Large oligosaccharide-based glycodendrimers.

Article abstract of DOI:10.1002/1521-3765(20020703)8:13<2988::AID-CHEM2988>3.0.CO;2-2

Carbohydrate-based dendritic structures composed of 21 and 27 monosaccharide residues have been synthesized in a convergent manner from trisaccharide building blocks. The oligosaccharide AB2 monomers are based on a maltosyl beta(1-->6)galactose structure,

Full text of DOI:10.1002/1521-3765(20020703)8:13<2988::AID-CHEM2988>3.0.CO;2-2

FULL PAPER
Large Oligosaccharide-Based Glycodendrimers**
W. Bruce Turnbull,[a, b] Stacey A. Kalovidouris,[a] and J. Fraser Stoddart*[a]
Abstract: Carbohydrate-based dendrit-
ic structures composed of 21 and 27
monosaccharide residues have been syn-
thesized in a convergent manner from
trisaccharide building blocks. The oligo-
saccharide AB2 monomers are based on
a maltosylb(1 ! 6)galactose structure,
which has been modified to include
two methylamino groups at the primary
positions of the glucosyl residues. Re-
ductive alkylation of the secondary ami-
no groups, with the innate formyl func-
tion of a second oligosaccharide mono-
mer, allows for the chemoselective
construction of dendritic wedges, while
employing a minimal number of protect-
ing groups. The first-generation dendron
can be coupled either to another AB2
monomer, to give a second-generation
dendron, or to a tris[2-(methylamino)-
ethyl]amine-based core moiety, to pro-
vide a carbohydrate-based dendrimer.
Alternating a- and b-glucosyl residues in
the monomers and dendrons, simplifies
1H NMR spectra as a consequence of
spreading out the anomeric proton sig-
nals. Monomers and dendrons were
characterized by extensive one- and
two-dimensional NMR spectroscopy in
addition to FAB, electrospray, and
MALDI-TOF mass spectrometry. Mo-
lecular dynamics simulations revealed
similar conformations in the dendrons as
in the isolated trisaccharide repeating
units.
Keywords: carbohydrates
¥
den-
drimers ¥ glycodendrimers ¥ oligo-
saccharides ¥ reductive amination
Introduction
Nature uses carbohydrates for a variety of functions, ranging
from energy storage, through structural materials, to infor-
mation transfer through a complex sugar code[1] or ™glyco-
code∫ based on stereochemistry and conformation. This
glycocode is usually read through molecular recognition of
the oligosaccharide by proteins called lectins,[2] although
sometimes by other oligosaccharides.[3] Although individual
interactions between lectins and their carbohydrate ligands
are relatively weak,[4] the multivalent presentation[5] of both
ligands and receptors at a cell surface remarkably enhances
both the affinity[6] and selectivity[7] of the interactions.
Chemists who have committed themselves to intervening in
these binding processes,[8] starting with Lee×s[9] glycoclusters[10]
(Figure 1a) and proceeding through glycopolymers[11] (Fig-
ure 1b) with many pendant saccharides to the structurally well
Figure 1. Graphical representation of multivalent neoglycoconjugates:
a) glycocluster, b) glycopolymer, c) carbohydrate-coated dendrimer,
d) carbohydrate-based dendrimer, and e) carbohydrate-centered den-
drimer.
[a] Prof. J. F. Stoddart, Dr. W. B. Turnbull, S. A. Kalovidouris
Department of Chemistry and Biochemistry
University of California, Los Angeles
defined, highly branched polymers called glycodendrimers
(Figure 1c and d),[12] have most successfully adopted Nature×s
multivalent approach to their work. These studies have not
only provided potent inhibitors of protein-carbohydrate
interactions,[9, 13] but they have also led to an increased
understanding of multivalent processes.[6, 7, 14]
In synthetic chemistry, by comparison, monosaccharides
have long been employed as sources of chirality[15] for the
synthesis of even more elaborate chiral compounds, and
additionally, in recent times, as scaffolds[16] for constructing
405 Hilgard Avenue, Los Angeles, CA 90095-1569 (USA)
Fax : (‡1)310-206-1843
E-mail: stoddart@chem.ucla.edu
[b] Dr. W. B. Turnbull
Current Address:
Astbury Centre for Structural Molecular Biology
School of Biochemistry and Molecular Biology
University of Leeds, Leeds, LS2 9JT (UK)
[**] Synthetic Carbohydrate Dendrimers, Part 9: for Part 8, see: W. B.
Turnbull, A. R. Pease, J. F. Stoddart, ChemBioChem 2000, 1, 70 74.
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2988 3000
peptidomimetics.[17] Oligosaccharides have been used[18] to
arrange guanidinium groups in arrays suitable for binding to
DNA, and various functionalities, including metal complexes
and coenzymes, have been appended onto cyclodextrins to
provide enzyme-like catalysts.[19] Carbohydrates, with their
multiple functional groups and structural diversity have also
been employed as scaffolds for the multivalent display of
other, biologically important, saccharides. While mono- and
disaccharides have been used as cores for glycoclusters,[13a, 20]
and for arranging sialyllactosamines[21] in varying orientations
for binding to influenza hemagglutinin, cyclodextrins have
been employed extensively for clustering mono- and oligo-
saccharides,[22] and glycodendrons.[23] Finally, the polysacchar-
ide, chitosan, has also been used recently as a scaffold for
attaching both monosaccharides and glycodendrons.[24]
and predictable conformations[34] that would provide the
dendrons and dendrimers with shapes which could be
controlled by the appropriate selection of their constituent
oligosaccharides.
One of our original aims in designing the synthesis of these
molecules was to try to minimize our use of protecting groups
during the coupling reactions, as we had previously found[27]
that the extra steric bulk associated with protecting groups
limits the yields of branched products and increases the
number of synthetic steps that need to be performed on the
growing dendrons. Thus, we decided to focus on using
reductive amination[35, 36] (Scheme 1) as a suitable chemo-
selective means of coupling the oligosaccharides together in
NaCNBH3
It has been noted that, conceptually, there are several
limiting designs for molecules described as glycodendrimers
(Figure 1c, d, and e).[25] Firstly, there are carbohydrate-coated
dendrimers in which the saccharides are present only around
the periphery of a non-carbohydrate dendritic scaffold,
secondly, at the other end of the spectrum, there are fully
carbohydrate dendrimers, in which saccharides form the
branching units of the dendrimer, and thirdly, there are
carbohydrate-centered dendrimers. Although considerable
interest has been shown in dendrimers constructed from
chiral building blocks,[26] only a few examples of glycoden-
drimers and glycodendrons that incorporate saccharides into
O
OH
O
OH
H
R
OH
OH
H
pH 6-7
N
H2O
R'
NaCNBH3
H+
– H+
R'
O
OH
N
OH
H
N
R
N R'
R
R'
R
Scheme 1. Reductive amination. The aldehyde function of a reducing
sugar reacts with a secondary amine to provide an iminium ion which is
selectively reduced by NaCNBH3, under neutral conditions to give a
tertiary amine. The aldehyde group is not susceptible to reduction by
NaCNBH3 under these conditions.
29]
their branched skeletons have been described to date.[27
Here we present an approach that we have been developing
for the synthesis of carbohydrate-based dendritic skeletons,[29]
which could be further elaborated with biologically important
oligosaccharides or proteins around their peripheries.
the presence of many unprotected hydroxyl groups in these
molecules. To make branched products employing this
reaction, we required an AB2 monomer that possesses either
1) one aldehyde and two amino groups or 2) two aldehydes
and one amine. As a reducing sugar already contains an
aldehyde function, we chose to introduce two complementary
amino groups into an existing oligosaccharide (Figure 2).
Although this approach would require several synthetic steps
in order to introduce, into the AB2 monomers, all of the
necessary functionality for chemoselective coupling, we
anticipated that these monomers could then be built into
large dendrons in only a few more additional steps.
Results and Discussion
Concept and synthetic design: Several important lessons for
designing glycodendrimers have emerged from literature
studies. Firstly, optimal biological activities are usually
attained[30] with glycodendrimers of moderate valency rather
than with high-generation dendrimers that have reached their
™starburst limit∫.[31] Secondly, small changes in the structure of
the underlying scaffold can have considerable effects on
binding affinities.[30b] Thirdly, the distances which must be
spanned in order to cross-link lectin binding sites can be on
the order of several nanometres, especially in systems
involving monovalent lectins arranged in multivalent arrays
at a cell surface.[32] Although glycopolymers[11d] and other
polyvalent glycoconjugates[33] may perform well in such
situations, they cannot readily provide information regarding
the optimal arrangement of both the ligands and lectins that is
required for efficient recognition at the cell surface–infor-
mation that well-defined, nanometer-scale, low valency den-
drimers may be able to provide.
Two important issues that also had to be addressed were 1)
how to purify the dendrons and dendrimers and 2) how to
simplify the NMR spectroscopic characterization of these
structurally complex molecules.
Although Nature constructs branched oligosaccharides in a
divergent fashion,[37] mediated by glycosyl transferases, this
process typically provides glycans exhibiting microheteroge-
neity,[37] that is, small ™defects∫ through incomplete glycosy-
lations. Similar problems that are associated[38] with the
divergent construction of dendrimers may be circumvented
by adopting a convergent synthesis,[39] that is, starting at the
dendrimer×s periphery and proceeding towards its core. Here,
typically only two or three reactions need to be performed at a
time on each molecule, allowing easier purification of the
reaction products. Unprotected oligosaccharides, especially
those containing amino functions, do not lend themselves well
to silica gel chromatography. However, hydrophobic glyco-
We chose to use oligosaccharides as the building blocks for
the structures of our dendrimers[29] on account of 1) their low
toxicity and immunogenicity, 2) the size advantage that they
offer over monosaccharide building blocks when it comes to
constructing large dendrons, and 3) their fairly well defined
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J. F. Stoddart et al.
FULL PAPER
mary positions of the glucosyl residues by a standard
protecting-group approach. The primary hydroxy groups of
the trisaccharide 6 were converted to tert-butyldimethylsilyl
(TBDMS) ethers, before acetylating the secondary positions
to give compound 7. This bis-silyl ether was converted directly
into the dibromide 8 by using triphenylphosphine and
bromine, and then sodium azide was used to introduce
nitrogen into the primary positions. It was found to be
preferable to remove the acetyl groups from the diazide 9
prior to reduction, otherwise the 4-O-acetyl group on the
terminal glucosyl residue migrates rapidly to give the
6-acetamido derivative. Following deprotection and azide
reduction by transfer hydrogenation with hydrazine and
Pearlman×s catalyst, the diamine 10 was treated with benzoyl
chloride in pyridine. The O-benzoates were removed under
NMe
O
NMe
O
HO
HO
HO
OH
O
O
HO
OH
OH
HO
HO
O
O
NH
O
NHMe
O
HO
HO
HO
HO
NH
O
NHMe
O
HO
¬
HO
O
Zemplen conditions to provide the N-protected trisaccharide
O
O
O
HO
HO
Me
11. This approach was found to give better reproducibility
than acylation using the Schotten Baumann procedure. The
isopropylidene acetals were hydrolyzed selectively with 90%
trifluoroacetic acid (TFA) to give the hemiacetal 1 in
good yield. Compound 1 provided very complex NMR
spectra on account of the mixture of isomers that a reducing
galactose residue can adopt. Consequently, the hemiacetal
was reduced with sodium borohydride to give a single alditol
derivative 12, allowing easier characterization of this com-
pound.
OH
HO
O
HO
HO
O
O
1
O
2
O
Me
OH
OH
O
Me
Me
Figure 2. Retrosynthetic analysis of an oligosaccharide-based glycoden-
drimer with a maltosylgalactityl trisaccharide repeating unit. The outer
portion of the dendrimer is derived from the benzamide-protected
reducing sugar 1, following reductive amination with the diamine 2, which
forms the internal branching units of the dendrimer.
Since preliminary reductive amination experiments[29] using
this masked aldehyde and primary diamine gave complex
product mixtures of secondary and tertiary amines that
proved tedious to separate, the bis-secondary amine AB2
sides can be handled easily on reversed-phase silica,[40] and so
we chose to use benzamide groups (Figure 2) at the periphery
of the dendrons to provide both 1) a degree of hydrophobicity
to the dendrons and also 2) a UV-active probe for following
the progress of reactions by
reversed-phase HPLC. We ad-
dressed the issue of NMR char-
OR
O
OAc
OH
O
OR
O
O
RO
Me
Me
OAc
AcO
O
RO
acterization by choosing a line-
ar b-maltosyl-(1 ! 6)-galactose
trisaccharide scaffold, in the
first instance, for developing
the coupling chemistry. A com-
bination of both a- and b-link-
ages in the monomer trisacchar-
ides helps to disperse the
anomeric signals, which allows
for easier identification of indi-
vidual spin systems in the
1H NMR spectra.
AcO
a
RO
O
O
AcO
O
O
RO
O
AcO
Me
O
RO
O
O
AcO
O
O
O
CCl3
NH
Me
3
4
5 R = Ac
6 R = H
O
Me
b
Me
O
Me
Me
c
R
O
NHBz
R
O
AcO
O
AcO
NHBz
O
HO
HO
AcO
O
O
HO
AcO
O
O
Me
HO
OH
AcO
O
O
OH
OH
O
Me
12
O
O
7 R = OTBDMS
8 R = Br
d
e
HO
Me
HO
Me
i
OH
Synthesis of AB2 monomers:
9 R = N3
The key trisaccharide scaffold
f
NHBz
O
was
readily
synthesized
NHBz
O
NHR
O
HO
HO
(Scheme 2) from maltose and
galactose through the coupling
of maltosyl trichloroacetimi-
date 3[41] and the diacetone
galactose derivative 4 to give
the protected trisaccharide 5 in
over 80% yield. Following de-
HO
O
NHR
HO
O
HO
HO
HO
O
OH
HO
O
O
O
1
HO
HO
Me
HO
O
OH
HO
O
10 R = H
O
Me
g
O
O
11 R = Bz
h
Me
Me
Scheme 2. Reagents: a) TMSOTf/CH2Cl2/4 ä MS (81%); b) NaOMe/MeOH (97%) c) 1. TBDMSCl/C5H5N,
2. Ac2O/C5H5N (75% over two steps); d) Br2/PPh3/CH2Cl2 (95%); e) NaN3/DMF (95%); f) 1. NaOMe/MeOH,
2. N2H4/Pd(OH)2 on C/MeOH (93% over two steps); g) 1. BzCl/C5H5N, 2. NaOMe/MeOH (80% over two
steps); h) TFA/H2O (9:1) (80%); i) NaBH4/MeOH (87%).
¬
acetylation under Zemplen
conditions, the amino functions
were introduced into the pri-
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Oligosaccharide Glycodendrimers
2988 3000
monomer 2 was also prepared (Scheme 3). The dibromide 8
was heated in N-benzylmethylamine to give, after replacing
any acetates that were lost in the aminolysis side reaction, the
bis-tertiary amine 13 in moderate yield. Again, it proved
advantageous to remove the acetates prior to hydrogenolytic
deprotection of the amino groups with hydrogen over Pearl-
man×s catalyst.
in aqueous solution, the reaction proceeds more rapidly in
MeOH, to give, almost exclusively,[29] the bis-tertiary amine
dendron 14. Reactions were followed by analytical HPLC as
all of the amino compounds streaked badly on TLC. Follow-
ing completion of the reaction, the product was isolated by
reversed-phase chromatography with a solvent gradient from
100% H2O to 100% MeOH (0.01% TFA throughout). The
product eluted with approximately 70% MeOH, but curi-
ously, beyond this solvent composition, any product still to be
eluted was retained on the column. Further elution with
approximately 70% MeOH allowed the remaining product to
be recovered from the column. The first-generation dendron
was found to be soluble in both H2O and MeOH.
a
NMe
8
O
NMe
O
AcO
AcO
AcO
O
O
AcO
Me
Although this first-generation dendron is already quite a
complex molecule, a number of features may be easily
identified in its NMR spectra (Figure 3). In addition to signals
corresponding to the acetonide and benzamide protecting
AcO
O
O
O
13
O
Me
O
Me
b
Me
1
groups, the portion of the H NMR spectrum from d ˆ 4.0
NHMe
O
5.5 ppm shows the expected five anomeric signals, indicating a
degree of ™pseudo-symmetry∫ in the molecule. The three a-
anomeric signals are well dispersed from the two b-anomeric
signals that overlap, in part, with two ring protons from the
reducing-terminal galactose residue. Broadening of the sig-
nals relating to the protons around the branching point of the
dendron is reflected in the DEPT-135 spectrum, in which low
intensity peaks are observed for the corresponding 13C nuclei,
suggesting some restricted motion around the branching a-
glycosidic linkage. Beyond these observations, severe overlap
of resonances prevented easy assignment of the rest of the
signals. The dendron was acetylated (Scheme 4) to try to
improve the dispersion of the ring proton signals, and aid in
their assignment. A combination of DEPT-135 and HMQC
spectra of the peracetate 15 (Figure 4) allowed the identi-
fication of the anomeric signals and also the methylene
signals, which conveniently fall into three groups–those
attached to 1) O-glucosyl residues, 2) N-methyl groups, and 3)
N-benzamide groups. With these introductions into each
monosaccharide spin system, a combination of COSY and
TOCSY 2D spectra allowed full assignment of all of the
pyranose ring protons. Although the protons attached to C6 of
the two galactityl residues appear as essentially equivalent in
the 1H NMR spectrum,[43] those attached to C1 of these
residues do not share this ™pseudo-symmetry∫. We hoped that
we could distinguish these positions directly using through-
space couplings across the amino linkages using a T-RO-
ESY[44] experiment, but all such correlations were too close to
the diagonal for clear identification. However, off diagonal
correlations (Figure 5) between the H5s of the glucopyranosyl
residues and the N-methyl groups and between the N-methyls
and H2s of the galactityl residues allowed unambiguous
identification of all carbohydrate proton signals, with the
exception of H3 and H4 in each of the two galactityl units.
Although no unambiguous correlations to these protons were
observed in the homonuclear experiments conducted, the
remaining four signals could be identified, by elimination, in
the HMQC spectrum (Figure 4).
NHMe
HO
HO
O
HO
O
O
HO
Me
HO
O
O
2
O
Me
O
O
Me
Me
Scheme 3. Reagents: a) 1. MeNHBn/D, 2. Ac2O/C5H5N (42% over two
steps); b) 1. NaOMe/MeOH, 2. H2/Pd(OH)2 on C/MeOH (92% over two
steps).
Synthesis and characterization of a first-generation dendron:
Reductive amination (Scheme 4) of the reducing sugar 1 with
the diamine 2 was conducted with NaCNBH3 at approxi-
mately pH 6 7. Under these conditions,[42] NaCNBH3 reduces
selectively iminium ions yet does not reduce aldehydes to
alcohols. However, as NaCNBH3 may also contain traces of
NaBH4, which does readily reduce aldehydes, it is preferable
to purify the NaCNBH3 according to a literature procedure[42]
prior to use. Although reductive aminations can be conducted
1
2
a
i
NHBz
O
NHBz
O
RO
NHBz
O
h
RO
NHBz
RO
O
RO
O
RO
RO
OR
O
RO
O
OR
OR
O
f
RO
OR
OR
e
RO
RO
RO
OR
g
RO
c
d
N
Me
O
N
14 R = H
RO
Me
b
RO
O
15 R = Ac
RO
O
O
RO
Me
RO
O
b
O
O
O
Me
a
O
Me
Me
Scheme 4. Reagents: a) NaCNBH3/AcOH/MeOH (78%); b) Ac2O/
C5H5N (86%). Monosaccharide residue labels (in squares) for compound
14 relate to 1H NMR spectra assignments given in Figures 3, 4, and 5 and in
the Experimental Section.
Synthesis and characterization of a second-generation den-
dron: With a first-generation dendron in hand, it was then
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J. F. Stoddart et al.
neous, albeit mixture of
FULL PAPER
a
anomers. Reductive amination
of this reducing sugar with the
bis-methylamino monomer 2,
under similar conditions to
those used previously, gave the
second-generation dendron 17
(Scheme 5), composed of 21
monosaccharide residues. On
this occasion, since the product
streaked quite badly on C-18
reversed-phase silica (Fig-
ure 6), it was separated from
excess of starting material and
under-substituted products by
gel-permeation chromatogra-
phy on Sephadex LH20 resin.
The electrospray mass spec-
trum (Figure 7a) showed sig-
nals
for
[M‡6H]6‡,
[M‡5H]5‡, [M‡4H]4‡, and
[M‡3H]3‡, in accord with the
six tertiary amino groups in the
structure. The 1H NMR spec-
trum gave good agreement be-
tween the integrals for the ace-
tonide and benzamide protect-
ing groups with the expected
ratio of 40:12 protons. The
anomeric region of this spec-
trum (Figure 7b) had seven
anomeric signals–three in the
™b-region∫ with integration ra-
tios of approximately 4:2:1 and
four signals in the ™a-region∫
with ratios 4:2:1:1. A full as-
signment of the signals was not
attempted as a consequence of
the severe overlap of resonan-
ces.
Figure 3. a) 1H NMR spectrum (500 MHz, CD3OD) of dendron 14 with an expansion of the anomeric region
inset. b) DEPT-135 spectrum (125 MHz, CD3OD) of the same compound, showing low intensity signals for nuclei
around the branching point of the dendron.
Synthesis and characterization
of a first-generation dendrimer:
Continuing with the reductive
amination theme, a trivalent
amine was identified as a suit-
able core for the synthesis of a
first-generation dendrimer. Re-
ductive alkylation of tris-pri-
mary amine 18 has been repor-
ted[35c] for the synthesis of small
glycoclusters, but, as with our[29]
earlier experiments using re-
ductive amination, mixtures of
Figure 4. HMQC and DEPT-135 spectra of acetylated dendron 15 (500 MHz, [D7]DMF), indicating the locations
of the anomeric and methylene signals.
secondary and tertiary amines
possible to synthesize a second-generation dendron. The
isopropylidene acetals in the dendron 14 were hydrolyzed
with TFA, as before, and both HPLC and electrospray mass
spectroscopy indicated that the product (16) was homoge-
resulted. Therefore, we opted to use the known tris[2-
(methylamino)ethyl]amine 20,[45] which was prepared from
commercially available tris(2-aminoethyl)amine 18 in two
steps (Scheme 6). Tris-amine 18 was treated with ethylchloro-
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Oligosaccharide Glycodendrimers
2988 3000
HMQC spectrum of 22 was also
in good agreement with the
proposed structure; however,
signals for the core moiety
could not be assigned unambig-
uously as a consequence of
increased line broadening (es-
pecially associated with the ter-
tiary amine groups), and many
of the signals in the DEPT-135
spectrum were also found to be
very weak.
Figure 5. Section from the TROESY spectrum of acetylated dendron 15 (500 MHz, [D7]DMF), showing
crosspeaks to the NMe groups.
formate under basic conditions, and then the resulting tris-
carbamate 19 was reduced with lithium aluminum hydride to
afford 20.
Reductive amination of the reducing sugar 16 with the
trivalent core 20 was conducted at about pH 6 7 with
NaCNBH3, as in previous examples, to give the first-
generation dendrimer 21 (Scheme 7). This water soluble
dendrimer 21 was separated from excess of the starting
material and under-substituted products by gel permeation
chromatography on Sephadex G-50 resin (Figure 8). The
‡
MALDI-TOF mass spectrum showed the expected [M‡K]
at m/z ˆ 5920.5. The 1H NMR spectrum of 21 displayed
considerable line broadening in D2O, even up to 858C, and
coupling constants were found only to begin to resolve in
[D7]DMF around 1008C. Therefore, 21 was acetylated to form
Figure 6. C-18 Reversed phase HPLC analysis (MeOH/H2O/TFA,
65:35:0.0001 ! 90:10:0.0001) of a) the reductive amination reaction mix-
ture and b) the 21-mer dendron 17, following isolation by gel permeation
chromatography.
22 with the aim of obtaining a better resolved spectrum. The
MALDI-TOF mass spectrum of 22 exhibited a molecular ion
peak that was consistent with the acetylated dendrimer. The
NHBz
u
r
O
HO
OH
NHBz
O
HO
NHBz
O
t
HO
l
NHBz
O
NHBz
HO
O
O
HO
HO
NHBz
O
O
HO
O
HO
NHBz
OH
O
HO
HO
O
HO
HO
NHBz
HO
O
OH
OH
OH
OH
O
q
HO
O
HO
O
HO
HO
OH
OH
O
k
HO
HO
i
OH
OH
OH
HO
p
h
HO
HO
HO
N
s
OH
j
g
Me
HO
O
N
O
HO
HO
N
Me
O
14
Me
HO
O
O
N
HO
HO
o
HO
a
Me
OH
O
HO
n
O
OH
O
HO
f
OH
HO
OH
NHBz
O
e
HO
OH
NHBz
O
NHBz
O
HO
HO
HO
m
OH
HO
HO
O
HO
NHBz
O
d
O
HO
N
HO
OH
HO
O
OH
OH
Me
O
O
HO
N
HO
HO
OH
Me
OH
OH
O
HO
HO
HO
O
O
HO
HO
c
OH
Me
O
HO
O
O
16
b
N
O
Me
a
Me
17
O
O
N
HO
Me
HO
Me
O
HO
Me
O
O
HO
OH
HO
2
b
O
HO
HO
OH
Scheme 5. Reagents: a) TFA/H2O (9:1) (60%); b) NaCNBH3/AcOH/MeOH (25%). Monosaccharide residue labels (in squares) for compound 17 relate to
1H NMR spectra assignments given in Figure 7 and the Experimental Section
Chem. Eur. J. 2002, 8, No. 13
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0813-2993 $ 20.00+.50/0
2993

J. F. Stoddart et al.
FULL PAPER
NHMe
NH2
NHCO2Et
NHCO2Et
a
b
N
MeHN
N
N
H2N
EtO2CHN
NHMe
NH2
18
19
20
Scheme 6. Reagents: a) ClCO2Et/C6H6/H2O/KOH (40%); b) LiAlH4/
THF (56%).
C3 C6 torsion angles. The galactityl units in 17 (Figure 9b),
by comparison, appeared to interconvert between these
conformers only slowly (in a 4 ns MD run at 300 K), typically
remaining close to their starting conformations. Therefore, to
generate a large variety of structures in search of a global
minimum conformation, a 4 ns MD run was conducted at
600 K, providing a full set of conformations similar to those
shown in Figure 9a, for each trisaccharide repeat unit in the
dendron. The lowest energy conformer obtained from min-
imization of 50 structures selected during the run is shown in
Figure 9c. The average distance from the reducing terminal
anomeric carbon to the peripheral benzamide nitrogen atoms
was 17.5 ä during both the 600 K MD run and in the final
minimized structure. Average distances between individual
pairs of benzamide nitrogen atoms varied from 5 ä for those
in the same maltosyl unit, to 18 ä separating N6q and N6l.
The slow interconversions of the galactityl units and consis-
tent conformations of the maltosyl branching points suggest
that it should be possible to vary the shapes of these molecular
Figure 7. Characterization of the second-generation dendron 17. a) Elec-
trospray mass spectrum and b) partial 1H NMR spectrum (500 MHz, D2O)
showing the anomeric region.
Molecular modeling of the second-generation dendron:
Oligosaccharides, although flexible about their glycosidic
linkages, are known[34] to adopt
OR
RO
certain preferred conforma-
O
RO
tions in solution. The solution
conformations of alditols have
been investigated, and it has
been reported[46] that galactitol
principally adopts an extended
zig-zag conformation in which
there are no unfavorable 1,3-
cis-interactions between the hy-
droxyl substituents. Molecular
dynamics (MD) simulations of
the second-generation dendron
17 were used to assess the
flexibility of the dendron and
to search for low energy con-
formations. Plots of the f ver-
sus y torsion angles for the
Glca(1 ! 4)Glc linkages (Fig-
ure 9a and b) showed a single
major conformation through-
out all of the MD runs. How-
ever, the galactityl portion of
the isolated trisaccharide re-
peat unit occupied several con-
formations (Figure 9a and b),
under the conditions of the
experiment, as evidenced from
the plots of the C1 C4 versus
C2 C5 and the C2 C5 versus
RO
O
NHBz
RO
O
O
NHBz
RO
RO
Me
RO
RO
OR
16
N
OR
a
O
OR
O
RO
OR
OR
OR
OR
RO
O
OR
O
Me
RO
N
O
O
BzHN
BzHN
20
O
OR OR
RO
O
OR
OR
RO
RO
RO
RO
NHBz
O
MeN
Me
RO
O
N
OR
NHBz
O
OR
N
RO
RO
OR
O
RO
O
RO
OR
O
O
OR
RO
OR
O
NMe
OR
OR
OR
NHBz
O
RO
NMe
OR
RO
RO
RO
MeN
RO
RO
NHBz
O
RO
RO
RO
O
OR
O
OR
OR
RO
O
OR
RO
RO
OR
MeN
O
OR
OR
OR
O
RO
OR
OR
O
RO
MeN
O
O
O
O
BzHN
O
OR
OR
OR
O
OR
OR
OR
BzHN
O
RO
OR
21
R = H
BzHN
b
OR
OR
O
R = Ac 22
BzHN
OR
OR
Scheme 7. Reagents: a) NaCNBH3/AcOH/MeOH (54%); b) Ac2O/C5H5N/DMAP (50%).
2994
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0947-6539/02/0813-2994 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 13

Oligosaccharide Glycodendrimers
2988 3000
building blocks has been accom-
plished. The chemoselective
coupling reaction allows den-
dron synthesis in the presence
of only a few protecting groups,
unlike earlier approaches to
making
carbohydrate-based
dendrimers through chemical
glycosylation. Furthermore, only
a few synthetic steps are re-
quired to construct large den-
drons, with the trisaccharide
building blocks in hand. Inter-
pretation of 1H and 13C NMR
spectra is facilitated by including
alternating a- and b-glycosidic
linkages in both the monomers
and dendrons, and acetylation of
a first-generation dendron al-
lowed for an almost complete
Figure 8. GPC traces of a) first-generation dendrimer 21, b) undersubstituted products of the reductive
amination reaction, and c) a MALDI-TOF mass spectrum of the product mixture isolated from the reaction.
scaffolds selectively, and thus influence the presentation of
bioactive saccharides attached at the dendrons× peripheries by
the appropriate selection of carbohydrate building blocks.
assignment of its 1H and 13C NMR spectra through a
combination of two-dimensional experiments. Molecular
modeling reveals that the key maltose-based branching points
on the dendrons retain the conformation of the parent
compound and that the galactityl chains appear less flexible
than in an isolated fragment of the dendron. Dendrons and
dendrimers, such as these, should find useful application as
scaffolds for the construction of large multivalent glycocon-
jugates. Studies to this effect will be reported in due course.
Conclusion
The synthesis of oligosaccharide-based dendrons and den-
drimers by the reductive amination of suitable trisaccharide
Figure 9. Plots of torsion angles, sampled during MD simulations, for the Glca(1 ! 4)Glc glycosidic linkage (f vs. y) and the carbon backbone of the
galactityl residue (C1-C4 vs. C2-C5 and C2-C5 vs. C3-C6) in a) an isolated trisaccharide repeat unit and b) representative examples from the second-
generation dendron 17. The MD simulations were conducted for a) 1 ns and b) 5 ns at 300 K as described in the Experimental section. c) Space-filling and
polytube representations of the second-generation dendron 17, highlighting the constituent trisaccharide residues in light grey through to dark grey and the
peripheral benzamide groups in black.
Chem. Eur. J. 2002, 8, No. 13
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0813-2995 $ 20.00+.50/0
2995

J. F. Stoddart et al.
FULL PAPER
1H; H1c), 5.49 (d, J1,2 ˆ 4.9 Hz, 1H; H1a); 13C NMR (100 MHz, CDCl3):
d ˆ 20.6 (3C), 20.7, 20.8, 20.9, 21.0, 24.4, 25.0, 25.9, 26.0, 61.5, 62.9, 67.7, 68.0,
68.5, 69.3, 69.4, 70.0, 70.4, 70.6, 71.2, 71.9, 72.1, 72.8, 75.2, 95.5, 96.2, 101.0,
108.6, 109.4, 169.4, 169.8, 169.9, 170.1, 170.4, 170.5, 170.6; FABMS: m/z
Experimental Section
General methods: All solvents were dried prior to use, according to
standard methods. Sodium cyanoborohydride (Aldrich) was purified[42] as
its dioxane complex prior to use. Otherwise, commercial reagents were
used, without further purification. Analytical TLC was performed on silica
gel 60-F254 (Merck) with detection by fluorescence and/or by charring
following immersion in 5% H2SO4/EtOH. During workup, organic
solutions were washed two or three times with equal volumes of each of
the aqueous solutions listed. All concentrations were performed in vacuo.
Flash chromatography was performed with silica gel 60 (Silicycle).
Analytical reversed-phase HPLC was conducted by using a Hypersil
5 mm BDS C-18 silica column (ThermoQuest; 4.6 Â 250 mm) under iso-
cratic elution (MeOH/H2O/TFA, 65:35:0.0001) and analytical GPC was
performed by using a Ultrahydrogel 250GPC column (7.8 Â 250 mm) in
water and UV detection with a Dynamax PDA-2 diode array detector.
Preparative reversed-phase chromatography was conducted on fully
endcapped C-18 or C-8 silica gel 100 (230 400 mesh, Fluka), and gel-
permeation chromatography was performed using a 25 Â 900 mm column
of Sephadex LH20 resin (Sigma), eluting with MeOH. Optical rotations
were measured at the sodium D-line with a Rudolph Research AUTOPOL
‡
‡
(%): 901.29 (100) [M‡Na] , 619.24 (20) [M À a] , 331.11 (20) [M À
‡
(a‡b)] ; elemental analysis calcd (%) for C38H54O23 (878.8): C 51.93, H
6.19; found: C 51.84, H 6.13.
a-d-Glucopyranosyl-(1 ! 4)-b-d-glucopyranosyl-(1 ! 6)-1,2:3,4-di-O-iso-
propylidene-a-d-galactopyranose (6): A solution of the heptaacetate 5
(13.6 g, 15.5 mmol) and NaOMe (3 mL, 0.5m in MeOH, 1.5 mmol) in
MeOH (250 mL) was kept overnight at RT. The solution was neutralized
‡
with Amberlite IR-120 (H form) ion-exchange resin, filtered, and
concentrated to afford 6 (8.8 g, 97%) as a white solid. [a]2D0 ˆ ‡11.6 (c ˆ
1 in H2O); 1H NMR (500 MHz, CD3OD): d ˆ 1.33, 1.34, 1.40, 1.52 (4s, 12H;
2CMe2), 3.24 3.29 (m, 2H; H2b, H4c), 3.39 (m, 1H; 5b), 3.44 (dd, J1,2
ˆ
3.7 Hz, J2,3 ˆ 9.7 Hz, 1H; H2c), 3.54 (pt, J3,4 ꢀ J4,5 ˆ 9.1 Hz, 1H; H4b),
3.59 3.71 (m, 5H, H6a, H3b, H3c, H5c, H6c), 3.79 3.85 (m, 2H; H6b,
H6c'), 3.90 (dd, J5,6 ˆ 1.6 Hz, J6,6' ˆ 12.2 Hz, 1H; H6b'), 4.02 4.08 (m, 2H;
H5a, H6a'), 4.28 4.33 (m, 2H; H4a, H1b), 4.38 (dd, J1,2 ˆ 4.9 Hz, J2,3
ˆ
2.4 Hz, 1H; H2a), 4.63 (dd, J2,3 ˆ 2.4 Hz, J3,4 ˆ 7.9 Hz, 1H; H3a), 5.16 (d,
J1,2 ˆ 3.7 Hz, 1H; H1c), 5.52 (d, J1,2 ˆ 4.9 Hz, 1H; H1a); 13C NMR
(125 MHz, CD3OD): d ˆ 24.5, 25.1, 26.3 (2C), 62.15, 62.7, 68.8, 69.9, 71.4,
71.8, 71.9, 72.4, 74.1, 74.6, 74.7, 75.0, 76.6, 77.5, 81.2, 97.7, 102.9, 104.6, 110.1,
IVautomatic polarimeter. [a]D values are given in units of 10À1 degcm2 gÀ1
.
1H and 13C NMR spectra were recorded on a Bruker ARX400 spectrometer
(at 400 MHz and 100 MHz, respectively) and on either Bruker ARX500 or
Bruker Avance 500 spectrometers (at 500 MHz and 125 MHz, respectively)
at ambient temperature unless stated otherwise. 1H NMR and 13C NMR
spectra were referenced using their residual solvent signals as internal
standards or to tBuOH for spectra run in D2O. Signals were assigned using
a combination of DEPT-135, dqf-COSY, and HMQC experiments, and
where appropriate TOCSY and T-ROESY experiments. Gradient-selected
versions of these 2D experiments were used on the Avance 500 spectrom-
eter. All 2D experiments were acquired in phase sensitive mode. TOCSY
and T-ROESY experiments used 70 ms and 500 ms mixing times, respec-
tively. For trisaccharides, the monosaccharide residues are labeled a, b, c
from the reducing terminus. For dendrons, the monosaccharide residues are
labeled a to u as described in Schemes 4 and 5. The following abbreviations
were used to explain the signal multiplicities or characteristics: s, singlet; d,
doublet; dd, double doublet; pt, pseudo-triplet; pdt, pseudo-double-triplet;
m, multiplet; br, broad. Fast atom bombardment (FAB) mass spectra were
recorded on a VG ZAB-SE spectrometer using a 3-nitrobenzyl alcohol
matrix. Fragment ions[47] resulting from loss of one or two monosaccharide
‡
‡
110.4; FABMS: m/z (%): 717.5 (100) [M‡Cs] , 607.5 (96) [M‡Na] ;
‡
HRFABMS: calcd for C24H40O16Na [M‡Na] : 607.2214; found: m/z:
607.2203.
2,3,4-Tri-O-acetyl-6-O-tert-butyldimethylsilyl-a-d-glucopyranosyl-(1 ! 4)-
2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl-b-d-glucopyranosyl-(1 ! 6)-
1,2:3,4-di-O-isopropylidene-a-d-galactopyranose (7): tert-Butyldimethyl-
silyl chloride (5.16 g, 34.2 mmol) was added to a stirred solution of the
trisaccharide 6 (8.0 g, 13.7 mmol) in C5H5N (80 mL) at 08C. After 1 h, more
tert-butyldimethylsilyl chloride (5.16 g, 34.2 mmol) was added and stirring
was continued at this temperature for a further 1 h and then at RT for 1 h
before quenching the reaction by adding MeOH. The mixture was
concentrated and then treated with Ac2O (40 mL) and C5H5N (40 mL) at
608C overnight. Following concentration and co-evaporation with toluene,
the mixture was dissolved in EtOAc (500 mL) and washed consecutively
with 1m HCl, saturated NaHCO3, and saturated NaCl solutions, before
drying over anhydrous Na2SO4 and concentrating to a foam. Column
chromatography (SiO2, hexanes/EtOAc, 7:3 to 6:4) gave the bis-silylether 7
(10.4 g, 75%) as a solid foam. [a]2D0 ˆ ‡15.0 (c ˆ 1 in CHCl3); 1H NMR
(500 MHz, CDCl3): d ˆ 0.02, 0.03, 0.08, 0.09 (4s, 12H; 4SiMe), 0.88, 0.90
(2s, 18H; 2SiCMe3), 1.32 (s, 6H; CMe2), 1.44, 1.49 (2s, 6H; CMe2), 1.98,
1.99, 2.01, 2.02, 2.04 (5s, 15H; 5COMe), 3.35 (m, 1H; H5b), 3.59 3.65 (m,
2H; H6a, H6c), 3.71 (dd, J5,6 ˆ 1.5 Hz, J6,6' ˆ 11.5 Hz, 1H; H6c'), 3.86 4.02
‡
residues from the reducing terminus are labeled as [M À a] and [M À
‡
(a‡b)] , respectively. For compounds that displayed only fragment ions in
their FAB mass spectra, the samples were doped with sodium acetate to
‡
give strong [M‡Na] , or they were studied by electrospray mass
spectrometry (ES-MS) recorded on a Sciex API IIIR triple quadrupole
electrospray mass spectrometer using H2O/MeCN/HCOOH, 50:50:0.1 as
the mobile phase. MALDI-TOF data were acquired with a DE-STR
instrument and the matrix used was a methanolic solution containing cyano
a-hydroxy cinnaminic acid and ammonium acetate. Elemental analyses
were performed by Quantitative Technologies, NJ (USA).
(m, 7H; H5a, H6a', H4b, H6b, H6b', H5c), 4.16 (dd, J3,4 ˆ 7.9 Hz, J4,5
ˆ
1.7 Hz, 1H; H4a), 4.27 (dd, J1,2 ˆ 4.9 Hz, J2,3 ˆ 2.4 Hz, 1H; H2a), 4.56 (m,
2H; H3a, H1b), 4.75 4.80 (m, 2H; H2b, H2c), 5.12 (pt, J3,4 ꢀ J4,5 ˆ 9.8 Hz,
1H; H4c), 5.24 (pt, J2,3 ꢀ J3,4 ˆ 9.4 Hz, 1H; H3b), 5.34 (pt, J2,3 ꢀ J3,4
ˆ
10.0 Hz, 1H; H3c), 5.37 (d, J1,2 ˆ 3.7 Hz, 1H; H1c), 5.48 (d, J1,2 ˆ 4.9 Hz,
1H; H1a); 13C NMR (100 MHz, CDCl3): d ˆ À5.5, À5.4, À5.1, À4.9, 18.4,
18.6, 20.6, 20.7, 20.8, 20.9, 21.0, 24.4, 25.0, 25.9 (2C), 26.0 (3C), 26.1 (3C),
61.5, 62.0, 67.5, 68.3, 68.5, 70.1, 70.4, 70.5, 70.6, 70.7, 71.0, 71.1, 72.1, 75.1,
75.3, 94.9, 96.2, 100.8, 108.6, 109.3, 169.2, 169.9, 170.2, 170.3, 170.4 ;
2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl-(1 ! 4)-2,3,6-tri-O-acetyl-b-d-
glucopyranosyl-(1 ! 6)-1,2:3,4-di-O-isopropylidene-a-d-galactopyranose
(5): A solution of the trichloroacetimidate 3[41] (15.0 g, 19.2 mmol) and the
acceptor 4 (10.0 g, 38.4 mmol) in CH2Cl2 (275 mL) was stirred with
powdered 4 ä molecular sieves (15.0 g) for 2 h under an argon atmosphere.
After cooling the mixture to 08C, trimethylsilyl trifluoromethanesulfonate
(695 mL, 3.8 mmol) was added dropwise over 5 min and stirring was
continued at this temperature for 30 min. Triethylamine (0.7 mL, 5.0 mmol)
was added to quench the reaction before filtering the solution through
Celite and concentrating. Column chromatography (SiO2, hexanes/EtOAc,
‡
‡
FABMS: m/z (%): 1045.23 (100) [M‡Na] , 763.40 (10) [M À a] , 403.21
‡
(80) [M À (a‡b)] ; elemental analysis calcd (%) for C46H78O21Si2 (1023.29):
C 53.99, H 7.68; found: C 54.10, H 7.28.
2,3,4-Tri-O-acetyl-6-bromo-6-deoxy-a-d-glucopyranosyl-(1 ! 4)-2,3-di-O-
acetyl-6-bromo-6-deoxy-b-d-glucopyranosyl-(1 ! 6)-1,2:3,4-di-O-isopro-
pylidene-a-d-galactopyranose (8): Bromine (1.25 mL, 24.4 mmol) was
added dropwise to a stirred solution of the bis-silylether
10.2 mmol) and triphenylphosphine (6.7 g, 25.5 mmol) in CH2Cl2 at 08C,
forming precipitate. The mixture was allowed to warm to room
7 (10.4 g,
6:4 to 1:1) gave the trisaccharide 5 (13.6 g, 81%) as a solid foam. [a]D20
ˆ
‡9.2 (c ˆ 1 in CHCl3); 1H NMR (400 MHz, CDCl3): d ˆ 1.32, 1.33, 1.45,
1.50 (4s, 12H; 2CMe2), 2.00, 2.01, 2.02, 2.03, 2.04, 2.09, 2.14 (7s, 21H;
7COMe), 3.65 3.71 (m, 2H; H6a, H5b), 3.89 4.06 (m, 5H; H5a, H6a',
H4b, H5c, H6c), 4.18 (dd, J3,4 ˆ 7.9 Hz, J4,5 ˆ 1.8 Hz, 1H; H4a), 4.21 4.27
(m, 2H; H6b, H6c'), 4.29 (dd, J1,2 ˆ 4.9 Hz, J2,3 ˆ 2.4 Hz, 1H; H2a), 4.48 (dd,
J5,6 ˆ 2.7 Hz, J6,6' ˆ 12.1 Hz, 1H; H6b'), 4.58 (dd, J2,3 ˆ 2.4 Hz, J3,4 ˆ 7.9 Hz,
1H; H3a), 4.64 (d, J1,2 ˆ 7.9 Hz, 1H; H1b), 4.81 4.88 (m, 2H; H2b, H2c),
5.05 (pt, J3,4 ꢀ J4,5 ˆ 9.7 Hz, 1H; H4c), 5.26 (pt, J2,3 ꢀ J3,4 ˆ 9.0 Hz, 1H;
H3b), 5.36 (dd, J2,3 ˆ 10.4 Hz, J3,4 ˆ 9.7 Hz, 1H; H3c), 5.41 (d, J1,2 ˆ 4.0 Hz,
a
temperature over 20 h, by which time, the precipitate had fully dissolved.
The solution was diluted with CH2Cl2 (400 mL) and was washed consec-
utively with saturated NaHCO3 solution and water, before drying over
anhydrous Na2SO4 and concentrating. Column chromatography (SiO2,
CH2Cl2/EtOAc, 9:1 to 85:15) gave the dibromide 8 (8.9 g, 95%) as a solid
foam. [a]2D0 ˆ ‡8.0 (c ˆ 1 in CHCl3); 1H NMR (400 MHz, CDCl3): d ˆ 1.32,
1.33, 1.45, 1.50 (4s, 12H; 2CMe2), 2.01, 2.02, 2.05, 2.06, 2.07 (5s, 15H;
5COMe), 3.43 (dd, J5,6 ˆ 5.0 Hz, J6,6' ˆ 11.5 Hz, 1H; H6c), 3.61 (dd, J5,6'
ˆ
2996
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Oligosaccharide Glycodendrimers
2988 3000
2.7 Hz, J6,6' ˆ 11.5 Hz, 1H; H6c'), 3.65 3.80 (m, 4H; H6a, H5b, H6b, H6b'),
a foam. Benzoate esters were removed using NaOMe (1 mL, 0.5m in
MeOH, 0.5 mmol) in MeOH (50 mL), followed by neutralization with
AcOH. Column chromatography (SiO2, CH2Cl2/MeOH, 9:1 to 85:15) gave
the bis-benzamide 11 (1.08 g, 80%) as an amorphous solid. [a]2D0 ˆ À9.0
(c ˆ 1 in MeOH); 1H NMR (500 MHz, CD3OD): d ˆ 1.26, 1.31, 1.32, 1.49
3.93 (m, 1H; H5c), 4.00 4.08 (m, 3H; H5a, H6a', H4b), 4.19 (dd, J3,4
ˆ
7.9 Hz, J4,5 ˆ 1.8 Hz, 1H; H4a), 4.29 (dd, J1,2 ˆ 5.0 Hz, J2,3 ˆ 2.4 Hz, 1H;
H2a), 4.59 (dd, J2,3 ˆ 2.4 Hz, J3,4 ˆ 7.9 Hz, 1H; H3a), 4.68 (d, J1,2 ˆ 7.8 Hz,
1H; H1b), 4.83 4.88 (m, 2H; H2b, H2c), 5.04 (pt, J3,4 ꢀ J4,5 ˆ 9.6 Hz, 1H;
H4c), 5.28 (pt, J2,3 ꢀ J3,4 ˆ 9.2 Hz, 1H; H3b), 5.37 (dd, J2,3 ˆ 10.5 Hz, J3,4
ˆ
( 4s, 12H; 2CMe), 3.20 (pt, J3,4 ꢀ J4,5 ˆ 9.5 Hz, 1H; H4c), 3.25 3.30 (m,
2
9.5 Hz, 1H; H3c), 5.42 (d, J1,2 ˆ 3.9 Hz, 1H; H1c), 5.50 (d, J1,2 ˆ 5.0 Hz, 1H;
H1a); 13C NMR, (100 MHz, CDCl3): d ˆ 20.5, 20.6, 20.7, 20.9, 21.0, 24.4,
25.0, 25.9, 26.0, 31.6, 32.9, 67.8, 69.0, 69.1, 69.4, 70.0, 70.4, 70.6, 70.7, 71.2,
71.9, 72.5, 73.8, 74.8, 95.1, 96.2, 100.9, 108.6, 109.4, 169.3, 169.7, 170.0, 170.2;
2H; H2b, H6b), 3.33 (pt, J3,4 ꢀ J4,5 ˆ 9.2 Hz, 1H; H4b), 3.49 3.54 (m, 2H;
H5b, H2c), 3.61 3.73 (m, 4H; H6a, H3b, H3c, H6c), 3.88 (dd, J5,6' ˆ 2.5 Hz,
J6,6' ˆ 14.0 Hz, 1H; H6c'), 3.97 4.07 (m, 4H; H5a, H6a', H6b', H5c), 4.18
(dd, J3,4 ˆ 8.0 Hz, J4,5 ˆ 1.4 Hz, 1H; H4a), 4.30 (d, J1,2 ˆ 7.9 Hz, 1H; H1b),
‡
‡
FABMS: m/z (%): 943.32 (100) [M‡Na] , 661.23 (35) [M À a] ; elemental
analysis calcd (%) for C34H48Br2O19 (920.55): C 44.36, H 5.26; found: C
44.60, H 5.22.
4.33 (dd, J1,2 ˆ 5.0 Hz, J2,3 ˆ 2.4 Hz, 1H; H2a), 4.55 (dd, J2,3 ˆ 2.4 Hz, J3,4 ˆ
7.9 Hz, 1H; H3a), 5.16 (d, J1,2 ˆ 3.8 Hz, 1H; H1c), 5.47 (d, J1,2 ˆ 5.0 Hz, 1H;
H1a), 7.26 (m, 2H; 2Ar-H), 7.35 (m, 1H; Ar-H), 7.43 (m, 2H; 2Ar-H), 7.52
(m, 1H; Ar-H), 7.72 7.77 (m, 4H; 4Ar-H); 13C NMR (125 MHz, CD3OD):
d ˆ 24.6, 25.1, 26.3 (2C), 42.4, 42.5, 68.9, 69.7, 71.8, 71.9, 72.5, 73.2, 73.6, 74.2,
74.4, 74.6, 74.8, 77.4, 84.5, 97.7, 103.4, 104.2, 110.0, 110.4, 128.3 (2C), 128.5
(2C), 129.4 (2C), 129.5 (2C), 132.5, 132.6, 135.4, 135.6, 170.2, 170.8;
2,3,4-Tri-O-acetyl-6-azido-6-deoxy-a-d-glucopyranosyl-(1 ! 4)-2,3-di-O-
acetyl-6-azido-6-deoxy-b-d-glucopyranosyl-(1 ! 6)-1,2:3,4-di-O-isopropyl-
idene-a-d-galactopyranose (9): A mixture of the dibromide 8 (7.00 g,
7.6 mmol), NaN3 (4.90 g, 76 mmol) and DMF (60 mL) was stirred at 758C
for 12 h, and then concentrated to about 25 mL. The mixture was diluted
with EtOAc (500 mL) and washed with saturated NaCl solution, before
drying over anhydrous Na2SO4, and concentrating to a syrup. Column
chromatography (SiO2, hexanes/EtOAc, 60:40 to 55:45) gave the diazide 9
(6.1 g, 95%) as white crystals. [a]2D0 ˆ ‡23.2 (c ˆ 0.5 in CHCl3); 1H NMR
(400 MHz, CDCl3): d ˆ 1.32, 1.33, 1.45, 1.50 (4s, 12H; 2CMe2), 2.00, 2.02,
2.03, 2.04, 2.05 (5s, 15H; 5COMe), 3.32 (dd, J5,6 ˆ 5.3 Hz, J6,6' ˆ 13.4 Hz,
‡
‡
FABMS: m/z (%): 813.30 (10) [M‡Na] , 791.31 (100) [M‡H] , 775.30 (5)
‡
‡
[M À 15] , 531.20 (20) [M À a] ; HRFABMS: calcd for C38H51N2O16
‡
[M‡H] : 791.3238; found: m/z: 791.3246.
6-Benzamido-6-deoxy-a-d-glucopyranosyl-(1 ! 4)-6-benzamido-6-deoxy-
b-d-glucopyranosyl-(1 ! 6)-d-galactose (1): The diacetonide 11 (620 mg,
785 mmol) was treated with 90% aqueous TFA (5 mL) at RT for 15 min
before concentration and co-evaporation with MeOH. Column chroma-
tography (C-8 reversed-phase silica, H2O to MeOH) gave the reducing
sugar 1 (445 mg, 80%) as an amorphous solid after freeze drying from H2O.
1H; H6c), 3.43 (dd, J5,6' ˆ 2.7 Hz, J6,6' ˆ 13.4 Hz, 1H; H6c'), 3.48 (dd, J5,6
ˆ
4.8 Hz, J6,6' ˆ 13.5 Hz, 1H; H6b), 3.65 3.75 (m, 3H; H5a, H6a, H6b'), 3.85
(ddd, J4,5 ˆ 10.0 Hz, J5,6 ˆ 5.3 Hz, J5,6' ˆ 2.7 Hz, 1H; H5c), 3.92 (m, 1H;
H5b), 4.01 4.07 (m, 2H; H6a', H4b), 4.18 (dd, J3,4 ˆ 7.9 Hz, J4,5 ˆ 1.9 Hz,
1
[a]2D0 ˆ ‡60.8 (c ˆ 1 in H2O); selected H NMR data (400 MHz, CD3OD):
d ˆ 4.32 4.43 (b-anomers), 5.10 5.16 (a-anomers), 7.22 (m, 2H), 7.34 (m,
1H), 7.41 (m, 2H), 7.50 (m, 1H), 7.70 (m, 4H); ESMS: m/z (%): 711.2 (100)
1H; H4a), 4.29 (dd, J1,2 ˆ 5.0 Hz, J2,3 ˆ 2.4 Hz, 1H; H2a), 4.58 (dd, J2,3
ˆ
‡
‡
2.4 Hz, J3,4 ˆ 7.9 Hz, 1H; H3a), 4.66 (d, J1,2 ˆ 7.9 Hz, 1H; H1b), 4.81 (dd,
J1,2 ˆ 4.0 Hz, J2,3 ˆ 10.5 Hz, 1H; H2c), 4.88 (dd, J1,2 ˆ 7.9 Hz, J2,3 ˆ 9.6 Hz,
1H; H2b), 4.98 (pt, J3,4 ꢀ J4,5 ˆ 9.6, 1H; H4c), 5.27 (pt, J2,3 ꢀ J3,4 ˆ 9.2 Hz,
[M‡H] , 733.1 (15) [M‡Na] .
6-Benzamido-6-deoxy-a-d-glucopyranosyl-(1 ! 4)-6-benzamido-6-deoxy-
b-d-glucopyranosyl-(1 ! 6)-d-galactitol (12): A portion of the hemiacetal 1
(40 mg, 56 mmol) was reduced with sodium borohydride (20 mg, 528 mmol)
in MeOH (1 mL) at RT for 2 h. The reaction was quenched by adding
Me2CO, and concentrated. Column chromatography (C-8 reversed-phase
silica, H2O to MeOH) gave the alditol 12 (35 mg, 87%) as a glassy solid.
[a]2D0 ˆ ‡13 (c ˆ 0.2 in H2O); 1H NMR (500 MHz, CD3OD): d ˆ 3.18 3.23
(m, 2H; H6b, H4c), 3.27 (dd, J1,2 ˆ 7.8 Hz, J2,3 ˆ 9.5 Hz, 1H; H2b), 3.33 (pt,
J3,4 ꢀ J4,5 ˆ 8.8 Hz, 1H; H4b), 3.48 3.56 (m, 2H; H5b, H2c), 3.60 3.72 (m,
8H; H1a, H1a', H3a, H4a, H6a, H3b, H3c, H6c), 3.87 (dd, J5,6 ˆ 2.6 Hz,
J6,6' ˆ 14.0 Hz, 1H; H6c'), 3.90 (ptd, J1,2 ꢀ J1',2 ˆ 6.3 Hz, J2,3 ˆ 1.3 Hz, 1H;
H2a), 3.98 (dd, J5,6' ˆ 4.9 Hz, J6,6' ˆ 9.8 Hz, 1H; H6a'), 4.06 4.10 (m, 2H;
1H; H3b), 5.32 (dd, J3,4 ˆ 9.5 Hz, J2,3 ˆ 10.5 Hz, 1H; H3c), 5.41 (d, J1,2
ˆ
4.0 Hz, 1H; H1c), 5.50 (d, J1,2 ˆ 5.0 Hz, 1H; H1a); 13C NMR (100 MHz,
CDCl3): d ˆ 20.5, 20.6 (2C), 20.9, 21.0, 24.3, 25.0, 25.9, 26.0, 50.9, 51.0, 67.7,
69.0, 69.1, 69.2, 69.5, 70.0, 70.4, 70.6, 71.2, 71.9, 72.1, 73.7, 75.0, 95.1, 96.2,
100.9, 108.6, 109.4, 169.5, 169.8, 170.0, 170.2, 170.4; FABMS: m/z (%):
‡
‡
866.84 (100) [M‡Na] , 585.33 (65) [M À a] ; HRFABMS: calcd for
‡
C34H48N6O19Na [M‡Na] : 867.2872; found: m/z: 867.2878.
6-Amino-6-deoxy-a-d-glucopyranosyl-(1 ! 4)-6-amino-6-deoxy-b-d-glu-
copyranosyl-(1 ! 6)-1,2:3,4-di-O-isopropylidene-a-d-galactopyranose
(10): A solution of the the pentaacetate 9 (1.60 g, 1.89 mmol) and NaOMe
(1 mL, 0.5m in MeOH, 0.5 mmol) in MeOH (20 mL) was kept overnight at
H5a, H5c), 4.12 (dd, J5,6' ˆ 2.0 Hz, J6,6' ˆ 14.0 Hz, 1H; H6b'), 4.30 (d, J1,2
ˆ
‡
RT. The solution was neutralized with Amberlite IR-120 (H form) ion-
7.8 Hz, 1H; H1b), 5.15 (d, J1,2 ˆ 3.7 Hz, 1H; H1c), 7.25 (m, 2H; 2Ar-H), 7.37
(m, 1H; Ar-H), 7.44 (m, 2H; 2Ar-H), 7.52 (m, 1H; Ar-H), 7.74 (m, 4H;
4Ar-H); 13C NMR (125 MHz, CD3OD): d ˆ 42.4, 42.7, 65.0, 70.2, 71.2, 71.3,
71.7, 73.1, 73.2, 73.6, 74.3, 74.5, 74.6, 75.1, 77.5, 84.6, 103.5, 104.5, 128.3 (2C),
128.5 (2C), 129.4 (2C), 129.5 (2C), 132.5, 132.7, 135.5 (2C), 170.3, 170.9;
exchange resin, filtered, and concentrated. A solution of this residue and
hydrazine hydrate (0.6 mL) in MeOH (50 mL) was heated under reflux for
4 h in the presence of 10% Pd(OH)2/C (1.0 g). The solution was filtered
through Celite and concentrated to give, after freeze-drying from H2O, the
diamine 10 (1.03 g, 93%) as an amorphous solid. [a]2D0 ˆ ‡12.0 (c ˆ 1 in
H2O); 1H NMR (500 MHz, CD3OD): d ˆ 1.33, 1.34, 1.39, 1.52 (4s, 12H;
‡
‡
ESMS: m/z (%): 713.3 (100) [M‡H] , 735.2 (70) [M‡Na] .
2,3,4-Tri-O-acetyl-6-N-benzylmethylamino-6-deoxy-a-d-glucopyranosyl-
(1 ! 4)-2,3-di-O-acetyl-6-N-benzylmethylamino-6-deoxy-b-d-glucopyra-
nosyl-(1 ! 6)-1,2:3,4-di-O-isopropylidene-a-d-galactopyranose (13): A sol-
ution of the dibromide 8 (1.90 g, 2.1 mmol) in N-benzylmethylamine
(5.7 mL) was stirred at 808C for 20 h. The reaction mixture was
concentrated and the crude product mixture was re-acetylated with
C5H5N (2 mL) and Ac2O (2 mL) at RT overnight. The solution was
concentrated, re-dissolved in EtOAc, and washed with saturated NaHCO3
and saturated NaCl solutions, before drying over anhydrous Na2SO4 and
concentrating to an oil. Column chromatography (SiO2, hexanes/EtOAc,
6:4 to 1:1) gave the protected diamine 13 (0.87 g, 42%) as an amorphous
solid. [a]2D0 ˆ ‡12.0 (c ˆ 1 in CHCl3); 1H NMR (500 MHz, CDCl3): d ˆ 1.34,
1.36, 1.43, 1.54 (4s, 12H; 2CMe2), 1.88, 2.02, 2.04, 2.07, 2.10 (5s, 15H;
5COMe), 2.28, 2.35 (2brs, 6H; 2NMe), 2.46 2.53 (m, 2H; H6c, H6c'), 2.92
(dd, J5,6 ˆ 7.7 Hz, J6,6' ˆ 13.5 Hz, 1H; H6b), 3.05 (bd, J6,6' ˆ 13.5 Hz, 1H;
H6b'), 3.52 (m, 2H; NCH2Ph), 3.66 (d, JA,B ˆ 13.4 Hz, 1H; NCH2Ph), 3.74
3.81 (m, 3H; NCH2Ph, H6a, H5b), 3.85 (pt, J3,4 ꢀ J4,5 ˆ 8.9 Hz, 1H; H4b),
3.96 (m, 1H; H5a), 4.01 4.06 (m, 2H; H6a', H5c), 4.21 (dd, J3,4 ˆ 7.9 Hz,
J4,5 ˆ 1.5 Hz, 1H; H4a), 4.33 (dd, J1,2 ˆ 4.9 Hz, J2,3 ˆ 2.4 Hz, 1H; H2a), 4.61
(dd, J2,3 ˆ 2.4 Hz, J3,4 ˆ 7.9 Hz, 1H; H3a), 4.67 (d, J1,2 ˆ 7.9 Hz, 1H; H1b),
4.85 (dd, J1,2 ˆ 3.9 Hz, J2,3 ˆ 10.6 Hz, 1H; H2c), 4.88 (pt, J1,2 ꢀ J2,3 ˆ 8.2 Hz,
1H; H2b), 4.96 (pt, J3,4 ꢀ J4,5 ˆ 9.7 Hz, 1H; H4c), 5.27 5.34 (m, 2H; H3b,
2CMe2), 2.76 2.83 (m, 2H, H6b, H6c), 3.04 (dd, J5,6' ˆ 2.9 Hz, J6,6'
ˆ
13.4 Hz, 1H; H6c'), 3.12 (dd, J5,6' ˆ 1.6 Hz, J6,6' ˆ 13.8 Hz, 1H; H6b'), 3.17
(pt, J3,4 ꢀ J4,5 ˆ 9.5 Hz, 1H; H4c), 3.26 (dd, J1,2 ˆ 7.9 Hz, J2,3 ˆ 9.3 Hz, 1H;
H2b), 3.35 3.42 (m, 2H; H4b, H5b), 3.45 (dd, J1,2 ˆ 3.7 Hz, J2,3 ˆ 9.5 Hz,
1H; H2c), 3.58 3.72 (m, 4H; H6a, H3b, H3c, H5c), 4.03 4.09 (m, 2H;
H5a, H6a'), 4.32 (dd, J3,4 ˆ 7.9 Hz, J4,5 ˆ 1.1 Hz, 1H; H4a), 4.34 (d, J1,2
ˆ
7.9 Hz, 1H; H1b), 4.38 (dd, J1,2 ˆ 5.0 Hz, J2,3 ˆ 2.4 Hz, 1H; H2a), 4.64 (dd,
J2,3 ˆ 2.4 Hz, J3,4 ˆ 7.9 Hz, 1H; H3a), 5.20 (d, J1,2 ˆ 3.7 Hz, 1H; H1c), 5.52
(d, J1,2 ˆ 5.0 Hz, 1H; H1a); 13C NMR (100 MHz, CD3OD): d ˆ 24.6, 25.1,
26.3 (2C), 43.6, 43.7, 68.8, 69.8, 71.9, 72.0, 72.4, 73.0, 74.0, 74.2, 74.6, 74.7,
76.0, 77.5, 82.9, 97.7, 102.8, 104.6, 110.0, 110.4; FABMS: m/z (%): 605.25
‡
‡
(100) [M‡Na] , 583.27 (50) [M‡H] ; HRFABMS: calcd for C24H43N2O14
‡
[M‡H] : 583.2714; found: m/z: 583.2737.
6-Benzamido-6-deoxy-a-d-glucopyranosyl-(1 ! 4)-6-benzamido-6-deoxy-
b-d-glucopyranosyl-(1 ! 6)-1,2:3,4-di-O-isopropylidene-a-d-galactopyra-
nose (11): Benzoyl chloride (2.77 mL, 24.0 mmol) was added dropwise to a
solution of the diamine 10 (1.00 g, 1.72 mmol) in C5H5N at 08C. After
stirring the mixture at RT for 24 h, the reaction was quenched by adding
MeOH and concentrated. The resulting oil was dissolved in EtOAc and
washed consecutively with 1m HCl, saturated NaHCO3, and saturated
NaCl solutions, before drying over anhydrous Na2SO4 and concentrating to
Chem. Eur. J. 2002, 8, No. 13
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0813-2997 $ 20.00+.50/0
2997

J. F. Stoddart et al.
FULL PAPER
H3c), 5.43 (d, J1,2 ˆ 3.9 Hz, 1H; H1c), 5.54 (d, J1,2 ˆ 5.0 Hz, 1H; H1a), 7.25
7.36 (m, 10H; Ar-H); 13C NMR (125 MHz, CDCl3): d ˆ 20.5, 20.6 (3C),
20.8, 24.3, 24.9, 25.8, 25.9, 42.6, 43.7, 57.2, 57.6, 62.2, 63.3, 67.6, 68.6, 69.6,
70.1, 70.2 (2C), 70.3, 70.5, 71.1, 72.1, 73.9, 74.7, 75.2, 95.1, 96.1, 100.6, 108.6,
109.3, 126.8, 126.9, 128.1 (2C), 128.2 (2C), 128.8 (2C), 128.9 (2C), 138.7,
138.8, 169.5, 169.8, 169.9, 170.1, 170.4; FABMS: m/z (%): 1001.45 (100)
(40 mg, 86%) as a solid foam. [a]2D0 ˆ ‡24.8 (c ˆ 1 in CHCl3); 1H NMR
(500 MHz, [D7]DMF, 350 K): d ˆ 1.32, 1.33, 1.40, 1.49 (4s, 12H; 2CMe2),
1.94 2.10 (m, 69H; 23COMe), 2.32, (s, 3H; NMe-c) 2.35 (s, 3H; NMe-b),
2.46 (dd, J1,1' ˆ 13.4 Hz, J1,2 ˆ 7.0 Hz, 1H; H1d), 2.55 2.62 (m, 3H, H6c,
H1d', H1g), 2.66 (dd, J5,6' ˆ 6.0 Hz, J6,6' ˆ 14.3 Hz, 1H; H6c'), 2.71 (dd, J1,1'
ˆ
13.6 Hz, J1,2 ˆ 5.4 Hz, 1H; H1g'), 2.84 (dd, J5,6 ˆ 7.2 Hz, J6,6' ˆ 14.0 Hz, 1H;
H6b), 2.92 (dd, J5,6' ˆ 1.9 Hz, J6,6' ˆ 14.0 Hz, 1H; H6b'), 3.50 3.56 (m, 2H;
H6e, H6h), 3.61 (dd, J5,6 ˆ 7.6 Hz, J6,6' ˆ 11.0 Hz, 2H; H6d, H6g), 3.65 3.78
(m, 6H; H6a, H5b, H6f, H6f', H6i, H6i'), 3.85 3.92 (m, 5H; H4b, H6d',
H5e, H6g', H5h), 3.94 3.98 (m, 3H; H5a, H4e, H4h), 4.00 4.04 (m, 2H;
‡
‡
‡
[M‡H] , 985.10 (10) [M À 15] , 741.20 (10) [M À a] , 392.17 (20) [M À
‡
‡
(a‡b)] ; HRFABMS: calcd for C50H68N2O19 [M‡H] : 1001.4494; found:
m/z: 1001.4486.
6-Deoxy-6-methylamino-a-d-glucopyranosyl-(1 ! 4)-6-deoxy-6-methyl-
amino-b-d-glucopyranosyl-(1 ! 6)-1,2:3,4-di-O-isopropylidene-a-d-galac-
topyranose (2): A solution of the pentaacetate 13 (870 mg, 0.87 mmol) and
NaOMe (0.5 mL, 0.5M in MeOH, 0.25 mmol) in MeOH (5 mL) was stirred
H6a', H5c), 4.10 4.16 (m, 2H; H6e', H6h'), 4.25 (dd, J3,4 ˆ 7.9 Hz, J4,5
ˆ
1.7 Hz, 1H; H4a), 4.29 4.33 (m, 2H; H5f, H5i), 4.35 (dd, J1,2 ˆ 4.9 Hz,
J2,3 ˆ 2.4 Hz, 1H; H2a), 4.63 (dd, J2,3 ˆ 2.4 Hz, J3,4 ˆ 7.9 Hz, 1H; H3a),
4.74 4.78 (m, 5H; H1b, H1e, H2e, H1h, H2h), 4.80 (pt, J1,2 ꢀ J2,3 ˆ 8.0 Hz,
‡
at RT for 4 h. The solution was neutralized with Amberlite IR-120 (H
1H; H2b), 4.87 4.91 (m, 2H; H2c, H4c), 4.97 (dd, J1,2 ˆ 3.9 Hz, J2,3
ˆ
form) ion exchange resin, filtered and concentrated. A solution of the
residue in MeOH (50 mL) was treated with 10% Pd(OH)2/C (300 mg)
under a hydrogen atmosphere for 2 h. The solution was filtered through
Celite and concentrated to give the bismethylamine 2 (490 mg, 92%) as an
amorphous solid after freeze-drying from H2O. [a]2D0 ˆ ‡15.6 (c ˆ 1 in
H2O); 1H NMR (500 MHz, CD3OD): d ˆ 1.33, 1.34, 1.40, 1.52 (4s, 12H;
2CMe2), 2.44, 2.46 (2brs, 6H; 2NMe), 2.73 2.81 (m, 2H; H6b, H6c),
2.91 3.10 (m, 2H; H6b', H6c'), 3.19 (pt, J3,4 ꢀ J4,5 ˆ 9.4 Hz, 1H; H4c), 3.25
(dd, J1,2 ˆ 7.9 Hz, J2,3 ˆ 9.1 Hz, 1H; H2b), 3.37 (pt, J3,4 ꢀ J4,5 ˆ 9.1 Hz, 1H;
H4b), 3.44 (dd, J1,2 ˆ 3.6 Hz, J2,3 ˆ 9.1 Hz, 1H; H2c), 3.54 (m, 1H; H5b),
3.58 3.70 (m, 3H; H6a, H3b, H3c), 3.77 (m, 1H; H5c), 4.01 4.08 (m, 2H;
10.5 Hz, 2H; H2f, H2i), 5.04 (pt, J3,4 ꢀ J4,5 ˆ 9.6, 2H; H4f, H4i), 5.09 (ptd,
J1,2 ꢀ J1',2 ˆ 7.0 Hz, J2,3 ˆ 1.8 Hz, 1H; H2d), 5.18 5.37 (m, 16H; H3b, H1c,
H3c, H3d, H4d, H5d, H3e, H1f, H3f, H2g, H3g, H4g, H5g, H3h, H1i, H3i),
5.49 (d, J1,2 ˆ 5.0 Hz, 1H; H1a), 7.39 (m, 4H; 4Ar-H), 7.46 7.50 (m, 6H;
6Ar-H), 7.53 7.56 (m, 2H; 2Ar-H), 7.90 7.94 (m, 8H; 8Ar-H), 8.11 8.17
(m, 4H; 4NHBz); 13C NMR (125 MHz, [D7]DMF, 350 K): d ˆ 20.3 20.9
(m, 23C), 24.6, 25.1, 26.2, 26.3, 40.8 (2C), 42.2 (2C), 43.8, 43.9, 58.9, 59.0,
59.3, 59.4, 67.9, 68.0, 68.1, 68.8, 68.9, 69.1, 69.2, 69.3, 69.4, 69.5, 69.8, 69.9,
70.2, 70.3, 70.4 (2C), 70.5 (2C), 70.7 (2C), 70.8, 70.9 (2C), 71.1, 71.3, 71.4,
71.8, 72.7 (2C), 72.9, 74.0 (2C), 74.5, 75.7 (3C), 75.8, 76.9, 77.0, 96.2, 96.9,
97.1 (2C), 100.4 (2C), 101.3, 108.8, 109.5, 127.9 (4C), 128.0 (4C), 128.6 (4C),
128.8 (4C), 131.4 (2C), 131.7 (2C), 135.4 (2C), 135.7 (2C), 167.6 (2C), 167.9
H5a, H6a'), 4.30 (dd, J3,4 ˆ 8.0 Hz, J4,5 ˆ 1.0 Hz, 1H; H4a), 4.33 (d, J1,2
ˆ
7.9 Hz, 1H; H1b), 4.37 (dd, J1,2 ˆ 4.9 Hz, J2,3 ˆ 2.4 Hz, 1H; H2a), 4.63 (dd,
J2,3 ˆ 2.4 Hz, J3,4 ˆ 8.0 Hz, 1H; H3a), 5.19 (d, J1,2 ˆ 3.6 Hz, 1H; H1c), 5.51
(d, J1,2 ˆ 4.9 Hz, 1H; H1a); 13C NMR (125 MHz, CD3OD): d ˆ 24.6, 25.1,
26.3, 26.4, 36.1, 36.4, 53.2, 53.9, 68.9, 69.8, 71.9, 72.0, 72.2, 72.5, 73.7, 74.0,
74.1, 74.7, 74.8, 77.4, 82.9, 97.8, 102.2, 104.4, 110.0, 110.5; FABMS: m/z (%):
‡
(2C), 169.8 170.7 (m, 23C); MALDI-TOF: m/z: 2990 [M‡Na] .
6,6'-N,N'-Bis[6,6'-bisbenzamido-6,6'-dideoxy-b-maltosyl-(1 ! 6)-d-galac-
tit-1-yl]-6,6'-dideoxy-6,6'-dimethylamino-b-maltosyl-(1 ! 6)-d-galactose
(16): The diacetonide 14 (104 mg, 47 mmol) was treated with 90% aqueous
TFA (5 mL) at RT for 15 min before concentration and co-evaporation
with MeOH. Column chromatography (C-18 reversed-phase silica, H2O to
MeOH) gave the reducing sugar 16 (60 mg, 60%) as an amorphous solid
after freeze drying from H2O. [a]2D0 ˆ ‡37.6 (c ˆ 1 in H2O); 1H NMR
(500 MHz, D2O, 323 K): d ˆ 3.00 3.06 (brm, 6H; 2NMe), 3.26 4.34 (m,
‡
‡
611.50 (100) [M‡H] , 351.33 (5) [M À a] ; HRFABMS: calcd for
‡
C26H46N2O14 [M‡H] : 611.3027; found: m/z: 611.3032.
6,6'-N,N'-Bis[6,6'-bis-benzamido-6,6'-dideoxy-b-maltosyl-(1 ! 6)-d-galac-
tit-1-yl]-6,6'-dideoxy-6,6'-dimethylamino-b-maltosyl-(1 ! 6)-1,2:3,4-di-O-
isopropylidene-a-d-galactopyranose (14): A solution of the reducing sugar
1 (192 mg, 270 mmol), the diamine 2 (55 mg, 90 mmol), acetic acid (5 mL,
90 mmol) and sodium cyanoborohydride (34 mg, 540 mmol) in MeOH
(4 mL) was heated under reflux for 8 h. The reaction mixture was cooled to
RT, concentrated to approximately 1 mL and diluted with H2O (8 mL),
before being subjected to column chromatography (C-8 reversed-phase:
MeOH/H2O/TFA, 0:100:0.0001 to 100:0:0.0001) to afford the dendron 14
58H), 4.38 4.41 (2d, J1,2 ˆ 8.0 Hz, 2H; H1e, H1h), 4.56 4.63 (3d, J1,2
ˆ
8.0 Hz, ca. 2.5H; H1ab, H1b), 5.25 (d, J1,2 ˆ 3.6 Hz, ca. 0.5H; H1aa), 5.27 (d,
J1,2 ˆ 3.8 Hz, 2H; H1f, H1i), 5.59 (d, J1,2 ˆ 3.7 Hz, 1H; H1c), 7.25 (m, 4H;
4Ar-H), 7.34 (m, 2H; 2Ar-H), 7.47 (m, 4H; 4Ar-H), 7.52 (m, 4H; 4Ar-H),
7.59 (m, 2H; 2Ar-H), 7.66 (m, 4H; 4Ar-H); ESMS: m/z (%): 960.7 (100)
‡
[M‡2H]2‡
,
1921.0 (5) [M‡H] ; calcd for C84H122N6O44
:
M ˆ 1919.9;
average of observed m/z: 1919.7.
1
(140 mg, 70%) as its bis-TFA salt. [a]2D0 ˆ ‡23.0 (c ˆ 1 in H2O); H NMR
(500 MHz, CD3OD): d ˆ 1.31, 1.33, 1.38, 1.50 (4s, 12H; 2CMe2), 2.74 (brs,
3H; NMe), 2.88 (brs, 3H; NMe), 2.93 3.06 (brm, 2H), 3.15 3.36 (m,
15H), 3.45 3.74 (m, 22H), 3.81 (pt, J ˆ 9.1, 1H), 3.87 (brd, J ˆ 13.9, 2H),
6,6'-N,N'-Bis{6,6'-N,N'-bis[6,6'-bisbenzamido-6,6'-dideoxy-b-maltosyl-
(1 ! 6)-d-galactit-1-yl]-6,6'-dideoxy-6,6'-dimethylamino-b-maltosyl-(1 !
6)-d-galactit-1-yl}-6,6'-dideoxy-6,6'-dimethylamino-b-maltosyl-(1 ! 6)-
1,2:3,4-di-O-isopropylidene-a-d-galactopyranose (17): Sodium cyanoboro-
hydride (2.9 mg, 46.1 mmol) was added in three portions over the course of
3 h, to a solution of the reducing sugar 16 (60 mg, 27.9 mmol), the diamine 2
(5.7 mg, 9.3 mmol), and acetic acid (0.5 mL, 9.1 mmol) in MeOH (300 mL)
heated under reflux. Heating was continued overnight. After cooling to
room temperature, the reaction mixture was diluted with MeOH (2 mL),
before being subjected to gel permeation chromatography (Sephadex
LH20: MeOH) to afford 17 (10 mg, 25%) as an amorphous solid after
freeze-drying from H2O. Selected 1H NMR data (500 MHz, D2O, 343 K):
d ˆ 1.34, 1.36, 1.45, 1.55 (4s, 12H; 2CMe2), 2.80 2.90 (m, 18H; 6NMe),
3.93 4.10 (m, 11H), 4.18 4.28 (brm, 2H), 4.30 (dd, J3,4 ˆ 7.9 Hz, J4,5
1.3 Hz, 1H; H4a), 4.33 (d, J1,2 ˆ 7.7 Hz, 2H; H1e, H1h), 4.36 (dd, J1,2
ˆ
ˆ
4.9 Hz, J2,3 ˆ 2.4 Hz, 1H; H2a), 4.43 (d, J1,2 ˆ 7.8 Hz, 1H; H1b), 4.61 (dd,
J2,3 ˆ 2.3 Hz, J3,4 ˆ 7.9 Hz, 1H; H3a), 5.16 (d, J1,2 ˆ 3.6 Hz, 1H; H1f, H1i),
5.32 (brd, J1,2 ꢀ 3.0 Hz, 1H; H1c), 5.52 (d, J1,2 ˆ 4.9 Hz, 1H; H1a), 7.25 (m,
4H; 4Ar-H), 7.35 (m, 2H; 2Ar-H), 7.43 (m, 4H; 4Ar-H), 7.52 (m, 2H; 2Ar-
H), 7.74 (m, 8H; 8Ar-H); 13C NMR (125 MHz, CD3OD): d ˆ 24.7, 25.1,
26.4, 26.5, 42.2 (2C), 42.7 (2C), 43.6, 44.4, 59.1, 60.8, 62.0, 62.6, 67.1, 67.3,
68.9, 69.9, 70.0 (2C), 70.9, 71.1, 71.4 (2C), 71.8, 71.9, 72.1, 72.5, 73.0 (2C),
73.2 (3C), 73.6 (2C), 73.7, 74.2 (2C), 74.4, 74.5 (2C), 74.6 (2C), 74.8, 75.1
(2C), 75.7, 77.6 (2C), 79.4, 80.8, 84.5, 84.6, 97.7, 99.3, 103.5 (2C), 104.0, 104.5
(2C), 110.1, 110.5, 128.3 (4C), 128.5 (4C), 129.4 (4C), 129.5 (4C), 132.5
4.82 (d, J1,2 ˆ 7.9 Hz, 4H; H1h, H1k, H1q, H1t), 4.87 (dd, J1,2 ˆ 4.9 Hz, J2,3
ˆ
2.4 Hz, 1H; H2a), 4.97 (d, J1,2 ˆ 8.0 Hz, 1H; H1b), 5.00 (d, J1,2 ˆ 8.1 Hz, 2H;
(2C), 132.7 (2C), 135.4 (2C), 135.5 (2C), 170.3 (2C), 170.8 (2C); ESMS:
H1e, H1n), 5.11 (dd, J2,3 ˆ 2.4 Hz, J3,4 ˆ 7.9 Hz, 1H; H3a), 5.69 (d, J1,2 ˆ
‡
m/z (%): 1001.0 (100) [M‡2H]2‡
,
2001.2 (5) [M‡H] ; calcd for
3.7 Hz, 4H; H1i, H1l, H1r, H1u), 5.91 (d, J1,2 ˆ 3.8 Hz, 1H; H1c), 5.93 (d,
J1,2 ˆ 3.7 Hz, 2H; H1f, H1o), 6.02 (d, J1,2 ˆ 4.9 Hz, 1H; H1a); ESMS: m/z
C90H130N6O44 [M]: 2000.0; average of observed m/z: 2000.1.
(%): 737.4 (20) [M‡6H]6‡, 884.7 (100) [M‡5H]5‡, 1105.8 (55) [M‡4H]4‡
,
2,3,2',3',4'-Penta-O-acetyl-6,6'-dideoxy-6,6'-dimethylamino-6,6'-N,N'-
bis[2,3,2',3',4'-penta-O-acetyl-6,6'-bisbenzamido-6,6'-dideoxy-b-maltosyl-
(1 ! 6)-2,3,4,5-tetra-O-acetyl-d-galactit-1-yl]-b-maltosyl-(1 ! 6)-1,2:3,4-
di-O-isopropylidene-a-d-galactopyranose (15): A solution of the dendron
14 (35 mg, 17 mmol) and DMAP (2 mg) in Ac2O (4 mL) and C5H5N (4 mL)
was stirred at RT for 36 h, and then concentrated. The mixture was diluted
with EtOAc (50 mL) and washed consecutively with 1m HCl, saturated
NaHCO3, and saturated NaCl solutions, before drying over anhydrous
Na2SO4 and concentrating to a foam. Column chromatography (SiO2,
CH2Cl2/MeOH, 95:5, then Sephadex LH20, MeOH) gave the peracetate 15
1473.6 (20) [M‡3H]3‡; calcd for C194H290N14O100: [M] 4418.5; average of
observed m/z: 4418.4.
Tris[2-(N-{6,6'-N,N'-bis[6,6'-bisbenzamido-6,6'-dideoxy-b-maltosyl-(1 !
6)-d-galactit-1-yl]-6,6'-dideoxy-6,6'-dimethylamino-b-maltosyl-(1 ! 6)-d-
galactit-1-yl}methylamino)ethyl]amine (21): Acetic acid (2.28 mL, 45 mmol)
was added to a solution of the reducing sugar 16 (90 mg, 47 mmol), tris[2-
(methylamino)ethyl]amine 20[45] (2 mg, 12 mmol), and sodium cyanoboro-
hydride (10 mg, 121 mmol) in MeOH (1 mL). The reaction mixture was
stirred and heated under reflux for 6 h. The mixture was allowed to cool to
2998
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0813-2998 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 13

Oligosaccharide Glycodendrimers
2988 3000
room temperature, diluted with H2O (2 mL) and purified by preparative
GPC Chromatography (Sephadex G-50, H2O) to afford 21 (38 mg, 54%).
[a]2D0 ˆ ‡32 (c ˆ 1 in H2O); selected 1H NMR data (500 MHz, [D7]DMF,
373 K): d ˆ 4.42 (d, J1,2 ˆ 7.2 Hz, 6H; H1e, H1h), 4.58 (d, J1,2 ˆ 7.5 Hz, 3H;
H1b), 5.20 (d, J1,2 ˆ 3.4 Hz, 6H; H1f, H1i), 5.52 (brd, J1,2 ꢀ 3.0 Hz, 3H;
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‡
H1c); MALDI-TOF: m/z: 5920.5 [M‡K] .
Tris[2-(N-methyl{2,2',3,3',4'-penta-O-acetyl-6,6'-dideoxy-6,6'-di-N-methyl-
6,6'-bis[2,2',3,3',4'-penta-O-acetyl-6,6'-bisbenzamido-6,6'-dideoxy-b-malto-
syl-(1 ! 6)-2,3,4,5-tetra-O-acetyl-d-galactit-1-yl]amino-b-maltosyl-(1 !
6)-2,3,4,5-tetra-O-acetyl-d-galactit-1-yl}amino)ethyl]amine (22): The first-
generation dendrimer 21 (38 mg, 6 mmol) was dissolved in a solution of
DMAP (2 mg) in Ac2O (4 mL) and C5H5N (4 mL) and was left to stir at
room temperature for 36 h. The solution was concentrated by co-
evaporation with PhMe, before diluting with EtOAc and washing with
1m HCl, saturated NaHCO3, and saturated NaCl solutions, drying
(Na2SO4), and concentrating to an oil. GPC chromatography (MeOH)
gave 22 (27 mg, 50%) as an oil. [a]2D0 ˆ ‡24 (c ˆ 1 in H2O); selected
1H NMR ([D7]DMF, 500 MHz, 350 K): d ˆ 1.98 2.17 (m, 243H;
81COMe), 2.37 (s, 9H; 3NMe), 2.43 (s, 9H; 3NMe), 3.57 (m, 6H; H6e,
H6h), 3.68 (m, 6H; H6f, H6i), 3.64 (m, 6H; H6f', H6i'), 3.65 (m, 9H; H6a,
H6d, H6g), 3.88 (m, 9H; H6a', H6d', H6g'), 3.93 (m, 6H; H5e, H5h), 3.99
(m, 6H; H4e, H4h), 4.02 (m, 3H; H5c), 4.13 (m, 6H; H6e', H6h'), 4.33 (m,
6H; H5f, H5i), 4.72 (m, 3H; H1b), 4.77 (m, 9H; H2b, H2e, H2h), 4.78 (m,
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6H; H1e, H1h), 4.90 (m, 6H; H2c, H4c), 4.97 (dd, J1,2 ˆ 3.9 Hz, J2,3
ˆ
10.5 Hz, 6H; H2f, H2i), 5.04 (pt, J3,4 ꢀ J4,5 ˆ 9.6, 6H; H4f, H4i), 5.28 (m,
12H; H1c, H3b, H3e, H3h), 5.38 (m, 6H; H1f, H1i), 7.41 (m, 4H; 12Ar-H),
7.50 (m, 6H; 18Ar-H), 7.57 (m, 2H; 6Ar-H), 7.94 (m, 8H; 24Ar-H);
selected DEPT-135 13C NMR ([D7]DMF, 125 MHz): d ˆ 40.1 (C6f, C6i),
42.0 (C6e, C6h), 72.4 (C2b, C2e, C2h), 73.4 (C5e, C5h), 75.5 (C3b, C3e,
C3h), 76.6 (C4e, C4h), 96.0 (C1c), 96.9 (C1f, C1i), 99.9 (C1e, C1h), 100.0
(C1b), 127.9 (C-Ar), 128.0 (C-Ar), 128.6 (C-Ar), 128.8 (C-Ar), 131.5 (C-
‡
Ar), 131.7 (C-Ar); MALDI-TOF: m/z: 9222 [M‡H] .
Molecular modeling: The molecular model of the second-generation
dendron 17 was developed in a ™convergent∫ fashion analogous to its
chemical synthesis. Each of the distinct trisaccharide building blocks were
constructed in the INPUT submode of Macromodel v5.0 and minimized by
using the PRCG algorithm[48] with the Amber* forcefield[49] and the GB/SA
solvation model for H2O,[50] to a final energy gradient of < 0.05 kJäÀ1. The
molecules were subjected to a molecular dynamics (MD) run (1 ns, 300 K,
1.5 fs timestep) and structures, sampled at regular intervals, were each
minimized as before, to select the conformer to be used in constructing the
dendron. Appropriate trisaccharides were then linked together to form a
first- and then a second-generation dendron. The process of minimization,
MD, and minimization was repeated at each stage. The picture of 17
(Figure 9c) shows the lowest energy conformer obtained after minimization
of 50 structures selected from a 4 ns MD run at 600 K. Conformational
flexibility of the building blocks and dendrons were assessed by monitoring
changes in selected torsion angels in both the maltosyl and galactityl units
during MD runs.
¬
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Acknowledgement
This research was supported by the Wellcome Trust through the award of
an International Prize Travelling Research Fellowship (054997/Z/98/Z) to
WBT, and by the National Science Foundation (equipment grant CHE-
9974928 and CHE-9808175). We thank Dr A. R. Pease for assistance with
molecular modeling experiments.
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