2, v/v) as eluent, in
a yield of 45 mg (49 mmol). 13C{1H} NMR (CDCl3): d
171.1±128.3 (CˆO acetyl, benzoyl, C-10), 133.2±128.3
(CHarom benzoyl), 128.9 (Cq benzoyl), 101.9 (C-1), 99.2
(C-20), 73.4, 72.3, 71.8, 70.0, 68.8, 67.8, 67.1 (C-3,4,5, C-
40,60,70,80), 64.6, 62.8 (C-6, C-90), 56.5 (OCH3), 52.6
(OCH3 ester), 52.2 (C-2), 48.9 (C-50), 37.3 (C-30), 23.1
(N-acetyl), 20.9, 20.6 (CH3 O-acetyl). b-Isomer d 98.0 (C-
50,60,90b), 3.82 (dd, 1H, H-6a, J5 6a=7.4 Hz, J6a 6b
12.2 Hz), 3.78±3.70 (m, 2H, H-5,6b), 3.75 (s, 3H, OCH3
=
ester), 2.33 (dd, 1H, H-30eq, J3 eq 4 =4.7 Hz, J3 eq 3 ax
=
0
0
0
0
12.8 Hz), 2.16, 2.14, 2.04, 1.95, 1.88, 1.86 (6Âs, 18H,
CH3 acetyl), 1.42 (dd, 1H, H-30ax, J3 ax 4 =12.3 Hz).
0
0
4-Nitrophenyl 2-acetamido-3,4-di-O-benzoyl-2-deoxy-6-
O-(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-D-neurami-
nate-2-yl)-ꢁ-D-glucopyranoside (29). Compound 29 was
prepared from acceptor 17 (180 mg; 327 mmol) and
donor 14 (130 mg; 218 mmol) as described above for the
synthesis of 27. Yield: 61 mg (60 mmol, 28%, a/b 6:1).
Isolated yield of a-isomer: 23 mg (22 mmol). 13C{1H}
NMR (CDCl3): d 171.9±164.9 (CˆO acetyl, benzoyl, C-
10), 161.6, 142.6 (Cq phenyl), 133.6±128.0 (CHarom ben-
zoyl), 125.6, 116.7 (CHarom phenyl), 99.9 (C-20), 98.5 (C-
1), 72.5, 71.7, 70.3, 68.5, 67.2 (C-3,4,5, C-40,60,70,80),
65.5, 63.7 (C-6, C-90), 54.2 (C-2), 52.8 (OCH3 ester),
48.7 (C-50), 37.0 (C-30), 23.2 (N-acetyl), 20.6 (CH3 O-
0
1
acetyl). b-Isomer d 98.1 (C-2 ). H NMR (CDCl3): d
8.21±7.29 (m, 14H, CHarom), 6.56 (d, 1H, NH acetyl,
JNH-2=9.4 Hz), 5.81 (dd, 1H, H-3, J3 4=9.7 Hz), 5.61
(dd, 1H, H-4, J4 5=9.6 Hz), 5.56 (ddd, 1H, H-80,
0
0
0
0
J8 9 a=2.8 Hz, J8 9 b=8.0 Hz), 5.50 (d, 1H, H-1,
0
J1 2=8.2 Hz), 5.23 (dd, 1H, H-70, J7 8 =9.8 Hz), 5.106
0
0
(d, 1H, NH acetyl, JNH-5 =9.6 Hz), 4.68 (ddd, 1H, H-4 ,
0
0
J4 5 =9.3 Hz), 4.66 (ddd, 1H, H-2, J2 3=10.3 Hz), 4.46
0
(dd, 1H, H-90a, J9 a 9 b=12.1 Hz), 4.33 (ddd, 1H, H-5,
0
0
2 ). H NMR (CDCl3): d 8.01±7.33 (m, 10H, CHarom),
6.19 (d, 1H, NH acetyl, JNH-2=9.3 Hz), 5.62 (dd, 1H, H-
3, J3 4=9.5 Hz), 5.55 (dd, 1H, H-4, J4 5=9.6 Hz), 5.33
J5 6a=6.7 Hz, J5 6b=5.0 Hz), 4.10 (ddd, 1H, H-50,
1
0
0
0
0
0
J5 6 =10.6 Hz), 4.03 (dd, 1H, H-6 , J6 7 =1.7 Hz),
3.90±3.82 (m, 2H, H-6a, H-90b), 3.71 (s, 3H, OCH3
ester), 3.68 (dd, 1H, H-6b, J6a 6b=13.0 Hz), 2.26 (dd,
(ddd, 1H, H-80, J8 9 a=2.6 Hz, J8 9 b=6.6 Hz), 5.24 (dd,
0
0
0
0
1H, H-70, J6 7 =1.2 Hz, J7 8 =10.0 Hz), 4.73 (ddd, 1H,
1H, H-30eq, J3 eq 4 =4.7 Hz, J3 eq 3 ax=12.9 Hz), 2.20,
2.19, 1.94, 1.88, 1.86 (5Âs, 18H, CH3 acetyl), 1.29 (dd,
0
0
0
0
0
0
0
0
H-40, J4 5 =9.9 Hz), 4.65 (d, 1H, H-1, J1 2=8.4 Hz),
0
0
4.29 (dd, 1H, H90a, J9 a 9 b=12.3 Hz), 4.23 (ddd, 1H,
H-2, J2 3=10.1 Hz), 4.05±3.95 (m, 3H, H-50,60,90b),
3.86±3.71 (m, 3H, H-5,6a,6b), 3.74 (s, 3H, OCH3 ester),
1H, H-30ax, J3 ax 4 =12.7 Hz).
0
0
0
0
Methyl 2-azido-3,4-di-O-benzoyl-2-deoxy-6-O-(methyl 4,
7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-D-neuraminate-2-yl)-ꢁ-D-
glucopyranoside (30). Compound 30 was prepared from
acceptor 18 (128mg; 300 mmol) and donor 14 (119mg;
200 mmol) as described above for the synthesis of 27.
Yield: 90mg (100mmol, 50%, a/b 5:1). Isolated yield of a-
isomer: 51mg (56 mmol). 13C{1H} NMR (CDCl3): d
170.7±164.8 (CˆO acetyl, benzoyl, C-10), 133.1±128.2
(CHarom benzoyl), 129.2, 129.0 (Cq benzoyl), 102.9 (C-
1), 98.6 (C-20), 73.0, 72.5, 72.4, 69.2, 69.0, 68.4, 67.1,
64.1 (C-2,3,4,5, C-40,60,70,80), 63.4, 62.1 (C-6, C-90), 57.1
(OCH3), 52.4 (OCH3 ester), 49.1 (C-50), 37.4 (C-30), 23.0
(N-acetyl), 20.6 (CH3 O-acetyl). b-Isomer d 98.1 (C-20).
1H NMR (CDCl3): d 8.04±7.29 (m, 10H, CHarom), 5.51
3.54 (s, 3H, OCH3), 2.39 (dd, 1H, H-30eq, J3 eq 4
0
0
=
4.6 Hz, J3 eq 3 ax=12.7 Hz), 2.11, 2.03, 1.96, 1.86, 1.85
0
0
(5Âs, 18H, CH3 acetyl), 1.55 (dd, 1H, H-30ax, J3 ax 4
0
0
=
12.3 Hz).
2-Acetamido-3,4-di-O-benzoyl-2-deoxy-6-O-(methyl
4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-D-neuraminate-2-yl)-
ꢁ-D-glucopyranosyl azide (28). Compound 28 was pre-
pared from acceptor 16 (149 mg; 327 mmol) and donor
14 (130mg; 218 mmol) as described above for the synthesis
of 27. Yield: 83mg (89 mmol, 41%, a/b 4:1). Isolated yield
of a-isomer: 54mg (58 mmol). 13C{1H} NMR (CDCl3): d
171.5±164.6 (CˆO acetyl, benzoyl, C-10), 133.3±128.3

92
L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97
(dd, 1H, H-4, J4 5=9.6 Hz), 5.40 (dd, 1H, H-3, J3 4=
9.6 Hz), 5.24 (dd, 1H, H-70, J7 8 =8.6 Hz), 5.19 (ddd,
4.48 (d, 1H, H-1, J1 2=8.3 Hz), 4.31 (dd, 1H, H-90a,
0
0
0 0
J9a 9 b=12.4 Hz), 4.04 (ddd, 1H, H-2, J2 3=10.4 Hz),
1H, H-80, J8 9 a=2.5 Hz, J8 9 b=5.0 Hz), 4.79 (ddd,
4.03 (dd, 1H, H-90b), 3.97 (dd, 1H, H-60, J5 6 =10.8 Hz,
0
0
0
0
0
0
1H, H-40, J4 5 =9.8 Hz), 4.45 (d, 1H, H-1, J1 2
=
0
(s, 3H, OCH3 ester), 3.46 (s, 3H, OCH3), 2.61 (dd, 1H,
0
0
0
0
J6 7 =2.0 Hz), 3.94±3.56 (m, 4H, H-5 , H-5,6a,6b), 3.79
8.0 Hz), 4.10 (dd, 1H, H-90a, J9 a 9 b=12.5 Hz), 3.99 (dd,
0
0
1H, H-60, J5 6 =6.0 Hz, J6 7 =1.4 Hz), 3.97±3.94 (m,
2H, H-50, H-6a), 3.86±3.82 (m, 1H, H-5), 3.80 (dd, 1H,
H-90b), 3.65 (dd, 1H, H-2, J2 3=10.1 Hz), 3.61 (dd, 1H,
H-6b, J5 6b=3.0 Hz, J6a 6b=12.1 Hz), 3.74, 3.63 (2Âs,
H-30eq, J3 eq 4 =4.7 Hz, J3 eq 3 ax=12.7 Hz), 2.14, 2.13,
2.05, 2.03, 2.02, 2.00, 1.86 (7Âs, 21H, CH3 acetyl), 1.94
0
0
0
0
0
0
0
0
(dd, 1H, H-30ax, J3 ax 4 =12.4 Hz), 1.56 (t, 2H, CH2
octanoyl), 1.25 (broad, 10H, CH2 octanoyl), 0.86 (t, 3H,
CH3 octanoyl).
0
0
6H, OCH3), 2.49 (dd, 1H, H-30eq, J3 eq 4 =4.7 Hz,
0
0
0
0
J3 eq 3 ax=12.6 Hz), 2.08, 2.02, 2.00, 1.99, 1.83 (5Âs,
15H, CH3 acetyl), 1.78 (dd, 1H, H-30ax, J3 ax 4
12.5 Hz).
Methyl 2-naphthoylamido-3,4-di-O-acetyl-2-deoxy-6-O-
(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-D-neurami-
nate-2-yl)-ꢁ-D-glucopyranoside (33). Compound 33 was
prepared from acceptor 21 (177 mg; 410 mmol) and
donor 14 (160 mg; 268 mmol) as described above for the
synthesis of 27. Yield: 63 mg (69 mmol, 25%, a/b 5:1).
Isolated yield of a-isomer: 39 mg (43 mmol). 13C{1H}
NMR (CDCl3): d 171.3±167.3 (CˆO acetyl, naphtoyl,
C-10), 134.5±131.0 (Cq naphtoyl), 128.6±123.3 (CHarom
naphtoyl), 101.6 (C-1), 98.4 (C-20), 73.0, 72.0, 71.8, 69.2,
68.9, 68.2, 67.2 (C-3,4,5, C-40,60,70,80), 63.0, 62.3 (C-6,
C-90), 56.3 (OCH3), 54.1 (C-2), 52.2 (OCH3 ester), 48.5
(C-50), 37.3 (C-30), 22.0 (N-acetyl), 20.1 (CH3 O-acetyl).
0
0
=
Methyl 2-benzoylamido-3,4-di-O-acetyl-2-deoxy-6-O-
(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-D-neurami-
nate-2-yl)-ꢁ-D-glucopyranoside (31). Compound 31 was
prepared from acceptor 19 (164 mg; 430 mmol) and
donor 14 (170 mg; 285 mmol) as described above for the
synthesis of 27. Yield: 102 mg (119 mmol, 42%, a/b 3:1).
Isolated yield of a-isomer: 70 mg (81 mmol). 13C{1H}
NMR (CDCl3): d 171.0±166.9 (CˆO acetyl, benzoyl, C-
10), 134.1 (Cq benzoyl), 131.5±126.9 (CHarom benzoyl),
102.0 (C-1), 98.7 (C-20), 73.0, 72.4, 72.3, 69.0, 68.3, 67.4
(C-3,4,5, C-40,60,70,80), 63.6, 62.6 (C-6, C-90), 56.5
(OCH3), 54.5 (C-2), 52.6 (OCH3 ester), 49.3 (C-50), 37.6
(C-30), 23.0 (N-acetyl), 20.7 (CH3 O-acetyl). b-Isomer d
98.3 (C-20). 1H NMR (CDCl3): d 7.75±7.38 (m, 5H,
CHarom), 6.59 (d, 1H, NH benzoyl, JNH-2=9.1 Hz), 5.40
b-Isomer d 98.1 (C-20). H NMR (CDCl3): d 8.27±7.48
1
(m, 7H, CHarom), 6.73 (d, 1H, NH naftanoyl, JNH-2=
=
9.0 Hz), 5.41 (ddd, 1H, H-80, J8 9 a=2.5 Hz, J8
6.3 Hz), 5.40 (dd, 1H, H-3, J3 4=9.6 Hz), 5.34 (d, 1H,
0
0
0
0
9 b
NH acetyl, JNH-5 =9.5 Hz), 5.30 (dd, 1H, H-70,
0
(ddd, 1H, H-80, J8 9 a=2.6 Hz, J8 9 b=6.1 Hz), 5.35
0
0
0
0
0
J7 8 =8.7 Hz), 5.23 (dd, 1H, H-4, J4 5=9.5 Hz), 4.88
0
(dd, 1H, H-3, J3 4=9.6 Hz), 5.28 (dd, 1H, H-70,
(ddd, 1H, H-40, J4 5 =9.8 Hz), 4.60 (d, 1H, H-1, J1 2
=
0
0
8.3 Hz), 4.37 (dd, 1H, H-90a, J9 a 9 b=12.3 Hz), 4.28
0
J7 8 =8.7 Hz), 5.20 (dd, 1H, H-4, J4 5=9.5 Hz), 4.88
0
0
0
(ddd, 1H, H-40, J4 5 =9.7 Hz), 4.57 (d, 1H, H-1,
(ddd, 1H, H-2, J2 3=10.2 Hz), 4.08 (ddd, 1H, H-500,
0
0
J1 2=8.3 Hz), 4.36 (dd, 1H, H-90a, J9 a 9 b=12.4 Hz),
0
0
0
0
J5 6 =10.7 Hz), 4.04 (dd, 1H, H-9 b), 4.02 (dd, 1H, H-6 ,
0
4.22 (ddd, 1H, H-2, J2 3=10.2 Hz), 40.07 (ddd, 1H, H-50,
J6 7 =1.9 Hz), 3.90 (dd, 1H, H-6a, J6a 6b=11.5 Hz,
J5 6a=4.1 Hz), 3.81 (s, 3H, OCH3 ester), 3.76±3.70 (m,
1H, H-5), 3.65 (dd, 1H, H-6b, J5 6b=3.0 Hz), 3.49 (s,
0
0
0
0
J5 6 =10.7 Hz), 4.02 (dd, 1H, H-9 b), 4.00 (dd, 1H,
H-60, J6 7 =1.9 Hz), 3.88 (dd, 1H, H-6a, J5 6a=4.1 Hz,
J6a 6b=11.4 Hz), 3.81 (s, 3H, OCH3 ester), 3.74±3.68
(m, 1H, H-5), 3.65 (dd, 1H, H-6b, J5 6b=3.0 Hz), 3.47
0
0
3H, OCH3), 2.64 (dd, 1H, H-30eq, J3 eq 4 =4.6 Hz,
0
0
0
0
J3 eq 3 ax=12.7 Hz), 2.16, 2.15, 2.09, 2.04, 2.02, 1.97,
(s, 3H, OCH3), 2.63 (dd, 1H, H-30eq, J3 eq 4 =4.6 Hz,
1.87 (7Âs, 21H, CH3 acetyl), 1.95 (dd, 1H, H-30ax,
0
0
0
0
0
J3 ax 4 =12.3 Hz).
0
J3 eq 3 ax=12.7 Hz), 2.15, 2.14, 2.08, 2.03, 2.02, 1.97 1.86
0
(7Âs, 21H, CH3 acetyl), 1.94 (dd, 1H, H-30ax, J3 ax 4
0
=
12.3 Hz).
Methyl 2-(4-nitrobenzoyl)amido-3,4-di-O-acetyl-2-deoxy-
6-O-(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-D-neura-
minate-2-yl)-ꢁ-D-glucopyranoside (34). Compound 34
was prepared from acceptor 22 (180 mg; 422 mmol) and
donor 14 (160 mg; 268 mmol) as described above for the
synthesis of 27. Yield: 109 mg (121 mmol, 44%, a/b 4:1).
Isolated yield of a-isomer: 67 mg (74 mmol). 13C{1H}
NMR (CDCl3): d 171.1±165.3 (CˆO acetyl, benzoyl, C-
10), 149.6, 139.6 (Cq benzoyl), 128.3, 123.6 (CHarom
benzoyl), 101.7 (C-1), 98.8 (C-20), 73.0, 72.5, 72.2, 69.0,
68.9, 68.3, 67.4 (C-3,4,5, C-40,60,70,80), 63.7, 62.7 (C-6,
C-90), 56.5 (OCH3), 54.8 (C-2), 52.6 (OCH3 ester), 49.3
(C-50), 37.6 (C-30), 22.9 (N-acetyl), 20.6 (CH3 O-acetyl).
Methyl 2-octanoylamido-3,4-di-O-acetyl-2-deoxy-6-O-
(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-D-neurami-
nate-2-yl)-ꢁ-D-glucopyranoside (32). Compound 32 was
prepared from acceptor 20 (122 mg; 302 mmol) and
donor 14 (119 mg; 200 mmol) as described above for the
synthesis of 27. Yield: 53 mg (60 mmol, 30%, a/b 3:1).
Isolated yield of a-isomer: 34 mg (38 mmol). 13C{1H}
NMR (CDCl3): d 173.3±167.7 (CˆO acetyl, octanoyl,
C-10), 101.8 (C-1), 98.7 (C-20), 72.9, 72.5, 72.3, 69.0,
68.4, 67.4 (C-3,4,5, C-40,60,70,80), 63.4, 62.5 (C-6, C-90),
56.5 (OCH3), 54.2 (C-2), 52.6 (OCH3 ester), 49.4 (C-50),
37.6, 36.7 (C-30, CH2 octanoyl), 31.6±22.5 (CH2 octa-
noyl), 23.0 (N-acetyl), 20.6 (CH3 O-acetyl), 13.9 (CH3
b-Isomer d 98.4 (C-20). H NMR (CDCl3): d 8.29 (m,
1
4H, CHarom), 7.17 (d, 1H, NH nitrobenzoyl, JNH-2=
9.2 Hz), 5.48 (d, 1H, NH acetyl, JNH-5 =9.5 Hz), 5.40
octanoyl). b-Isomer d 98.3 (C-20). H NMR (CDCl3): d
1
0
(ddd, 1H, H-80, J8 9 a=2.4 Hz, J8 9 b=6.4 Hz), 5.34
0
0
0
0
5.64 (d, 1 H, NH octanoyl, JNH-2=9.0 Hz), 5.36 (ddd,
1H, H-80, J8 9 a=2.7 Hz, J8 9 b=5.9 Hz), 5.31 (d, 1H,
(dd, 1H, H-3, J3 4=9.6 Hz), 5.30 (dd, 1H, H-70, J7
0
0
0
0
0
0
=
8
0
8.6 Hz), 5.21 (dd, 1H, H-3, J3 4=9.4 Hz), 5.12 (dd, 1H,
0
NH acetyl, JNH-5 =8.4 Hz), 5.29 (dd, 1H, H-7 , J7
0
0
0 0
8.6 Hz, J6 7 =1.4 Hz), 5.18 (dd, 1H, H-4, J4 5=9.5 Hz),
=
8
4.88 (ddd, 1H, H-40, J4 5 =9.8 Hz), 4.62 (d, 1H, H-1,
0 0
H-4, J4 5=9.4 Hz), 4.85 (ddd, 1H, H-40, J4 5 =9.7 Hz),
J1 2=8.3 Hz), 4.40 (dd, 1H, H-90a, J9 a 9 b=12.3 Hz),
0 0
0
0

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97
93
4.25 (ddd, 1H, H-2, J2 3=10.1 Hz), 4.07 (ddd, 1H, H-50,
1H, H-30, J3 4 =3.4 Hz), 5.37 (dd, 1H, H-2, J2 3
=
=
0
0
9.5 Hz), 5.33 (dd, 1H, H-700, J6 =1.7 Hz, J7
00 00 00
7
0
0
J5 6 =10.6 Hz), 4.06±4.03 (m, 1H, H-6 ), 4.02 (dd, 1H,
0
00
8
H-90b), 3.86 (dd, 1H, H-6a, J5 6a=4.5 Hz, J6a 6b
11.5 Hz), 3.81 (s, 3H, OCH3 ester), 3.78±3.74 (m, 1H,
=
8.3 Hz), 5.04 (d, 1H, H-10, J1 2 =7.8 Hz), 4.85 (d, 1H,
0
0
H-1, J1 2=8.9 Hz), 4.79 (ddd, 1H, H-400, J4
9.6 Hz), 4.68 (dd, 1H, H-6a, J5 6a=1.9 Hz, J6a 6b
=
=
00
00
5
H-5), 3.64 (dd, 1H, H-6b, J5 6b=3.0 Hz), 3.50 (s, 3H,
OCH3), 2.63 (dd, 1H, H-30eq, J3 eq 4 =4.6 Hz, J3 eq 3 ax
0
0
0
0
=
12.8 Hz), 2.15, 2.12, 2.08, 2.05, 2.01, 1.97, 1.88 (7Âs,
12.3 Hz), 4.59 (dd, 1H, H-6b, J5 6b 5.5 Hz), 4.44 (dd,
H-900a, J9 a 9 b=12.1 Hz), 4.39 (dd, H-4, J4 5=9.6 Hz),
00
00
21H, CH3 acetyl), 1.94 (dd, 1H, H-30ax, J3 ax 4
12.3 Hz).
4.12 (dd, 1H, H-900b), 4.16 (m, 4H, H-5005,600, H-50, H-5),
0
0
=
3.53 (dd, 1H, H-60a, J5
0
0 0
60a
5.6 Hz, J6 a 6 b=10.0 Hz),
3.43 (s, 3H, OCH3 ester), 2.90 (dd, 1H, H-60b,
J5 6 b=9.6 Hz), 2.42 (dd, 1H, H-300eq, J3 eq 4 =4.5 Hz,
0
0
0
0
0
0
Methyl 2,3,6-tri-O-benzoyl-4-O-(2,3,4-tri-O-benzoyl-6-O-
(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-D-neuraminate
-2-yl)-ꢁ-D-galactopyranosyl)-ꢁ-D-glucopyranoside (35).
Compound 35 was prepared from acceptor 23 (131 mg;
129 mmol) and donor 14 (149 mg; 250 mmol) as described
above for the synthesis of 27. Yield: 49 mg (34 mmol,
26%, a/b 2:1). Isolated yield of a-isomer: 23 mg
(15 mmol). 13C{1H} NMR (CDCl3): d 170.8±164.9 (CˆO
acetyl, benzoyl, C-100), 133.6±128.2 (CHarom benzoyl),
129.0 (Cq benzoyl), 101.7, 100.6 (C-1, C-10), 98.9 (C-20),
75.8, 73.4, 72.9, 72.1, 71.9, 71.8, 70.2, 68.6, 67.6, 67.3
(C-2,3,4,5, C-20,30,40,50, C-40,60,70,80), 62.9, 62.7, 61.6 (C-
6, C-60, C-90), 56.7 (OCH3), 52.5 (OCH3 ester), 49.5 (C-
50), 37.9 (C-30), 23.1 (N-acetyl), 20.7 (CH3 O-acetyl).
00
00
J3 eq 3 ax=13.0 Hz), 2.20, 2.17, 2.09, 2.00, 1.88 (5Âs,
15H, CH3 acetyl), 1.83 (dd, 1H, H-300ax, J3 ax 4
12.5 Hz).
00
00
=
Methyl 2,3,4-tri-O-benzoyl-6-O-(methyl 4,7,8,9-tetra-O-
acetyl-N-acetyl-ꢀ-D-neuraminate-2-yl)-ꢁ-D-galactopyra-
noside (37). Compound 37 was prepared from acceptor
25 (152 mg; 300 mmol) and donor 14 (119 mg; 200 mmol)
as described above for the synthesis of 27. Yield: 92 mg
(94 mmol, 47%, a/b 6:1). 13C{1H} NMR (CDCl3): d
170.8±165.1 (CˆO acetyl, benzoyl, C-10), 133.8±128.1
(CHarom benzoyl), 129.0 (Cq benzoyl), 102.3 (C-1), 99.0
(C-20 a-isomer), 72.4, 71.9, 71.7, 69.9, 68.7, 68.4, 67.7,
67.4 (C-2,3,4,5, C-40,60,70,80), 62.7, 62.3 (C-6, C-90), 57.1
(OCH3), 52.5 (OCH3 ester), 49.4 (C-50), 37.9 (C-30), 23.0
(N-acetyl), 20.6 (CH3 O-acetyl). b-Isomer d 98.8 (C-20).
b-Isomer d 99.1 (C-20). H NMR (CDCl3): d 8.10±7.16
1
(m, 30H, CHarom), 5.78 (dd, 1H, H-3, J3 4=9.5 Hz),
5.75 (d, 1H, H-40), 5.61 (dd, 1H, H-20, J2 3 =10.2 Hz),
0
0
5.49 (ddd, 1H, H-800, J8 9 a=2.9 Hz, J8 9 b=6.7 Hz),
00
00
00
00
5.44 (dd, 1H, H-30, J3 4 =3.1 Hz), 5.38 (dd, 1H, H-2,
Methyl 2,3,4-tri-O-acetyl-6-O-(methyl 4,7,8,9-tetra-O-
acetyl-N-acetyl-ꢀ-D-neuraminate-2-yl)-ꢁ-D-galactopyra-
noside (39). Compound 37 (78 mg; 79 mmol) was dis-
solved in a methanolic solution of sodium methanolate
(0.05 M; 2 mL), and stirred overnight. The solution was
neutralised by the addition of Dowex/H+ and ®ltered.
The ®ltrate was evaporated to dryness, and dissolved in
a mixture of pyridine and acetic anhydride (1:1, v/v;
10 mL). This mixture was stirred overnight and then
concentrated, evaporated with toluene (2Â10 mL) and
ethanol (10 mL), and applied to a silica gel column.
Elution with ethanol:toluene (0:1!1:9, v/v) a€orded the
pure a-isomer of 39. Isolated yield of a-isomer: 37 mg
(47 mmol). 1H NMR (CDCl3): d 5.44 (dd, 1H, H-4,
J4 5=0.8 Hz, J3 4=3.3 Hz), 5.36 (ddd, 1H, H-80,
0
0
J2 3=9.6 Hz), 5.32 (dd, 1H, H-700, J7 =8.4 Hz), 5.21
00
00
8
0
J1 2 =7.8 Hz), 4.78 (ddd, 1H, H-4 , J4 =10.1 Hz),
00
(d, 1H, NH acetyl, JNH-5 = 9.7 Hz), 5.00 (d, 1H, H-1 ,
00
4.66 (dd, 1H, H-6a, J5 6a= 1.7 Hz, J6a 6b=12.0 Hz),
0
0
00
00
5
4.60 (d, 1H, H-1, J1 2= 7.9 Hz), 4.55 (dd, 1H, H-6b,
J5 6b=5.7 Hz), 4.43 (dd, 1H, H-900a, J9 a 9 b=12.3 Hz),
00
00
4.35 (dd, 1H, H-4, J4 5=9.6 Hz), 4.11 (dd, 1H, H-900b),
00
4.10 (dd, 1H, H-600, J6 =1.8 Hz), 4.03 (dd, 1H, H-5 ,
00
00
7
0
00
00
J5 =10.6 Hz), 3.91±3.86 (m, 2H, H-5, H-5 ), 3.51 (dd,
6
1H, H-60a, J5 6 a=5.5 Hz, J6 a 6 b=9.9 Hz), 3.42, 3.41
0
0
0
0
(2Âs, 6H, OCH3), 2.87 (dd, 1H, H-60b, J5 6 b=9.5 Hz),
0
0
H-300eq,
00 00
J3 eq 4 =4.6 Hz,
2.40
00
(dd,
1H,
00
J3 eq 3 ax=12.8 Hz), 2.18, 2.16, 2.09, 2.00, 1.89 (5Âs,
15H, CH3 acetyl), 1.81 (dd, 1H, H-300ax, J3 ax 4
12.5 Hz).
00
00
J8 9 a=2.7 Hz, J8 9 b=5.7 Hz), 5.28 (dd, 1H, H-70,
0
0
0
0
0
0
0
J7 8 =8.8 Hz), 5.23 (d, 1H, NH acetyl, JNH-5 =9.6 Hz),
5.18 (dd, 1H, H-2, J2 3=10.5 Hz), 5.05 (dd, 1H, H-3,
2,3,6-Tri-O-benzoyl-4-O-(2,3,4-tri-O-benzoyl-6-O-
(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-D-neuraminate-
2-yl)-ꢁ-D-galactopyranosyl)-ꢁ-D-glucopyranosyl azide
(36). Compound 36 was prepared from acceptor 24
(131 mg; 129 mmol) and donor 14 (149 mg; 250 mmol) as
described above for the synthesis of 27. Yield: 98 mg
(67 mmol, 33%, a/b 3:1). Isolated yield of a-isomer:
59 mg (40 mmol). 13C{1H} NMR (CDCl3): d 170.8±164.9
(CˆO acetyl, benzoyl, C-100), 133.3±128.3 (CHarom ben-
zoyl), 128.9 (Cq benzoyl), 100.3 (C-10), 98.9 (C-20), 87.9
(C-1), 75.6, 75.3, 72.8, 72.4, 71.9, 71.5, 70.1, 68.6, 68.5,
67.6, 67.2 (C-2,3,4,5, C-20,30,40,50, C-40,60,70,80), 62.8,
61.6 (C-6, C-60, C-90), 52.5 (OCH3 ester), 49.4 (C-500),
37.8 (C-300), 23.0 (N-acetyl), 20.7 (CH3 O-acetyl). b-Iso-
J3 4=3.3 Hz), 4.85 (ddd, 1H, H-40, J4 5 =9.7 Hz), 4.46
0
0
(d, 1H, J1 2=7.9 Hz), 4.33 (dd, 1H, H-90a, J9 a 9 b
0
0
=
12.4 Hz), 4.09 (dd, 1H, H-60, J6 7 =1.9 Hz), 4.06 (dd,
0
0
1H, H-90b), 4.03 (ddd, 1H, H-50, J5 6 =10.8 Hz), 3.95±
3.91 (bt, 1H, H-5, J5 6a=6.0 Hz, J5 6b=7.6 Hz), 3.83
(dd, 1H, H-6a, J6a 6b=10.1 Hz), 3.79 (s, 3H, OCH3
ester), 3.53 (s, 3H, OCH3), 3.40 (dd, 1H, H-6b), 2.53
0
0
(dd, 1H, H-30eq, J3 eq 4 =4.6 Hz, J3 eq 3 ax=12.9 Hz),
2.18, 2.14, 2.13, 2.06, 2.03, 2.02, 1.97, 1.88 (8Âs, 24H,
0
0
0
0
CH3 acetyl), 1.91 (dd, 1H, H-30ax, J3 ax 4 =12.3 Hz).
0
0
4-Nitrophenyl 2,3,4-tri-O-benzoyl-6-O-(methyl 4,7,8,9-
tetra-O-acetyl-N-acetyl-ꢀ-D-neuraminate-2-yl)-ꢁ-D-
galactopyranoside (38). Compound 38 was prepared
from acceptor 26 (184 mg; 300 mmol) and donor 14
(119 mg; 200 mmol) as described above for the synthesis
of 27. Yield: 114 mg (105 mmol, 52%, a/b 4:1). 13C{1H}
mer d 99.1 (C-20). H NMR (CDCl3): d 8.11±7.16 (m,
1
30H, CHarom), 5.84 (dd, 1H, H-3, J3 4=9.6 Hz), 5.77 (d,
1H, H-40), 5.62 (dd, 1H, H-20, J2 3 =10.3 Hz), 5.50
0
0
(ddd, 1H, H-800, J8 9 a=3.1 Hz, J8 9 b 6.8 Hz), 5.46 (dd,
00
00
00
00

94
L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97
1
NMR (CDCl3): d 170.9±165.0 (CˆO acetyl, benzoyl, C-
10), 161.6, 142.8 (CHarom phenyl), 133.8±128.2 (CHarom
benzoyl), 129.2 (Cq benzoyl), 125.8, 117.0 (Cq phenyl),
99.8 (C-20 a-isomer), 98.4 (C-1), 72.8, 72.4, 71.5, 69.5,
68.5, 67.7, 67.4 (C-2,3,4,5, C-40,60,70,80), 63.7, 62.9 (C-6,
C-90), 52.7 (OCH3 ester), 49.2 (C-50), 37.7 (C-30), 23.0
(N-acetyl), 20.7 CH3 O-acetyl). b-Isomer d 98.8 (C-20).
(41 mmol, 51%). H NMR (D2O): d 7.78±7.49 (m, 5H,
CHarom), 4.57 (d, 1H, J1 2=8.5 Hz), 3.50 (s, 3H,
OCH3), 2.76 (dd, 1H, H-30eq, J3 eq 4 =4.4 Hz,
0
0
0
0
J3 eq 3 ax=12.3 Hz), 2.03 (s, 3H, CH3 acetyl), 1.73 (dd,
0
1H, H-30ax, J3 ax 4 =12.0 Hz). HRMS: calculated for
0
[C25H36N2O14+Na+] 611.2064, found 611.2079.
Methyl 2-octanoylamido-2-deoxy-6-O-(N-acetyl-ꢀ-D-
neuraminate-2-yl)-ꢁ-D-glucopyranoside (5). Yield: 17 mg
4-Nitrophenyl 2,3,4-tri-O-acetyl-6-O-(methyl 4,7,8,9-
tetra-O-acetyl-N-acetyl-ꢀ-D-neuraminate-2-yl)-ꢁ-D-
galactopyranoside (40). Compound 38 (93 mg, 85 mmol)
was treated with sodium methanolate and then acetyl-
ated as described above for the synthesis of 39. Silica gel
chromatography, with ethanol:toluene (1:0!1:9, v/v) as
eluent, a€orded the pure a-isomer of 40. Isolated yield
of a-isomer: 38 mg (42 mmol). 1H NMR (CDCl3): d 8.21±
7.21 (m, 4H, CHarom), 5.59 (d, 1H, H-4), 5.52 (dd, 1H,
1
(28 mmol, 74%). H NMR (D2O): d 4.39 (d, 1H, H-1,
J1 2=8.4 Hz), 3.48 (s, 3H, OCH3), 2.72 (dd, 1H, H-30eq,
0
0
0
0
J3 eq 4 =4.6 Hz, J3 eq 3 ax=12.4 Hz), 2.25 (t, 2H,
CH2CˆO octanoyl, J=7.2 Hz), 2.01 (s, 3H, CH3 ace-
tyl), 1.70 (dd, 1H, H-30ax, J3 ax 4 =12.0 Hz), 1.58 (bt,
2H, CH2 octanoyl), 1.27 (bs, 8H, CH2 octanoyl), 0.84 (t,
3H, CH3 octanoyl, J=6.8 Hz). HRMS: calculated for
[C26H46 N2O14+Na+] 633.2847, found 633.2863.
0
0
H-2, J2 3=10.0 Hz), 5.50 (ddd, 1H, H-80, J8 9 a=3.0Hz,
0
0
0
0
J8 9 b=7.1 Hz), 5.44 (d, 1H, H-1, J1 2=7.9Hz), 5.29
Methyl 2-naphthoylamido-2-deoxy-6-O-(N-acetyl-ꢀ-D-
neuraminate-2-yl)-ꢁ-D-glucopyranoside (6). Yield: 19 mg
(dd, 1H, H-3, J3 4=3.5 Hz), 5.24 (dd, 1H, H-70,
0
1
0
0
0
0
J7 8 =9.9Hz), 4.85 (ddd, 1H, H-4 , J4 5 =10.0 Hz), 4.37
0
(30 mmol, 70%). H NMR (D2O): d 8.32±7.61 (m, 7H,
CHarom), 4.65 (d, 1H, H-1, J1 2=8.5 Hz), 3.56 (s,
(dd, 1H, H-90a, J9 a 9 b=12.2 Hz), 4.34 (dd, 1H, H-5,
0
J5 6a=6.5 Hz, J5 6b=8.5 Hz), 4.16 (dd, 1H, H-60,
3H, OCH3), 2.80 (dd, 1H, H-30eq, J3 eq 4 =4.5 Hz,
0
0
0
0
0
0
0
0 0
J3 eq 3 ax=12.3 Hz), 2.07 (s, 3H, CH3 acetyl), 1.78 (dd,
J5 6 =10.7 Hz, J6 7 =1.8 Hz), 4.14±4.05 (m, 1H, H-5 ),
3.98 (dd, 1H, H-90b), 3.81 (s, 3H, OCH3 ester), 3.67 (dd,
1H, H-6a, J6a 6b=11.5 Hz), 3.55 (dd, 1H, H-6b), 2.53
1H, H-30ax, J3 ax 4 =12.0 Hz). HRMS: calculated for
0
0
[C29H38 N2O14+Na+] 661.2221, found 661.2201.
(dd, 1H, H-30eq, J3 eq 4 =4.6 Hz, J3 eq 3 ax=12.9 Hz),
2.24, 2.22, 2.17, 2.08, 2.03, 2.01, 1.91, 1.90 (8Âs, 24H,
0
0
0
0
Methyl 2-(4-nitrobenzoylamido)-2-deoxy-6-O-(N-acetyl-
ꢀ-D-neuraminate-2-yl)-ꢁ-D-glucopyranoside (7). Yield:
CH3 acetyl), 1.93 (dd, 1H, H-30ax, J3 ax 4 =12.3 Hz).
0
0
1
35 mg (55 mmol, 74%). H NMR (D2O): d 8.33 (d, 2H,
CHarom, J=9.0 Hz), 7.95 (d, 2H, CHarom), 4.58 (d, 1H,
Methyl 2-acetamido-2-deoxy-6-O-(N-acetyl-ꢀ-D-neura-
minate-2-yl)-ꢁ-D -glucopyranoside (1). Compound 27
(45 mg; 49mmol) was dissolved in a methanolic solution of
sodium methanolate (0.05 M; 2 mL), and stirred over-
night. TLC analysis (2-propanol:water 4:1, v/v) revealed
the formation of a single product. The solution was neu-
tralised by the addition of Dowex/H+ and ®ltered. The
®ltrate was evaporated to dryness and dissolved in a
solution of NaOH (0.1 M) in 1,4-dioxane:water (3:1, v/v;
2 mL). The solution was carefully neutralised by the
addition of Dowex/H+ and ®ltered. The ®ltrate was
evaporated to dryness and applied to an HW40 S gel
®ltration column, with water as eluent. The product
fractions were pooled, concentrated and lyophilised.
Yield of 1: 23 mg (44 mmol, 90%). Characteristic 1H
NMR (D2O) signals: d 4.40 (d, 1H, H-1, J1 2=8.4 Hz),
H-1, J1 2=8.5 Hz), 3.51 (s, 3H, OCH3), 2.75 (dd, 1H,
0
H-30eq, J3 eq 4 =4.6 Hz, J3 eq 3 ax=12.3 Hz), 2.02 (s, 3H,
0
0
0
CH3 acetyl), 1.73 (dd, 1H, H-30ax, J3 ax 4 =12.0 Hz).
Mass: calculated for C25H35N3O16 m/e 633.2, found
656.6 [M+Na+].
0
0
2-Acetamido-2-deoxy-6-O-(N-acetyl-ꢀ-D-neuraminate-2-
yl)-ꢁ-D-glucopyranosyl azide (8). Yield: 27 mg (50 mmol,
=
90%). 1H NMR (D2O): d 4.73 (d, 1H, H-1, J1 2
9.2 Hz), 2.73 (dd, 1H, H-30eq, J3 eq 4 =4.4 Hz,
0
0
0
0
J3 eq 3 ax=12.3 Hz), 2.04, 2.03 (2Âs, 6H, CH3 acetyl),
1.69 (dd, 1H, H-30ax, J3 ax 4 =11.9 Hz). HRMS: calcd
0
0
for [C19 H31N5O13+Na+] 560.1816, found 560.1823.
4-Nitrophenyl 2-acetamido-2-deoxy-6-O-(N-acetyl-ꢀ-D-
neuraminate-2-yl)-ꢁ-D-glucopyranoside (9). Yield: 11 mg
(17 mmol, 79%). 1H NMR (D2O): d 8.24, 7.18 (2Âd, 4H,
CHarom, J=9.3 Hz), 5.28 (d, 1H, H-1, J1 2=8.4 Hz),
3.47 (s, 3H, OCH3), 2.72 (dd, 1H, H-30eq, J3 eq 4
0
0
=
0
0
4.7 Hz, J3 eq 3 ax=12.4 Hz), 2.01, 2.00 (2Âs, 6H, CH3
acetyl), 1.69 (dd, 1H, H-30ax, J3 ax 4 =12.0 Hz). HRMS:
calculated for [C20H34N2O14+Na+] 549.1908, found
549.1918.
0
0
2.73 (dd, 1H, H-30eq, J3 eq 4 =4.4 Hz, J3 eq 3 ax
=
0
0
0
0
12.3 Hz), 2.00 (s, 6H, CH3 acetyl), 1.65 (dd, 1H, H-30ax,
0
0
J3 ax 4 =12.0 Hz). HRMS: calcd for [C25H35N3O16+
Methyl 2-azido-2-deoxy-6-O-(N-acetyl-ꢀ-D-neuraminate-
2-yl)-ꢁ-D-glucopyranoside (2). Yield: 13 mg (25 mmol,
=
Na+] 656.1915, found 656.1931.
89%). 1H NMR (D2O): d 4.41 (d, 1H, H-1, J1 2
Methyl 4-O-(6-O-(N-acetyl-ꢀ-D-neuraminate-2-yl)-ꢁ-D-
galactopyranosyl)-ꢁ-D-glucopyranoside (10). Yield: 10 mg
8.2 Hz), 3.57 (s, 3H, OCH3), 3.28 (dd, 1H, H-2,
J2 3=9.4 Hz), 2.72 (dd, 1H, H-30eq, J3 eq 4 =4.5 Hz,
(15 mmol, 99%). H NMR (D2O): d 4.40 (d, 1H, H-10,
1
0
0
0
0
0 0
J1 2 =7.8 Hz), 4.39 (d, 1H, H-1, J1 2=8.0 Hz), 3.55 (s,
J3 eq 3 ax=12.3 Hz), 2.01 (s, 3H, CH3 acetyl), 1.69 (dd,
0
1H, H-30ax, J3 ax 4 =12.1 Hz). HRMS: calculated for
3H, OCH3), 3.51 (dd, 1H, H-20, J2 3 =10.0 Hz), 3.31 (dd,
0
0
0
[C18H30 N4O13+Na+] 533.1707, found 533.1679.
1H, H-2, J2 3=8.6 Hz), 2.69 (dd, 1H, H-300eq, J3 eq 4
00
00
=
4.7 Hz, J3 eq 3 ax=12.3 Hz), 2.00 (s, 3H, CH3 acetyl),
00
00
1.72 (dd, 1H, H-300ax, J3 ax 4 =12.1 Hz). HRMS: calcd
00
00
Methyl 2-benzoylamido-2-deoxy-6-O-(N-acetyl-ꢀ-D-neur-
aminate-2-yl)-ꢁ-D-glucopyranoside (4). Yield: 24 mg
for [C24H41NO19+Na+] 670.2170, found 670.2165.

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97
95
4-O-(6-O-(N-Acetyl-ꢀ-D-neuraminate-2-yl)-ꢁ-D-galacto-
pyranosyl)-ꢁ-D-glucopyranosyl azide (11). Yield: 26 mg
(10.9 mmol, 72%). 13C{1H} NMR (CDCl3): d 170.4±168.8
(CˆO acetyl), 99.9 (C-1), 71.8, 70.6, 68.1 (C-3,4,5), 69.1,
69.0, 68.0 (CH2O glycol, C-6), 53.3 (C-2), 49.7 (CH2N3),
21.7 (CH3 N-acetyl), 19.4 (CH3 O-acetyl).
1
(39 mmol, 99%). H NMR (D2O): d 4.75 (d, 1H, H-1,
J1 2=8.8 Hz), 4.40 (d, 1H, H-10, J1 2 =7.8 Hz), 3.51
0
0
(dd, 1H, H-20, J2 3 =9.9 Hz), 3.32 (dd, 1H, H-2, J2 3
=
=
0
0
8.9 Hz), 2.68 (dd, 1H, H-30eq, J3 eq 4 =4.7 Hz, J3 eq 3 ax
12.3 Hz), 2.01 (s, 3H, CH3 acetyl), 1.71 (dd, 1H, H-30ax,
1-Azido-3-oxopentyl 2-amino-2-deoxy-ꢁ-D-glucopyrano-
side (43). A solution of the azido derivative 42 (1.38 g;
3.0 mmol) in aq NaOH (1 M; 150 mL) was re¯uxed over-
night. The mixture was allowed to cool to room tempera-
ture, and was neutralised by the addition of aq HCl (1 M).
The mixture was concentrated, evaporated with methanol
(3Â50mL) and ®ltered. The ®ltrate, containing the crude
amine 43, was concentrated and led over a silica gel col-
umn, with ethyl acetate:methanol:water (7:2:1, v/v/v) as
eluent. The product, still contaminated with salts, was
used immediately for the synthesis of 45.
0
0
0
0
0
0
J3 ax 4 =12.1 Hz). HRMS: calcd for [C22H38N4O18+
Na+] 681.2079, found 681.2118.
Methyl 6-O-(N-acetyl-ꢀ-D-neuraminate-2-yl)-ꢁ-D-galac-
topyranoside (12). Yield: 13 mg (26 mmol, 55%). 1H
NMR (D2O): d 4.27 (d, 1H, H-1, J1 2=7.9 Hz), 3.53 (s,
3H, OCH3), 3.45 (dd, 1H, H-2, J2 3=10.0 Hz), 2.69 (dd,
1H, H-30eq, J3 eq 4 =4.6 Hz, J3 eq 3 ax=12.3 Hz), 1.99 (s,
0
0
0
0
3H, CH3 acetyl), 1.66 (dd, 1H, H-30ax, J3 ax 4
12.0 Hz). HRMS: calcd for [C18H31NO14+Na+]
508.1642, found 508.1639.
0
0
=
1-Azido-3-oxopentyl 2-(4-nitrobenzoyl)amido-3,4-di-O-
acetyl-2-deoxy-ꢁ-D-glucopyranoside (45). The crude
amine 43 (ꢁ3 mmol) was evaporated with dry toluene
(2Â20 mL), and dissolved in pyridine (30 mL). The
solution was placed in an ice bath. Triethylamine
(0.42 mL; 3.0 mmol) and 4-nitrobenzoyl chloride (2.22 g;
12 mmol) were added, and the mixture was stirred
overnight. TLC analysis (ethanol:toluene 1:9, v/v)
revealed the absence of 43. The reaction was quenched
by the addition of water (1 mL), the mixture was con-
centrated, taken up in DCM (50 mL), washed with aq
NaHCO3 (50 mL) and water (50 mL), dried (MgSO4)
and evaporated with toluene (2Â20 mL) and ethanol
(20 mL). The product mixture, obtained as a viscous oil,
was dissolved in a methanolic solution of sodium
methanolate (0.1 M; 50 mL), and stirred overnight.
According to TLC analysis (toluene:methanol 1:1, v/v)
one product was formed. The solution was neutralised
by the addition of Dowex/H+ and ®ltered. The ®ltrate,
containing the crude 4-nitrobenzoylamido glucopyr-
anoside 44, was concentrated, evaporated with dry
toluene (2Â20 mL), and dissolved in pyridine (30 mL).
tBDMS-Cl (0.46 g; 3.0 mmol) was added and the mix-
ture was stirred for 3 h. TLC analysis (toluene:methanol
1:1, v/v) indicated complete conversion of the starting
material. Acetic anhydride (2 mL) was added and the
mixture was stirred overnight. TLC analysis (hexanes/
ethyl acetate) showed the acetylation to be complete.
The mixture was concentrated, taken up in DCM
(100 mL), washed with aq NaHCO3 (1 M, 100 mL) and
water (100 mL), dried over MgSO4 and evaporated with
toluene (2Â10 mL) and ethanol (10 mL). The fully
protected product thus obtained was dissolved in
acetonitrile:water (7:1, v/v; 16 mL) and the pH of the
mixture was adjusted to 3 by the addition of para-toluene-
sulfonic acid monohydrate. After 2 h, TLC analysis
(DCM:methanol 95:5, v/v) showed the removal of the
tBDMS group to be complete. The reaction mixture was
diluted with ethyl acetate (50 mL), washed with aq
NaHCO3 (1 M; 50 mL) and water (50 mL), dried
(MgSO4) and concentrated. The crude product was
applied to a silica gel column, and eluted with DCM:
MeOH (1:0!9:1, v/v). Yield: 0.51 g (0.97 mmol, 32%
based on 42). 13C{1H} NMR (CDCl3): d 170.6±166.1
(CˆO acetyl, benzoyl), 149.1, 139.5 (Cq benzoyl), 127.9,
123.1 (CHarom benzoyl), 100.2 (C-1), 73.9, 72.5, 68.7
4-Nitrophenyl 6-O-(N-acetyl-ꢀ-D-neuraminate-2-yl)-ꢁ-D-
galactopyranoside (13). Yield: 12 mg (20 mmol, 91%).
1H NMR (D2O): d 8.25 (d, 2H, CHarom, J=9.3 Hz),
7.23 (d, 2H, CHarom), 5.16 (d, 1H, H-1, J1 2=7.3 Hz),
2.73 (dd, 1H, H-30eq, J3 eq 4 =4.2 Hz, J3 eq 3 ax
=
0
0
0
0
12.3 Hz), 1.97 (s, 3H, CH3 acetyl), 1.61 (dd, 1H, H-30ax,
0
0
J3 ax 4 =11.8 Hz). HRMS: calcd for [C23H32N2O16+
Na+] 615.1650, found 615.1627.
Methyl 2-amino-2-deoxy-6-O-(N-acetyl-ꢀ-D-neuraminate-
2-yl)-ꢁ-D-glucopyranoside (3). Compound 2 (14 mg;
28 mmol) was dissolved in dry methanol (140 mL). To
this solution were added triethylamine (20 mL; 140 mmol)
and 1,3-propanedithiol (14 mL; 140 mmol). The mixture
was left at room temperature, until TLC analysis (2-
propanol:water 4:1, v/v) indicated complete conversion of
the starting material into a ninhydrine-positive product.
The mixture was ®ltered, the ®ltrate was concentrated and
then evaporated with 1,4-dioxane (1Â10 mL). The crude
product thus obtained was lyophilised from 1,4-dioxane,
and then applied to an HW40 S gel ®ltration column,
with water as eluent. The product fractions were pooled,
concentrated and lyophilised. Yield: 2.4 mg (5.0 mmol,
18%). 1H NMR (D2O): d 4.61 (d, 1H, H-1, J1 2=8.1 Hz),
3.57 (s, 3H, OCH3), 2.73 (dd, 1H, H-30eq,
0
0
0
0
J3 eq 4 =4.5 Hz, J3 eq 3 ax=12.3 Hz), 2.02 (s, 3H, CH3
0
acetyl), 1.70 (dd, 1H, H-30ax, J3 ax 4 =12.1 Hz). HRMS:
calcd for [C18H32N2O13+H+] 485.1983, found 485.1984.
0
1-Azido-3-oxopentyl 2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-ꢁ-D-glucopyranoside (42). To a solution of the
known30 oxazoline of glucosamine (41) (4.94 g;
15 mmol) and 2-(2-azidoethoxy)ethanol (2.95 g; 22.5
mmol) in 1,2-dichloroethane (DCE, 50 mL) was added,
under an argon atmosphere, a catalytic amount of
TfOH (130 mL, 1.5 mmol). The reaction was stirred
overnight at 40 ꢀC. According to TLC analysis (DCM:
methanol 9:1, v/v), the starting material was completely
converted into a product with lower mobility. The mix-
ture was taken up in DCM (50 mL), washed with aq
NaHCO3 (1 M, 50 mL) and water (50 mL), dried over
MgSO4 and concentrated. The crude oil thus obtained
was puri®ed by silica gel column chromatography, with
DCM:methanol (100:0!96:4, v/v) as eluent. Yield: 5.02g

96
L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97
(C-3,4,5), 69.7, 69.3, 68.4 (OCH2 glycol), 60.6 (C-6),
54.4 (C-2), 50.1 (CH2N3), 19.8 (CH3 O-acetyl). 1H
NMR (CDCl3): d 8.00±7.67 (m, 4H, CHarom benzoyl),
5.13 (dd, 1H, H-3, J3 4=9.6 Hz), 4.81 (dd, 1H, H-4,
J4 5=8.9 Hz), 4.59 (d, 1H, H-1, J1 2=8.6 Hz), 3.93 (dd,
1H, H-2, J2 3=10.5 Hz), 3.78 (m, 1H, H-5), 3.55±2.95
(m, 10H, H-6a,6b, CH2 glycol), 1.79, 1.71 (2Âs, 6H,
CH3 acetyl).
20 mL; 250 mmol) was added, followed by 1,4-dioxane
(10 mL). The mixture was concentrated, and evaporated
with 1,4-dioxane (2Â10 mL). The crude amine 48 thus
obtained was used immediately for the synthesis of the
cluster 50.
[Ac4NeuAcMeAc2GlcN(BzNO2)O(CH2)2O(CH2)2NHC
(O)(CH2)2OCH2]3CNHC(O)CH2NHZ (50). The amine
48 (90 mmol) and the tris-carboxylic acid 49 (11 mg;
20 mmol) were dissolved in DMF (5 mL), and DIPEA
(41 mL; 0.24 mmol) and HOBT (10 mg; 75 mmol) were
added. To this mixture was added HBTU (28 mg;
75 mmol). The reaction was stirred for 3 h, when TLC
analysis (DCM:methanol 9:1, v/v) revealed the reaction to
be complete. The mixture was taken up in DCM (40 mL),
washed with dil. H3PO4 (1 M; 50 mL), aq NaHCO3 (1 M;
50 mL), and water (50 mL), dried (Mg SO4), concentrated
and evaporated with toluene (2Â20mL) and ethanol
(20 mL). The crude product was puri®ed by silica gel
column chromatography, with DCM:methanol (1:0!
92:8, v/v) as eluent, followed by Sephadex LH-20 gel
®ltration, with DCM:methanol (2:1, v/v) as eluent.
Yield: 44 mg (13.0 mmol, 65%). 13C{1H} NMR (CDCl3):
d 171.7±165.8 (CˆO acetyl, benzoyl, C-10), 156.7 (CˆO
Z), 149.6, 140.0 (Cq benzoyl), 136.3 (Cq Z), 128.5±123.7
(CHarom benzoyl, Z), 101.0 (C-1), 98.8 (C-20), 72.9, 72.5,
69.2, 69.0, 68.3, 67.5 (C-3,4,5, C-40,60,70,80), 70.1 69.7,
69.4, 68.6 66.9 (OCH2 TRIS, propanoyl, glycol, CH2 Z),
63.6, 62.6 (C-6, C-90), 60.0 (Cq TRIS), 55.1 (C-2), 52.7
(OCH3 ester), 49.4 (C-50), 44.7 (CH2 Gly), 39.3, 37.6,
36.5 (C-3, CH2CˆO propanoyl, CH2NH), 23.0 (CH3 N-
1-Azido-3-oxopentyl 2-(4-nitrobenzoyl)amido-3,4-di-O-
acetyl-2-deoxy-6-O-(methyl 4,7,8,9-tetra-O-acetyl-N-ace-
tyl-ꢀ-D-neuraminate-2-yl)-ꢁ-D-glucopyranoside (47). A
mixture of acceptor 45 (262 mg; 0.500 mmol) and donor
46 (521 mg; 1.00 mmol)31 and powdered molecular
sieves (3 A) in acetonitrile (10 mL) was stirred for 1 h
under argon atmosphere. The temperature of the mix-
ture was lowered to 50 ꢀC, and a solution of NIS
(225 mg; 1.00 mmol) and TfOH (10 mL; 0.11 mmol) in
acetonitrile (1 mL) was added. The mixture was gradu-
ally warmed to room temperature over a period of 3 h.
The reaction was quenched by the addition of triethyl-
amine (0.1 mL). The mixture was ®ltered over Hy¯o,
diluted with ethyl acetate (50 mL), washed with aq
Na2S2O3 (1 M; 50 mL), aq NaHCO3 (1 M; 50 mL) and
water (50 mL), dried (MgSO4), concentrated, and
applied to a silica gel column. Elution was performed
with DCM:methanol (1:0!96:4, v/v). The fractions
containing the product were pooled, concentrated, and
applied to a Sephadex LH-20 column. The disaccharide
47 was obtained as a/b mixture. Yield: 214 mg
(214 mmol, 45%, a/b 3:1). The a-isomer was isolated
from the latter mixture by silica gel chromatography,
with toluene:ethanol (1:0!9:1, v/v) as eluent. Yield:
143 mg (143 mmol). 13C{1H} NMR (CDCl3): d 171.0±
165.4 (CˆO acetyl, benzoyl, C-10), 149.5, 139.5 (Cq
benzoyl), 128.2, 123.6 (CHarom benzoyl), 101.2 (C-1),
98.7 (C-20), 72.9, 72.3, 68.9, 68.3, 67.4 (C-3,4,5, C-
40,60,70,80), 70.6, 69.6, 68.5 (OCH2 glycol), 63.6, 62.5 (C-
6, C-90), 54.8 (C-2), 52.6 (OCH3 ester), 50.7 (CH2N3),
49.2 (C-50), 37.5 (C-30), 22.9 (CH3 N-acetyl), 20.6 (CH3
1
acetyl), 21.0, 20.6 (CH3 O-acetyl). H NMR (CDCl3):
d 8.30±7.19 (m, 12H, CHarom benzoyl), 7.31 (bs, 5H,
CHarom Z), 6.87 (d, 3H, NH benzoyl, J=9.3 Hz), 5.39
(ddd, 3H, H-80, J8 9 a=2.7 Hz, J8 9 b=6.2 Hz), 5.30±
0
0
0
5.17 (m, 11H, CH2 Z, H-3,4, H-70), 4.87 (ddd, 3H, H-40,
0
0
0
0
J4 5 =9.7 Hz, J3 ax 4 =12.3 Hz), 4.76 (d, 3H, H-1,
J1 2=8.4 Hz), 4.35 (dd, 3H, H-90a, J9 a 9 b=12.3 Hz),
4.27 (ddd, 3H, H-2, J2 3=9.3 Hz, J3 4=9.4 Hz), 4.10±
3.18 (m, 62H, H-5,6a,6b, H-50,60,90b, CH2 Gly, OCH2
propanoyl, CH2 glycol, OCH2 TRIS, CH2CˆO propa-
noyl), 3.81 (s, 9H, OCH3 ester), 2.63 (dd, 3H, H-30eq,
0
0
O-acetyl). b-Isomer: d 98.3 (C-20). H NMR (CDCl3): d
1
8.25±7.96 (m, 4H, CHarom benzoyl), 5.61 (dd, 1H, H-3,
J3 4=9.6 Hz), 5.38 (ddd, 1H, H-80, J8 9 a=2.5 Hz,
0
0
0
0
0
0
J3 eq 4 =4.6 Hz, J3 eq 3 ax=12.8 Hz), 2.15, 2.09, 2.04,
2.02, 1.98, 1.88 (6Âs, 63H, CH3 acetyl), 1.94 (dd, 3H,
H-30ax).
J8 9 b=6.0 Hz), 5.30 (dd, 1H, H-70, J7 8 =8.7 Hz,
0
0
0
0
0
0
J6 7 =11.5 Hz), 5.17 (dd, 1H, H-4, J4 5=9.6 Hz), 4.88
(m, 1H, H-40), 4.84 (d, 1H, H-1, J1 2=8.5 Hz), 4.34 (dd,
1H, H-90a, J9 a 9 b=9.9 Hz), 4.15 (ddd, 1H, H-2,
J2 3=9.0 Hz), 4.07±3.16 (m, 14H, H-5,6a,6b, H-50,60,90b,
CH2 glycol), 3.80 (s, 3H, OCH3 ester), 2.62 (dd, 1H, H-
[NeuAcGlcN(BzNO2)O(CH2)2O(CH2)2NHC(O)(CH2)2
OCH2]3CNHC(O)CH2NHZ (52). Compound 50
(44 mg; 13 mmol) was dissolved in a methanolic solution
of sodium methanolate (0.05 M; 2 mL), and stirred
overnight. According to TLC analysis (2-propanol:
water 4:1, v/v) one product was formed. The solution
was neutralised by the addition of Dowex/H+ and ®l-
tered. The ®ltrate was evaporated to dryness and dis-
solved in a solution of NaOH (0.1 M) in 1,4-dioxane:
water (3:1, v/v; 2 mL). The solution was carefully neutral-
ised by the addition of Dowex/H+ and ®ltered. The ®l-
trate was evaporated to dryness and applied to an
HW40 S gel ®ltration column, with water as eluent. The
product fractions were pooled, concentrated and
lyophilised. Yield: 16 mg (6 mmol, 47%). 1H NMR
(D2O): d 8.26±7.91 (m, 12 H, CHarom benzoyl), 7.30 (bs,
5H, CHarom Z), 5.02 (s, 2H, CH2 Z), 4.68 (d, 3H, H-1,
0
0
30eq, J3 eq 4 =4.5 Hz, J3 eq 3 ax=12.7 Hz), 2.14, 2.13,
2.07, 2.03, 2.01, 1.96, 1.88 (7Âs, 21H, CH3 acetyl), 1.93
(dd, 1H, H-30ax).
0
0
0
0
1-Amino-3-oxopentyl 2-(4-nitrobenzoylamido)-2-deoxy-6-
O-(N-acetyl-ꢀ-D-neuraminate-2-yl)-ꢁ-D-glucopyranoside
(48). The azido disaccharide 47 (122 mg; 122 mmol) was
dissolved in dry THF (2 mL). To the solution was added
triphenylphosphine (40 mg; 0.15 mmol), and the mixture
was stirred for 48 h. Water (6 mL; 350 mmol) was added,
and the mixture was stirred for another 24 h. According
to TLC analysis (toluene:methanol 1:1, v/v), the starting
material was completely converted into a lower-running
ninhydrine-positive product. Tri¯uoroacetic acid (TFA,

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97
97
J1 2=8.4 Hz), 2.73 (dd, 3H, H-30eq, J3 eq 4 =4.5Hz,
6. Doody, G. M.; Dempsey, P. W.; Fearon, D. T. Curr. Opin.
Immun. 1996, 8, 378.
7. Sgroi, D.; Varki, A.; Braesch Andersen, S.; Stamenkovic, I.
J. Biol. Chem. 1993, 268, 7011.
8. Powell, L. D.; Sgroi, D.; Sjoberg, E. R.; Stamenkovic, I.;
Varki, A. J. Biol. Chem. 1993, 268, 7019.
0
0
0
0
J3 eq 3 ax= 12.3 Hz), 2.01 (s, 9H, CH3 acetyl), 1.71 (dd,
0
3H, H-30ax, J3 ax 4 =12.0 Hz). Mass: calcd for
C107H152N14O60 m/e 2592.9, found 2592.7.
0
9. Powell, L. D.; Varki, A. J. Biol. Chem. 1994, 269, 10628.
10. Powell, L. D.; Jain, R. K.; Matta, K. L.; Sabesan, S.;
Varki, A. J. Biol. Chem. 1995, 270, 7523.
11. Sjoberg, E. R.; Powell, L. D.; Klein, A.; Varki, A. J. Cell
Biol. 1994, 126, 549.
Erythrocyte solid-phase binding assay
The anities of the glycosides for CD22 were deter-
mined in a solid phase binding assay, based on the
binding of porcine erythrocytes to immobilised murine
CD22D1-3-IgG (Costar, high binding). In short, multi-
well plates were coated with Goat anti Human Fc
(0.75 mg/well) by overnight incubation in 0.1 M sodium
bicarbonate bu€er (pH=9.6) at 4 ꢀC. Subsequently, the
wells were blocked with 5% skimmed milk (w/w) for 1 h
at 37 ꢀC. After thorough washing, the wells were incu-
bated with mCD22 for 3 h at 4 ꢀC at a concentration of
0.01 mg/well in PBS+0.25% BSA. The wells were again
thoroughly washed and incubated for 1 h at 20 ꢀC with
porcine erythrocytes (1% dilution in PBS+0.25% BSA)
in the absence or presence of 1.0±1500 mM of the inhi-
bitor. The peroxidase activity of haemoglobulin in the
bound erythrocytes was determined using o-phenylene-
diamine hydrochloride as substrate in 50 mM phosphate
citrate bu€er (pH=5)+0.014% H2O2 after thorough
washing with PBS+0.25% BSA. CD22-speci®c binding
was de®ned as the di€erential binding in the absence
and the presence of an excess of Orosomucoid (Sigma)
(3mM). The CD22-speci®c erythrocyte binding amounted
to a reproducible 40±60% of the total binding. From
the competition curves, the IC50 (de®ned as the con-
centration of inhibitor giving 50% reduction of speci®c
erythrocyte binding) and the Hill coecient (which is
indicative of the cooperativity of ligand binding) could
be calculated by non-linear regression analysis (Prism,
GraphPad Software Inc.). Data are means of a triplicate
experiment.
12. Kelm, S.; Schauer, R.; Manuguerra, J.-C.; Gross, H.-J.;
Crocker, P. R. Glycoconjugate J. 1994, 11, 576.
13. Kelm, S.; Brossmer, R.; Isecke, R.; Gross, H. J.; Strenge,
K.; Schauer, R. Eur. J. Biochem. 1998, 255, 663.
14. Brandley, B. K.; Kiso, M.; Abbas, S.; Nikrad, P.; Sriva-
satava, O.; Foxall, C.; Oda, Y.; Hasegawa, A. Glycobiology
1993, 3, 633.
15. Sliedregt, L. A. J. M.; Rensen, P. C. N.; Rump, E. T.; van
Santbrink, P. J.; Bijsterbosch, M. K.; Valentijn, A. R. P. M.;
Van der Marel, G. A.; Van Boom, J. H.; van Berkel, T. J. C.;
Biessen, E. A. L. J. Med. Chem. 1999, 42, 609.
16. Marra, A.; Sinay, P. Carbohydr. Res. 1989, 187, 35.
17. Veeneman, G. H.; Van Leeuwen, S. H.; Van Boom, J. H.
Tetrahedron Lett. 1990, 31, 1331.
18. Nifant'ev, N. E.; Tsvetkov, Y. E.; Shashkov, A. S.;
Kononov, L. O.; Menshov, V. M.; Tuzikov, A. B.; Bovin, N.
V. J. Carbohydr. Chem. 1996, 15, 939.
19. Bayley H.; Standring D. N.; Knowles J. R. Tetrahedron
Lett. 1978, 3633.
20. Nath, D.; Van der Merwe, P. A.; Kelm, S.; Brad®eld, P.;
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21. Van der Merwe, P. A.; Crocker, P. R.; Vincent, M.;
Barclay, A. N.; Kelm, S. J. Biol. Chem. 1996, 271, 9273.
22. Nelson, R. M.; Dolich, S.; Aru€o, A.; Cecconi, O.;
Bevilacqua, M. P. J. Clin. Invest. 1993, 91, 1157.
23. Toogood, P. L.; Galliker, P. K.; Glick, G. D.; Knowles, J.
R. J. Med. Chem. 1991, 34, 3138.
24. Liang, R.; Loebach, J.; Ge, M.; Uozumi, Y.; Sekanina,
K.; Horan, N.; Gildersleeve, J.; Thompson, C.; Smith, A.;
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25. Nikrad, P. V.; Kashem, M. A.; Wlasichuk, K. B.; Alton,
G.; Venot, A. P. Carbohydr. Res. 1993, 250, 145.
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Acknowledgement
The ®nancial support from Snow Brand Milk Products
(Japan) is gratefully acknowledged
27. Hayashi, M.; Tanaka, M.; Itoh, M.; Miyauchi, H. J. Org.
Chem. 1996, 61, 2938.
28. Miyauchi, H.; Yuri, M.; Tanaka, M.; Kawamura, N.;
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H.; Kiso, M.; Kelm, S. Eur. J. Biochem. 1998, 258, 677.
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