Novel template-assembled oligosaccharide clusters as epitope mimics for HIV-neutralizing antibody 2G12. Design, synthesis, and antibody binding study

Article abstract of DOI:10.1039/b702961f

The synthesis of a new class of template-assembled oligomannose clusters as the mimics of the epitope of the HIV-neutralizing antibody 2G12 is described. The novel oligomannose clusters were successfully assembled on a cyclic decapeptide template using th

Full text of DOI:10.1039/b702961f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Novel template-assembled oligosaccharide clusters as epitope mimics for
HIV-neutralizing antibody 2G12. Design, synthesis, and antibody binding
study†
Jingsong Wang, Hengguang Li, Guozhang Zou and Lai-Xi Wang*
Received 27th February 2007, Accepted 26th March 2007
First published as an Advance Article on the web 17th April 2007
DOI: 10.1039/b702961f
The synthesis of a new class of template-assembled oligomannose clusters as the mimics of the epitope
of the HIV-neutralizing antibody 2G12 is described. The novel oligomannose clusters were successfully
assembled on a cyclic decapeptide template using the Cu(I)-catalyzed 1,3-dipolar cycloaddition of
azides to alkynes by introducing four units of a synthetic D1 arm tetrasaccharide
(Mana1,2Mana1,2Mana1,3Mana-) of high-mannose N-glycan on one face of the template and two
T-helper epitope peptides on the other face of the template. Their binding to human antibody 2G12
was studied using surface plasmon resonance (SPR) technology. It was found that while the synthetic
monomeric D1 arm oligosaccharide and its fluorinated derivative interacted with 2G12 only weakly,
the corresponding template-assembled oligosaccharide clusters showed high affinity to antibody 2G12,
indicating a clear clustering effect in 2G12 recognition. Interestingly, the fluorinated D1 arm cluster, in
which the 6-OH of the terminal mannosyl residue was replaced with a fluorine atom, showed a distinct
kinetic model in 2G12 binding as compared with the cluster of the natural D1 arm oligosaccharides.
The oligosaccharide clusters with varied length of spacer demonstrated different affinity to 2G12,
suggesting that an appropriate spatial orientation of the sugar chains in the cluster was crucial for high
affinity binding to the antibody 2G12. It was also found that the introduction of two T-helper epitopes
onto the template did not affect the structural integrity of the oligomannose cluster. The novel synthetic
glycoconjugates represent a new type of immunogen that may be able to raise carbohydrate-specific
neutralizing antibodies against HIV-1.
suggests the presence of unique neutralizing epitopes on the viral
Introduction
envelope that can be explored for immunogen design.9 Among
AIDS is caused by the infection of the human immunodeficiency
them, the human antibody 2G12 was revealed to target a novel
virus (HIV).1 It is considered that the best hope to control the
worldwide epidemic of HIV/AIDS is an effective preventive HIV-
1 vaccine. However, development of an effective HIV-1 vaccine
has faced many difficulties.2–4 Under the pressure of host immune
surveillance, HIV-1 has evolved a number of defense mechanisms
to evade immune attacks, including frequent mutation of neu-
tralizing epitopes; conformational masking of receptor binding
sites; heavy glycosylations to form an evolving glycan shield;
and formation of envelope glycoprotein complexes to occlude
conserved epitopes.5–8 Therefore, a major challenge in HIV vaccine
design is to identify unique neutralizing epitopes on the HIV-1
envelope capable of eliciting broadly neutralizing antibodies to
break down the strong viral defense. The existence of a few human
monoclonal antibodies such as 2F5, 4E10, b12, and 2G12 that
are able to neutralize a broad range of HIV-1 primary isolates
oligomannose cluster present on HIV-1 gp120.10
Initial biochemical and mutational studies have demonstrated
that the epitope of antibody 2G12 involves a novel oligomannose
cluster consisting of several high-mannose type N-glycans with
terminal Mana1,2Man residues.10–12 Subsequent 2G12-binding
studies with defined oligosaccharides and synthetic clusters have
indicated that the terminal Mana1,2Man unit was essential for
2G12 recognition but a Mana1,2Man unit alone was not sufficient
for an effective binding to 2G12.13–17 The full-size Man9 was shown
to have the highest affinity to 2G12 among several natural high-
mannose oligosaccharides.14 Interestingly, the synthetic mannose
tetrasaccharide (Man4) corresponding to the D1 arm of Man9
was revealed to have comparable affinity to the antibody as
that of the Man9 moiety.13 The structural requirement of the
oligosaccharide subunit for 2G12 binding was confirmed by recent
X-ray structural studies on the 2G12-oligomannose complexes.18,19
Since 2G12 recognizes an oligomannose cluster with at least
two sugar chains, we have initiated a project aiming to create
template-assembled oligomannose clusters to mimic the 2G12
epitope found on gp120.14,15 For example, we have demonstrated
that the galactose-based tetravalent Man9-cluster (Tetra-Man9)
was 73-fold and 5000-fold more effective in binding to 2G12 than
the monomeric Man9GlcNAc and Man6GlcNAc, respectively.14
Institute of Human Virology, University of Maryland, 725 W. Lombard
Street, Baltimore, MD, 21201. E-mail: wangx@umbi.umd.edu; Fax: 001-
410-706-5068; Tel: 001-410-706-4982
† Electronic supplementary information (ESI) available: Experimental
details for the synthesis of the fluorinated glycosyl donor 6, the glycosyl
acceptor 11, and the succinimidyl 8-azido-3,6-dioxaoctanoate 17, and the
ESI-MS and HPLC profiles of the synthetic glycopeptides 21, 22, 23, 24,
25 and 27. See DOI: 10.1039/b702961f
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These binding data suggest that synthetic oligomannose clusters
such as the Tetra-Man9 provides a mimic to the 2G12 epitope.
We have recently conjugated the oligomannose cluster Tetra-
Man9 to a carrier protein keyhole limpet hemocyanin (KLH),
and our immunization studies with the KLH conjugate have
demonstrated that the glycoconjugate could induce moderate
carbohydrate-specific antibodies in rabbits, which were weakly
cross-reactive to HIV-1 gp120.20 However, it was observed that the
majority of antibody responses were directed to the linkers and
the template, which drastically dilutes the immune response to the
oligomannose cluster. Indeed, the undesired immune responses
to the linkers and/or template is of a particular concern when
dealing with weakly immunogenic carbohydrate antigens such as
those found on HIV and human cancers.21 As a continuous effort
in developing a carbohydrate-based HIV-1 vaccine,22 we report
in this paper the synthesis of a new class of template-assembled
oligomannose clusters using a novel cyclic peptide as the template
that are expected to have low immunogenicity and the D1 arm
tetramannose (Man4) oligosaccharide as the 2G12-recognizing
subunits. The oligomannose clusters were efficiently assembled
using the Cu(I)-catalyzed 1,3-dipolar cycloaddition between azides
and alkynes. The binding of the synthetic clusters and human
antibody 2G12 were studied using surface plasmon resonance
(SPR) technology.
The immunization data suggest that the choice of appropriate
linkers and template is of paramount importance in dealing with
weakly immunogenic epitopes such as the self-like high-mannose
type oligosaccharides. As a continuous effort in designing a
better immunogen, we have chosen the regioselectively addressable
functionalized template (RAFT) as the template for oligosaccha-
ride cluster assembling and the D1 arm tetramannose (Man4)
oligosaccharide (Mana1,2Mana1,2Mana1,3Mana-) of the high-
mannose type N-glycan (Fig. 1A) as the 2G12 recognizing sub-
units. RAFT is a novel cyclic decapeptide containing two proline-
glycine moieties as beta-turn inducers that stabilize the template
conformations and up to 6 selectively protected lysine residues.23–26
Thus, the cyclic decapeptide provides two functional faces, one for
the attachment of oligosaccharide moieties and the other for the
attachment of T-helper peptide epitopes. The conjugation of the
oligosaccharide cluster to a T-helper epitope or a carrier protein
is important to create an effective immunogen capable of raising
long-lasting IgG type antibodies.27–33 Recent immunization studies
have demonstrated that the cyclic peptide template could serve as
a non-immunogenic scaffold for assembling multiepitopic anti-
cancer vaccines bearing clustered tumor-associated carbohydrate
antigens.33 Based on these considerations, we have designed a novel
template-assembled oligomannose cluster immunogen, in which
four a-linked Man4 oligosaccharides corresponding to the D1
arm of Man9GlcNAc2 were introduced at one face of the cyclic
peptide template to form a novel cluster, and two universal T-
helper epitopes corresponding to the sequence (aa830–844) of
tetanus toxoid34 were introduced at the other face of the cyclic
template (Fig. 1B).
Results and discussion
Design
Our previous work on the 2G12 binding of synthetic oligomannose
clusters has demonstrated that an appropriate template-assembled
oligomannose cluster could exhibit much higher affinity to
2G12 than the monomeric oligomannose, showing a clustering
effect in the antibody–antigen interactions.14,15 In addition, our
preliminary immunization studies indicated that it was possible
to induce carbohydrate-specific antibodies using glycoconjugates
containing an oligomannose cluster, but the nature of the linker
and template was crucial in determining the immune responses
because the maleimide linkers and/or the template in the first
generation conjugate vaccines were found to induce predom-
inantly linker/template-specific antibodies, which significantly
suppresses the antibody responses to the oligomannose epitope.20
The D1 arm tetrasaccharide was chosen because its binding
affinity to 2G12 was shown to be comparable to the largest
oligomannose Man9GlcNAc2.13 X-Ray structural studies have
also confirmed that the D1 arm residues account for more than
85% of the 2G12 Fab contacts to Man9GlcNAc2.18,19 Moreover,
considering the weak immunogenicity of the high-mannose type
N-glycans that may be regarded as “self” antigens by the immune
system, we also decided to selectively modify the terminal mannose
residue of the D1 arm Man4 at the 6-position with a fluorine
atom, which may still behave as a hydrogen-bonding acceptor
but the unnatural modification may enhance the immunogenicity
of the oligomannose cluster. To assemble the designed immuno-
gen, we decided to attach the multiple functionality to the
Fig. 1 Structures of Man9GlcNAc2 (A) and the designed template-assembled glycopeptide vaccine (B).
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cyclic template by the highly efficient copper(I)-catalyzed 1,3-
dipolar azide–alkyne cycloaddition.35–39 This will introduce the
1,2,3-triazole linkers in the glycoconjugate. Compared to the
hydrophobic aminohexyl and maleimide linkers, the hydrophilic
1,2,3-triazole linkers are expected to be less immunogenic. The
successful synthesis of such triazole-linked glycoconjugates will
allow the evaluation of whether the triazole-linker is truly an
immune-compatible linker for conjugate vaccines when applied
for immunization studies.
was prepared in several steps (see ESI†), gave the trisaccharide
derivatives 7 and 8, respectively. After conversion to the cor-
responding trisaccharide trichloroacetimidates 9 and 10, these
glycosyl donors were coupled with the glycosyl acceptor 11 to
afford the tetrasaccharide derivatives 12 and 13, respectively, with
the desired a-glycosidic linkages. Treatment of compounds 12 and
13 with 80% aqueous acetic acid at elevated temperature to remove
the benzylidene group, followed by de-O-acylation with MeONa–
MeOH gave the D1 arm tetrasaccharide 14 and the fluorinated
D1 arm tetrasaccharide 15, respectively. The compounds were
purified and further characterized as their per-O-acetylated forms.
The tetrasaccharide derivatives contain an azido functionality at
the aglycon portion that will be used for conjugation to the cyclic
peptide template through click chemistry. To prepare a longer
spacer at the aglycon for comparative studies, the azido group in
compound 14 was reduced to an amino group by hydrogenation
to give 16, which was then coupled with the azido-containing
derivative 17 to provide another tetrasaccharide derivative 18
(Scheme 1).
Synthesis
The synthesis of the D1 arm oligosaccharide and its derivatives
is summarized in Scheme 1. TMSOTf-catalyzed glycosidation
between the mannosyl trichloroacetimidate 1 and acceptor 3 gave
the disaccharide 4. The 2ꢀ-O-acetyl group in 4 was selectively
removed by mild acidic hydrolysis to give the intermediate 5.
Glycosidation of 5 with the mannosyl trichloroacetimidate 1
or the 6-deoxy-6-fluoro-mannosyl trichloroacetimidate 6, which
Scheme 1 Reagents and conditions: a) 1) TMSOTf, CH2Cl2, 82%; 2) MeONa, MeOH, 95%; b) TMSOTf, CH2Cl2, 83%; c) CH3COCl–MeOH, 72%;
d) TMSOTf, CH2Cl2, 82% for 7, 78% for 8; e) 1) PdCl2, MeOH, 90%; 2) CCl3CN, DBU, 85% for 9, 88% and 81% for 10; f) TMSOTf, CH2Cl2, 77% for
12, 73% for 13; g) 1) 80% AcOH, 60 ◦C; 2) NaOCH3–CH3OH; 3) Ac2O–Py; 4) NaOCH3–CH3OH, 76% for 14 over 4 steps, 75% for 15 over 4 steps;
h) H2, Pd/C, 98%; i) 0.05M NaHCO3, CH3CN–H2O 1 : 1, 74%.
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Scheme 2 Reagents and conditions: a) 50% AcOH, DCM, r.t., 95%; b) HBTU, DIPEA, 88%; c) 2% hydrazine, DMF, 84%; d) 1) propynoic acid, DCC,
77%; 2) 90% TFA, 93%; e) CuSO4 (0.2 mol. equiv.), sodium ascorbate (0.4 mol. equiv.), t-BuOH–H2O (1 : 1), over 90% for 21, 22, 23, and 24.
To synthesize the cyclic decapeptide template 19, a distinctly
protected linear decapeptide, in which two Lys residues were
protected with Boc groups and the remaining four Lys residues
were protected with Dde protecting groups, was prepared by au-
tomated peptide synthesizer on an acidic sensitive resin, NovaSyn
TGT. After being released from the resin by mild acidic hydrolysis,
the fully protected linear peptide was cyclized by treatment with
HBTU–DIPEA in DMF to give the corresponding cyclic peptide
in excellent yield. Then the Dde protecting groups were selectively
removed by treatment with 2% hydrazine40 in DMF to give the
cyclic peptide 19 with four free amino groups on one face of
the template. Introduction of alkynyl groups to the template was
achieved by coupling four propynoic acid moieties to compound
19 using DCC as the coupling reagent to give the corresponding
conjugate in 77% yield, from which the two Boc groups were
selectively removed by treatment with TFA to afford the alkynyl
derivative 20 in 93% yield (Scheme 2).
BuOH–H2O, 1 : 1), following the reported procedures.41,42 The
reaction was monitored by HPLC analysis. It was found that the
reaction between 2 and 20 at rt proceeded very slowly, resulting
in a mixture of mono-, di-, tri- and tetra-substituted products as
revealed by ESI-MS analysis of the HPLC peaks (data not shown).
Only a fraction of starting materials was consumed even after
2 days. However, it was observed that the cycloaddition proceeded
smoothly and quickly when the reaction mixture was heated and
stirred at 60 ◦C. As revealed by HPLC, all of the alkynyl template
◦
was consumed in 30 min at 60 C to give a mixture of di- (peak
2), tri- (peak 3) and tetra-substituted (peak 4) products (Fig. 2).
After 6 h, the reaction went to completion to give the single final
product (peak 4), which was characterized by MS and NMR as the
1
desired tetra-substituted compound 21. H NMR of 21 revealed
only a singlet signal at 8.5 ppm for the proton of the resulting 1,2,3-
triazole, suggesting the sole formation of the 1,4-substituted 1,2,3-
triazole, rather than a mixture with the 1,5-substituted isomer
(which would appear at ca. 8.3 ppm as a singlet). The results
confirm that the Cu(I)-catalyzed 1,3-dipolar cycloaddition results
in a regioselective formation of the 1,4-substituted derivative even
at an elevated temperature.41,43,44 Similarly, the Cu(I)-catalyzed
After successful preparation of the alkynyl cyclic decapeptide
template, the copper(I)-catalyzed dipolar cycloaddition was first
examined using the monosaccharide azide derivative 2 in the
presence of CuSO4 and sodium ascorbate in a mixed solvent (t-
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Antibody binding studies
With the synthetic oligosaccharide clusters in hand, their binding
to the human antibody 2G12 was analyzed utilizing surface
plasmon resonance (SPR) technology. Binding experiments were
carried out with a Biacore 3000 system in a HBS-P buffer. The
immobilization of antibody 2G12 to the CM5 sensor chip was
performed based on a previously reported procedure.17 Briefly,
the chip surface was activated by injection of EDC–NHS for
7 min at 5 ll min−1, followed by injection of 20 lg ml−1 2G12
solution in an acetate buffer (10 mM pH 5.5) until the target
level of 10 000 response units (RU) was reached, and then the
surface was saturated by 7 min pulse of ethanol amine HCl (1 M,
pH 8.5) at 5 ll min−1. A reference surface was prepared by a
similar procedure, but without the injection of antibody solution.
Each compound was injected to reference and the antibody-
activated surface channels, and the binding profile was obtained
by an automatic subtraction of the reference surface signal from
the 2G12-activated surface signal. The sensor surface between
runs was regenerated with a short pulse of 3.5 M MgCl2. It was
found that, at 10 lM concentrations, all the synthetic monomeric
D1 arm oligosaccharides 14, 15, and 18, as well as the natural
high-mannose type N-glycan Man9GlcNAc2Asn, did not show
apparent binding to antibody 2G12, with the signal responses
being around the detection threshold (below 5 RU). The results
are in agreement with the previous observation by Danishefsky
and co-workers that synthetic monomeric glycopeptides carrying
a single high-mannose type N-glycan did not bind to 2G12 at
10 lM as measured by SPR technology.17 The template-assembled
mannose cluster 21 did not show detectable 2G12 binding at
10 lM, either (data not shown). The results indicate that neither
single oligomannose nor the cluster of terminal a-mannosides
is sufficient for 2G12 binding. However, it was found that the
synthetic oligosaccharide clusters carrying four units of the D1
arm tetrasaccharide and its fluorinated derivative, namely clusters
22, 23, and 24, have demonstrated apparent affinity to antibody
Fig. 2 Monitoring the reaction of 1,3-dipolar cycloaddition between
azido sugar 2 and the alkynyl template 20. Peak 1, cyclic peptide 20;
peaks 2 and 3, reaction intermediates; and peak 4, the final adduct 21.
cycloaddition of the tetrasaccharide derivatives 14, 15, and 18
with the tetra-alkynyl template 20 proceeded very efficiently at an
elevated temperature to give the corresponding oligosaccharide
clusters 22–24 in more than 90% isolated yields. The results
indicate that the Cu(I)-catalyzed dipolar cycloaddition of azides to
alkynes provides a neat and highly efficient means for constructing
even large oligosaccharide clusters.
Finally, to construct an effective immunogen capable of eliciting
T-cell dependent immune responses such as IgG type antibodies,
two T-helper peptides derived from tetanus toxoid (aa830–844)34
were successfully introduced into the fluorinated oligosaccharide
cluster 23. Thus, the oligosaccharide cluster 23 that contains two
free amino groups at the down face was reacted with the activated
ester 17 to give the azido-containing oligosaccharide cluster 25.
The azido groups in 25 were then reacted with the alkynylated
T-helper peptide 26, again through Cu(I)-catalyzed cycloaddition,
affording the fully synthetic vaccine candidate 27 in 70% isolated
yield (Scheme 3).
Scheme 3 Reagents and conditions: a) 0.05 M NaHCO3, CH3CN–MeOH 1 : 1, 90%; b) CuSO4 (0.2 mol. equiv.), sodium ascorbate (0.4 mol. equiv.),
t-BuOH–H2O (1 : 1), 70%.
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2G12. Under the measuring conditions (10 lM, at 30 sec), for
example, the binding response units (RU) for the oligosaccharide
clusters 22, 23, and 24 are 105, 43, 142 RU, respectively. The
fluorinated derivative 23 also exhibits significant affinity to 2G12,
despite at a reduced efficiency in comparison with the natural D1
arm cluster 22. It was observed that the cluster 24 with an extended
spacer showed higher affinity to 2G12 than the cluster 22 that has
a relatively short spacer. The data suggest that an appropriate
spatial orientation of the sugar chains in the cluster is crucial for
high affinity binding to the antibody 2G12.
In order to probe the binding nature of synthetic oligosaccha-
ride clusters, we performed kinetic studies with compounds 22,
23, and 24. The experiments were carried out at six different
concentrations at 10.0, 5.00, 2.50, 1.25, 0.625, and 0.312 lM
(Fig. 3). The observed binding profiles for compounds 22 and
24 are similar, but both of them showed rather complex kinetics
that do not fit into a simple 1 : 1 Langmuir model.45 The observed
profile implies multiple association and dissociation (fast and slow)
steps that may involve a required conformational adjustment for
the binding of subsequent oligosaccharide chains in the cluster.46
These experimental data are similar to the kinetic studies for the
2G12-binding of a bivalent Man9GlcNAc2-containing glycopep-
tide reported by Danishefsky and co-workers.17 Interestingly, it
was found that the binding profile of the fluorinated oligomannose
cluster 23 was different from the non-fluorinated clusters, and
could fit perfectly to the 1 : 1 Langmuir model. Kinetic studies
gave Rmax = 47.2 RU, Ka = 3.79 e5, Kd = 2.64 e−6, whereas Chi2 =
0.49. The data suggested the existence of a typical first order kinetic
process in the antigen–antibody binding. Considering the fact that
the only structural difference between the clusters 22 and 23 is
the replacement of the 6-OH group in the terminal mannosyl
residue in 22 by a fluorine atom in 23, the different binding
kinetic outcome between 22 and 23 may implicate a different
2G12-binding model for the two compounds. Since fluorine and
hydroxyl groups have different capabilities in hydrogen bonding,
it is likely that the interaction of the fluorinated compound 23
with antibody 2G12 does not induce significant conformational
changes in the antibody, thus resulting in an apparent first
order binding, whereas the interaction between the cluster 22
and antibody 2G12 may cause conformational changes in the
antibody and shows a complex kinetic process. An alternative
explanation for this difference may be the existence of many
preexisting antibody conformations (conformational diversity).46
Thus, different antigens may interact preferably with different
conformational isomers with or without induced fit isomerization
that would lead to high-affinity interactions.
Finally, the 2G12-binding of the fully synthetic vaccine can-
didate 27 containing two T-helper peptides was examined. It
was found that compound 27 demonstrated almost identical
binding profiles and kinetic outcome to those of the corresponding
fluorinated oligosaccharide cluster 23 (data not shown). This result
suggested that the introduction of the T-helper epitopes onto the
cyclic decapeptide template did not affect the structural integrity
of the fluorinated oligosaccharide cluster formed at the other face
of the template. Therefore, the novel synthetic glycoconjugate
represents a valuable immunogen that may be able to raise
carbohydrate-specific neutralizing antibodies against HIV-1.
Conclusion
The synthesis of a new class of template-assembled oligomannose
clusters as the mimics of the epitope of human antibody 2G12
was described. The experimental data indicate that the Cu(I)-
catalyzed 1,3-dipolar cycloaddition of azides and alkynes is highly
efficient for constructing conjugate vaccines containing large
oligosaccharide clusters and free T-helper epitopes. The binding
studies with the synthetic oligosaccharide clusters have revealed
an interesting, distinct kinetic outcome for the different synthetic
antigens. The data implicate a distinct binding mechanism for the
fluorinated and natural D1 arm clusters that may involve antibody
conformational changes during the antigen–antibody interactions.
Oligosaccharide cluster 24, with an extended spacer, showed
higher affinity to 2G12 than the cluster 22, that has a relatively
short spacer; these results suggest that an appropriate spatial
Fig. 3 SPR kinetic studies on the 2G12-binding to compounds 22–24
at 10.0 (A), 5.00 (B), 2.50 (C), 1.25 (D), 0.625 (E), 0.312 (F) lM
concentrations, respectively.
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ꢀ
=
orientation of the sugar chains in the cluster is crucial for high
affinity binding to antibody 2G12. The successful construction
of the fully synthetic immunogens that contain both the novel
oligosaccharide clusters and T-helper epitopes has now laid the
foundation for further immunization studies in animals to evaluate
whether the vaccine candidates are able to elicit carbohydrate-
specific neutralizing antibodies against HIV-1.
(m, 4 H, -CH CH2, H-3, H-3 , H-4), 5.76 (t, 1 H, J = 2.5 Hz,
H-2ꢀ), 5.33 (dd, 1 H, J = 1.0, 17.0 Hz, 1/2 CH2 CHCH2), 5.26
=
=
(dd, 1 H, J = 1.0, 10.0 Hz, 1/2 CH2 CHCH2), 5.21 (d, 1 H, J =
1.7 Hz, H-1ꢀ), 5.15 (d, 1 H, J = 1.4 Hz, H-1), 4.70–4.50 (m, 5 H,
H-5ꢀ, 4 H-6), 4.46–4.40 (m, 2 H, H-2, H-5), 4.20–3.89 (m, 2 H,
=
CH2CH CH2), 2.08 (s, 3H, COCH3); dC (CDCl3, 125 MHz):
169.4, 166.4, 166.1, 165.7, 165.6, 165.3, 165.0, 133.6, 133.5, 133.4,
133.3, 133.2, 133.2, 133.2, 133.1, 130.2, 130.0, 130.0, 129.9, 129.9,
129.9, 129.8, 129.8, 129.7, 129.7, 129.3, 129.1, 128.9, 128.8, 128.6,
128.5, 128.5, 128.5, 128.4, 118.1, 99.6, 98.0, 70.9, 69.8, 69.6,
69.0, 68.8, 67.6, 67.2, 63.7, 63.4, 20.6; ESI-MS: m/z: calcd for
C59H52O18: 1048.3; found: 1049.2 [M + H]+, 1071.2 [M + Na]+.
Experimental
General procedures
TLC was performed on aluminium plates coated with silica gel
60 with detection by charring with 10% (v/v) sulfuric acid in
methanol or by UV detection. Flash column chromatography was
performed on silica gel 60 (230–400 mesh). 1H and 13C NMR, and
2D NMR spectra were recorded on a 500 NMR spectrometer
in CDCl3, D2O, or CD3OD, as specified. Chemical shifts are
expressed in ppm. The ESI-MS spectra were measured on a single
quadruple mass spectrometer. Analytical RP-HPLC was carried
out on a C18 column (3.9 × 150 mm) at 40 ◦C. The column
was eluted with a linear gradient of 0–90% MeCN containing
0.1% TFA at a flow rate of 1 mL min−1 over 25 minutes, unless
otherwise specified. Peptides and glycopeptides were detected at
double wavelengths (214 and 280 nm). Preparative RP-HPLC was
performed on a preparative C18 column (19 × 300 mm). The
column was eluted with a suitable gradient of MeCN containing
0.1% TFA at 12 mL min−1.
Allyl 3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1 → 2)-3,4,6-tri-
O-benzoyl-a-D-mannopyranoside (5). To a solution of 4 (500 mg,
0.476 mmol) in anhydrous CH3OH (30 mL) was added acetyl
chloride (1.0 mL). The mixture was stirred at 40 ◦C for 4 h,
when TLC (hexanes–EtOAc 2 : 1) indicated that the selective
de-O-acetylation was complete to give a less mobile spot. The
mixture was concentrated under reduced pressure, and the residue
was purified by flash silica gel column chromatography (hexanes–
EtOAc 3 : 1) to give 5 (348 mg, 72%) as a white foam. dH (500 MHz,
CDCl3): 8.14–7.36 (m, 30 H, Ph), 6.05 (t, 1 H, J = 9.9 Hz, H-4ꢀ),
=
5.99 (t, 1 H, J = 9.8 Hz, H-4), 5.94 (m, 1 H, CH CH2), 5.89 (dd,
1 H, J = 3.1, 9.9 Hz, H-3ꢀ), 5.85 (dd, 1 H, J = 3.1, 9.8 Hz, H-3),
=
5.33 (dd, 1 H, J = 1.0, 17.0 Hz, 1/2 CH2 CHCH2), 5.25 (dd, 1 H,
=
J = 1.0, 10.0 Hz, 1/2 CH2 CHCH2), 5.24 (d, 1 H, J = 1.1 Hz,
H-1ꢀ), 5.22 (d, 1 H, J = 1.2 Hz, H-1), 4.68–4.50 (m, 6 H), 4.48
(t, 1 H, J = 3.0 Hz, H-2), 4.45 (m, 1 H, H-5), 4.21–3.91 (m, 2 H,
2-(2-Azidoethoxy)ethyl-a-D-mannopyranoside (2). 2-O-Acetyl-
3,4,6-tri-O-benzoyl-a-D-mannopyranosyl trichloroacetimidate 147
(200 mg, 0.295 mmol) and 2-(2-azidoethoxy)-ethanol (100 mg,
0.76 mmol) were dried together under high vacuum for 2 h. The
mixture was dissolved in anhydrous CH2Cl2 (20 mL). TMSOTf
(10 ll, 0.05 mmol) was added and the mixture was stirred under
an argon atmosphere at rt overnight. The mixture was neutralized
with triethylamine and concentrated under vacuum. The residue
was purified by flash silica gel column chromatography (hexanes–
EtOAc 1 : 1) to give a white solid (167 mg). The solid was
dissolved in MeOH (10 mL) and a solution of MeONa in MeOH
(0.5 M, 0.3 mL) was added. The mixture was stirred at rt for 1 h,
then neutralized by adding Dowex 50W (H+). After filtration, the
filtrate was concentrated to give compound 2 (68 mg, 78% over
two steps) as a white solid. dH (500 MHz, CD3OD): 4.84 (d, 1 H,
J = 1.5 Hz, H-1), 3.91–3.59 (m, 12 H), 3.42 (t, 2 H, J = 5.0 Hz,
CH2N3); dC (CD3OD, 125 MHz): d = 100.4, 73.2, 71.2, 70.7, 69.9,
69.8, 67.2, 66.5, 61.5, 50.4.
=
CH2CH CH2); dC (CDCl3, 125 MHz): 166.4, 166.2, 165.7, 165.6,
165.4, 165.3, 133.5, 133.4, 133.3, 133.1, 133.1, 129.9, 129.8, 129.8,
129.7, 129.7, 129.3 129.1, 129.0, 129.0, 128.6, 128.5, 128.4, 128.4,
128.3, 128.3, 118.1, 101.6, 98.1, 72.3, 71.4, 69.7, 69.4, 68.9, 68.8,
67.5, 67.0, 63.7, 63.6; ESI-MS: m/z: calcd for C57H50O17: 1007.0;
found: 1007.5.
Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1 →
2)-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1 → 2)-3,4,6-tri-O-
benzoyl-a-D-mannopyranoside (7). To a solution of 1 (485 mg,
0.72 mmol) and 5 (600 mg, 0.6 mmol) in anhydrous CH2Cl2
(20 mL) was added TMSOTf (5 lL, 0.026 mmol). The reaction
mixture was stirred at rt overnight and was then neutralized with
triethylamine. The mixture was concentrated under reduced pres-
sure, and the residue was subject to flash column chromatography
(hexanes–EtOAc 2 : 1) to give compound 7 (750 mg, 82%) as
a white foam. dH (500 MHz, CDCl3): 8.15–7.30 (m, 45 H, Ph),
ꢀꢀ
ꢀ
ꢀꢀ
=
Allyl
2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-
6.09–5.90 (m, 3 H, H-3 ,H-4 , H-4 ), 5.95 (m,1 H, -CH CH2),
(1 → 2)-3,4,6-tri-O-benzoyl-a-D-mannopyranoside (4). To a
solution of allyl-3,4,6-tri-O-benzoyl-a-D-mannopyranoside 348
(532 mg, 1.0 mmol) and 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-
mannopyranosyl trichloroacetimidate 1 (810 mg, 1.20 mmol)
in anhydrous CH2Cl2 (10 mL) was added TMSOTf (10 ll,
0.05 mmol) under an argon atmosphere. The mixture was stirred
at rt for 10 h and neutralized with triethylamine. The mixture was
then concentrated under vacuum and the residue was purified by
flash silica gel column chromatography (hexanes–EtOAc 5 : 1)
to give 4 (870 mg, 83%) as a white foam. dH (500 MHz, CDCl3):
8.11–7.33 (m, 30 H, Ph), 6.03 (t, 1 H, J = 9.9 Hz, H-4ꢀ), 5.97–5.88
5.90–5.80 (m, 3 H, H-3, H-3ꢀ, H-4), 5.63 (t, 1 H, J = 2.0 Hz, H-2ꢀꢀ),
5.47 (s,1 H, H-1ꢀꢀ ), 5.37–5.27 (m, 2 H, CH2 CHCH2), 5.20 (s, 1 H,
=
H-1ꢀ), 4.92 (s, 1 H, H-1), 4.70–4.30 (m, 10 H), 4.28–3.96 (m, 2 H,
=
CH2CH CH2), 4.18 (m, 1 H, H-6), 2.09 (s, 3 H, COCH3); dC
(CDCl3, 125 MHz): 169.1, 166.3, 166.2, 165.8, 165.7, 165.6, 165.4,
165.3, 165.2, 165.0, 133.4, 133.3, 133.3, 133.3, 133.2, 133.1, 133.1,
130.1, 130.0, 130.0, 129.7, 129.6, 129.5, 129.1, 129.1, 129.0, 128.9,
128.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.3, 128.2, 128.2,
118.0, 100.1, 99.8, 98.0, 71.1, 70.4, 69.7, 69.6, 69.5, 68.9, 68.8, 67.4,
67.0, 63.9, 63.7, 63.4, 20.7; ESI-MS: m/z: calcd for C86H74O26:
1522.5; found: 1523.6.
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Allyl 2,3,4-tri-O-benzoyl-6-deoxy-6-fluoro-a-D-mannopyrano-
syl-(1 → 2)-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1 → 2)-
3,4,6-tri-O-benzoyl-a-D-mannopyranoside (8). To a solution of 5
(650 mg, 0.646 mmol) and 6 (500 mg, 0.707 mmol) in anhydrous
CH2Cl2 (20 mL) was added TMSOTf (5 lL, 0.026 mmol). The
mixture was stirred at rt under an argon atmosphere overnight
and was then neutralized with triethylamine. The mixture was
concentrated under reduced pressure, and the residue was purified
by flash column chromatography (hexanes–EtOAc 2 : 1) to give
compound 8 (750 mg, 78%) as a white foam. dH (500 MHz,
CDCl3): 8.15–7.20 (m, 45 H, Ph), 6.09–5.90 (m, 5 H, H-2ꢀꢀ, H-3ꢀꢀ,
133.4, 133.3, 133.2, 133.1, 133.0, 130.0, 130.0, 129.9, 129.9, 129.8,
129.8, 129.7, 129.7, 129.6, 129.3, 129.0, 128.9, 128.9, 128.8, 128.8,
128.6, 128.5, 128.5, 128.4, 128.4, 128.3, 99.7, 96.3, 90.7, 71.7, 70.5,
70.0, 69.6, 69.5, 69.5, 67.1, 67.0, 63.5, 63.3, 63.2, 20.6.
2,3,4-tri-O-Benzoyl-6-deoxy-6-fluoro-a-D-mannopyranosyl-(1
→
2)-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1 → 2)-3,4,6-tri-O-
benzoyl-a-D-mannopyranosyl trichloroacetimidate (10). To a so-
lution of 8 (600 mg, 0.40 mmol) in anhydrous MeOH (80 mL)
was added PdCl2 (0.30 g) and the mixture was stirred at 40 ◦C for
4 h, when TLC (hexanes–EtOAc 2 : 1) indicated the completion of
de-O-allylation. The mixture was filtered through a pad of celite
and the filtrate was concentrated. The residue was purified by flash
silica gel column chromatography (hexanes–EtOAc 2 : 1) to give
the corresponding hemiacetal (510 mg, 88%) as a white foam. dH
(500 MHz, CDCl3): 8.15–7.20 (m, 45 H, Ph), 6.10–5.80 (m, 7 H,
H-2ꢀꢀ , H-3, H-3ꢀ, H-3ꢀꢀ, H-4, H-4ꢀ, H-4ꢀꢀ), 5.64 (s, 1 H, H-1ꢀꢀ ), 5.46
(d, 1 H, J = 1.3 Hz, H-1ꢀ), 5.04 (d, 1 H, J = 1.5 Hz, H-1), 4.74–4.30
(m, 11 H); dC (CDCl3, 125 MHz): 166.5, 166.3, 165.9, 165.7, 165.6,
165.5, 165.3, 165.0, 164.9, 133.6, 133.5, 133.4, 133.3, 133.3, 133.2,
133.1, 133.0, 130.1, 130.0, 130.0, 129.9, 129.8, 129.7, 129.7, 129.6,
129.4, 129.2, 129.1, 129.0, 128.9, 128.6, 128.4, 128.4, 128.3, 100.3,
99.7, 93.5, 81.1 (d, JC–F = 175 Hz, C-6ꢀꢀ), 71.1, 70.5, 70.4, 70.0,
69.7, 68.8, 67.4, 67.0, 65.9, 63.7, 60.5.
ꢀ
ꢀꢀ
ꢀ
=
H-4 , H-4 , CH CH2), 5.90–5.80 (m, 3 H, H-3, H-3 , H-4), 5.48
(s,1 H, H-1ꢀꢀ ), 5.37–5.27 (m, 2 H, CH2 CHCH2), 5.24 (s, 1 H,
=
H-1ꢀ), 4.92 (s, 1 H, H-1), 4.70–4.30 (m, 9 H), 4.57 (m, 2 H, H-6ꢀꢀ),
=
4.28–3.96 (m, 2 H, CH2CH CH2); dC (CDCl3, 125 MHz): 166.4,
166.3, 165.8, 165.6, 165.5, 165.4, 165.3, 165.1, 164.9, 133.6, 133.5,
133.3, 133.3, 133.2, 133.1, 133.1, 130.1, 130.0, 130.0, 129.9, 129.8,
129.8, 129.2, 129.2, 129.1, 129.0, 128.9, 128.8, 128.7, 128.6, 128.5,
128.4, 128.3, 128.3, 128.2, 128.2, 118.1, 100.4, 99.8, 98.1, 81.3 (d,
JC–F = 175.0 Hz, C-6ꢀꢀ), 71.4, 70.5, 70.4, 70.3, 70.0, 69.7, 69.6,
69.0, 68.8, 67.5, 67.4, 65.9, 65.9, 63.7; ESI-MS: m/z: calcd for
C84H71FO24: 1482.4; found: 1483.5.
2-O-Acetyl-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1 → 2)-
3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1 → 2)-3,4,6-tri-O-ben-
zoyl-a-D-mannopyranosyl trichloroacetimidate (9). To a solution
of 7 (500 mg, 0.33 mmol) in anhydrous CH3OH (30 mL) was
added PdCl2 (0.30 g), the mixture was stirred at 40 ◦C for 4 h,
when TLC (2 : 1 hexanes–EtOAc) indicated the completion of the
de-O-allylation. The mixture was filtered through a pad of celite.
The filtrate was concentrated, and the residue was purified by flash
silica gel column chromatography (hexanes–EtOAc 2 : 1) to give
the corresponding hemiacetal (440 mg, 90%) as a white foam. dH
(500 MHz, CDCl3): 8.10–7.29 (m, 45 H, Ph), 6.00–5.88 (m, 3 H,
H-3ꢀꢀ , H-4ꢀ, H-4ꢀꢀ ), 5.84–5.78 (m, 3 H, H-3, H-3ꢀ, H-4), 5.58 (t, 1
H, J = 2.0 Hz, H-2ꢀꢀ), 5.54 (s, 1 H, H-1ꢀꢀ ), 5.42 (s, 1 H, H-1ꢀ), 4.90
(s, 1H, H-1), 4.64–4.50 (m, 7 H), 4.50-4.20 (m, 4 H), 2.08 (s, 3 H,
COCH3); dC (CDCl3, 125 MHz): 169.1, 166.5, 166.2, 166.2, 165.9,
165.7, 165.6, 165.4, 165.3, 165.0, 133.3, 133.2, 133.0, 130.1, 130.0,
130.0, 129.9, 129.8, 129.7, 129.7, 129.6, 129.4, 129.2, 129.1, 129.0,
128.9, 128.6, 128.4, 128.4, 128.3, 100.2, 99.8, 93.3, 69.6, 69.5, 68.9,
67.7, 67.0, 63.9, 63.7, 20.7; ESI-MS: m/z: calcd for C83H79O26:
1482.3; found: 1483.6 [M + H]+, 1505.8 [M + Na]+.
The above hemiacetal (450 mg, 0.312 mmol) was dissolved in
CH2Cl2 (20 mL), then trichloroacetonitrile (0.32 mL, 3.12 mmol)
and DBU (0.06 mL) were added. The reaction mixture was
stirred at room temperature overnight. The reaction mixture was
concentrated under vacuum and the residue was purified by flash
silica gel column chromatography (hexanes–EtOAc 4 : 1) to give
imidate 10 (400 mg, 81%) as a white foam. dH (500 MHz, CDCl3):
=
8.78 (s, 1 H, HN CCCl3), 8.18–7.20 (m, 45 H, Ph), 6.68 (d, 1 H,
J = 2.4 Hz, H-1), 6.17–6.13 (t, 2 H, J = 10.0 Hz, H-4ꢀ, H-4ꢀꢀ), 6.03
(dd, 1 H, J = 3.5, 10 Hz, H-3ꢀꢀ), 6.00 (dd, 1 H, J = 3.5, 9.5 Hz,
H-3ꢀ), 5.91 (dd, 1 H, J = 3.0, 9.0 Hz, H-3), 5.90 (t, 1H, J = 9.5 Hz,
H-4), 5.85 (dd, 1 H, J = 1.3, 3.5 Hz, H-2ꢀꢀ), 5.57 (s, 1 H, H-1ꢀꢀ ), 5.10
(s, 1 H, H-1ꢀ), 4.81 (t, 1 H, J = 3.0 Hz, H-2ꢀ), 4.77-4.62 (ddd, 2 H,
J = 2.3, 11.7, 44.0 Hz, H-6ꢀꢀ ), 4.76–4.60 (m, 3 H), 4.57 (dd, 1 H,
J = 1.0, 3.0 Hz, H-2), 4.48-4.28 (m, 4 H); dC (CDCl3, 125 MHz):
166.3, 166.2, 165.8, 165.6, 165.5, 165.3, 165.2, 165.0, 164.9, 160.1,
133.6, 133.5, 133.5, 133.4, 133.2, 133.1, 130.1, 130.0, 130.0, 129.9,
129.9, 129.8, 129.8, 129.7, 129.7, 129.6, 129.3, 129.0, 128.9, 128.9,
128.8, 128.8, 128.6, 128.5, 128.5, 128.4, 128.4, 128.3, 99.7, 96.3,
90.7, 81.2 (d, JC–F = 175 Hz, C-6ꢀꢀ), 71.6, 70.5, 70.0, 70.0, 69.6,
67.1, 65.8, 63.5, 63.5, 63.2.
The above prepared hemiacetal (450 mg, 0.303 mmol) was
dissolved in CH2Cl2 (40 mL), then trichloroacetonitrile (3 mL)
and DBU (0.3 mL) were added. The reaction mixture was
stirred at room temperature overnight. The reaction mixture was
concentrated under vacuum and the residue was purified by flash
silica gel column chromatography (hexanes–EtOAc 4 : 1) to give
imidate 9 (420 mg, 85%) as a white foam. dH (500 MHz, CDCl3):
2-(2-Azidoethoxy)ethyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-man-
nopyranosyl-(1
→
2)-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-
(1 → 2)-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1 → 3)-4,6-O-
benzylidene-2-O-benzoyl-a-D-mannopyranoside (12). To a solu-
tion of 9 (197 mg, 0.12 mmol) and 11 (42 mg, 0.0866 mmol)
in anhydrous CH2Cl2 (10 mL) was added TMSOTf (3 lL,
0.02 mmol). The mixture was stirred under an argon atmosphere
at rt overnight and was neutralized by triethylamine. The mixture
was concentrated under reduced pressure, and the residue was
purified by column chromatography (hexanes–EtOAc 2 : 1) to
give compound 12 (130 mg, 77%) as a white foam. dH (500 MHz,
CDCl3): 8.38–6.90 (m, 55 H, Ph), 6.10–5.95 (m, 2 H, H-4ꢀꢀ, H-4ꢀꢀꢀ),
5.93–5.70 (m, 3 H, H-3ꢀꢀ, H-3ꢀꢀꢀ, H-4ꢀ), 5.83 (s, 1 H, PhCH), 5.76
(dd, 1 H, J = 1.0, 3.5 Hz, H-2), 5.63 (s, 1 H, H-1ꢀꢀꢀ), 5.60–5.40
=
8.79 (s, 1 H, HN ), 8.16–7.29 (m, 45 H, Ph), 6.68 (d, 1 H, J =
2.4 Hz, H-1), 6.12–6.06 (t, 2 H, J = 9.5 Hz, H-4ꢀ, H-4ꢀꢀ), 6.00 (dd,
1 H, J = 3.5, 10 Hz, H-3ꢀꢀ), 5.93 (dd, 1 H, J = 2.5, 8.5 Hz, H-3),
5.88–5.85 (m, 2 H, H-3ꢀ, H-4), 5.68 (s, 1 H, H-2ꢀꢀ ), 5.62 (s, 1 H,
H-1ꢀꢀ), 5.03 (s, 1 H, H-1ꢀ), 4.79 (t, 1 H, J = 3.0 Hz, H-2ꢀ), 4.74 (dd,
1 H, J = 2.3, 11.7 Hz, H-6ꢀꢀ ), 4.70–4.58 (m, 5 H), 4.56 (t, 1 H, J =
2.0 Hz, H-2), 4.45 (m, 1H), 4.32 (m, 1 H, H-5), 4.17 (m, 1H, H-6),
2.08 (s, 3 H, COCH3); dC (CDCl3, 125 MHz): 169.1, 166.3, 166.2,
165.9, 165.6, 165.5, 165.4, 165.3, 165.2, 165.0, 160.0, 133.5, 133.4,
1536 | Org. Biomol. Chem., 2007, 5, 1529–1540
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(m, 2 H), 5.39 (s, 1 H, H-1ꢀꢀ), 5.15 (s, 1 H, H-1), 4.91 (s, 1 H,
H-1ꢀ), 4.83–4.60 (m, 5 H), 4.50–3.67 (m, 17 H), 3.44 (t, 2 H, J =
5.0 Hz, CH2N3), 2.01 (s, 3 H, CH3CO); dC (CDCl3, 125 MHz):
169.1, 166.4, 166.0, 165.7, 165.5, 165.2, 164.9, 137.0, 133.6, 133.2,
133.2, 133.1, 132.8, 130.1, 130.0, 130.0, 129.9, 129.9, 129.8, 129.8,
129.7, 129.6, 129.4, 129.2, 129.2, 129.1, 129.0, 128.8, 128.7, 128.5,
128.5, 128.4, 128.4, 128.4, 128.3, 128.2, 125.8, 110.0, 103.6, 101.7,
99.7, 99.0, 72.1, 71.7, 70.6, 70.3, 70.2, 69.6, 69.5, 69.4, 68.8, 67.0,
66.7, 63.7, 63.6, 63.6, 62.9, 62.9, 50.7, 20.6.
20.8, 20.8, 20.7, 20.7, 20.6; ESI-MS: m/z: calcd for C54H75N3O35:
1326.2, found: 1327.2 [M + H]+.
To a solution of the above compound (80 mg, 0.0603 mmol)
in anhydrous MeOH (5 mL) was added a solution of MeONa in
MeOH (0.5 M, 0.2 mL). The mixture was stirred at rt for 2 h, and
then neutralized by Dowex 50W (H+). The mixture was filtered
and the filtrate was concentrated to give 14 (45 mg, 98%) as a
white solid. dH (500 MHz, CD3OD): 5.42 (d, 1 H, J = 1.5 Hz, H-
1ꢀꢀꢀ), 5.34 (d, 1 H, J = 1.5 Hz, H-1ꢀꢀ), 5.03 (d, 1 H, J = 2.0 Hz, H-1),
4.82 (d, 1 H, J = 1.5 Hz, H-1ꢀ), 4.08–4.01 (m, 4 H), 3.98–3.59 (m,
26 H), 3.42 (t, 2 H, J = 5.0 Hz, CH2N3); dC (CD3OD, 125 MHz):
102.7, 100.9, 100.6, 100.5, 79.1, 78.9, 78.8, 73.7, 73.6, 73.4, 71.1,
70.7, 70.5, 70.1, 69.9, 67.7, 67.4, 66.6, 66.3, 61.8, 61.6, 61.4, 50.4;
ESI-MS: m/z: calcd for C28H49N3O22: 779.7; found: 780.5 [M +
H]+, 803.1 [M + Na]+.
2-(2-Azidoethoxy)ethyl 2,3,4,-tri-O-benzoyl-6-deoxy-6-fluoro-a-
D-mannopyranosyl-(1 → 2)-3,4,6-tri-O-benzoyl-a-D-mannopyra-
nosyl-(1 → 2)-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1 → 3)-
4,6-O-benzylidene-2-benzoyl-a-D-mannopyranoside (13). To a so-
lution of 10 (240 mg, 0.15 mmol) and 11 (61 mg, 0.125 mmol)
in anhydrous CH2Cl2 (10 mL) was added TMSOTf (3 lL,
0.02 mmol). The mixture was stirred under an argon atmosphere
at rt overnight and was then neutralized by triethylamine. The
mixture was concentrated under reduced pressure, and the residue
was purified by column chromatography (hexanes–EtOAc 2 : 1) to
give compound 13 (175 mg, 73%) as a white foam. dH (500 MHz,
CDCl3): 8.32–6.82 (m, 55 H, Ph), 6.11–5.98 (m, 2 H, H-4ꢀꢀ, H-4ꢀꢀꢀ),
5.97–5.78 (m, 3 H, H-3ꢀꢀ, H-3ꢀꢀꢀ, H-4ꢀ), 5.79 (s, 1 H, PhCH), 5.72
(dd, 1 H, J = 1.0, 3.5 Hz, H-2), 5.64 (s, 1 H, H-1ꢀꢀꢀ), 5.64–5.58 (m,
1 H), 5.42 (s, 1 H, H-1ꢀꢀ), 5.14 (s, 1 H, H-1), 4.93 (s, 1 H, H-1ꢀ),
4.80–4.56 (m, 5 H), 4.48–3.67 (m, 17 H), 3.44 (t, 2 H, J = 5.0 Hz,
CH2N3); dC (CDCl3, 125 MHz): 166.5, 166.1, 166.0, 165.5, 165.4,
165.3, 165.2, 165.1, 165.0, 164.9, 137.1, 133.6, 133.5, 133.3, 133.2,
133.0, 132.8, 130.1, 130.1, 130.0, 130.0, 129.9, 129.8, 129.7, 129.2,
129.2, 129.2, 129.1, 129.1, 128.9, 128.7, 128.7, 128.6, 128.6, 128.6,
128.5, 128.5, 128.4, 128.4, 128.2, 125.9, 101.7, 100.1, 99.7, 99.0,
81.2 (d, JC–F = 175 Hz, C-6ꢀꢀꢀ), 72.2, 70.5, 70.3, 70.2, 70.0, 69.7,
69.5, 69.4, 68.9, 67.1, 63.7, 63.0, 50.7.
2-(2-Azidoethoxy)ethyl 6-deoxy-6-fluoro-a-D-mannopyranosyl-
(1 → 2)-a-D-mannopyranosyl-(1 → 2)-a-D-mannopyranosyl-(1 →
3)-a-D-mannopyranoside (15).
A solution of 13 (180 mg,
0.0941 mmol) in aqueous acetic acid (80%, 10 mL) was stirred
at 60 ◦C. After 10 h, TLC indicated the completion of de-
benzylidenation. The mixture was concentrated under reduced
pressure, and the residue was dissolved in MeOH (10 mL), to
which a solution of MeONa in MeOH (0.5 M, 0.5 mL) was
added. The mixture was stirred overnight and then neutralized
with Dowex 50W (H+). The mixture was filtered and the filtrate was
concentrated. The residue was dissolved in pyridine–Ac2O (10 mL,
1 : 1) and the resulting solution was stirred overnight. The solvent
was removed under reduced pressure and the residue was purified
by flash column chromatography to give the corresponding O-
acetylated compound (90 mg, 78%) as a white solid. dH (500 MHz,
CDCl3): 5.45 (dd, 1 H, J = 3.5, 10.0 Hz, H-3ꢀꢀꢀ), 5.39–5.21 (m, 7 H),
5.21 (dd, 1 H, J = 3.0, 9.5 Hz, H-3ꢀꢀ), 5.14 (d, 1 H, J = 1.8 Hz,
H-1ꢀꢀꢀ), 5.04 (d, 1 H, J = 2.0 Hz, H-1ꢀꢀ), 4.99 (dd, 1 H, J = 1.8 Hz,
H-1), 4.92 (dd, 1 H, J = 1.8 Hz. H-1ꢀ), 4.56–4.45 (m, 2 H, H-6ꢀꢀꢀ),
4.30–4.08 (m, 10 H), 4.05–3.95 (m, 2 H), 3.90 (t, J = 2.5 Hz, 1 H),
3.90–3.84 (m, 1 H), 3.72–3.68 (m, 5 H), 3.42 (t, 2 H, J = 5.5 Hz,
CH2N3), 2.42–2.06 (12 s, 36 H, 12 CH3CO); dC (CDCl3, 125 MHz):
170.9, 170.8, 170.7, 170.4, 170.0, 169.9, 169.8, 169.7, 169.7, 169.5,
169.5, 169.4, 100.3, 99.9, 99.3, 97.5, 81.8 (d, JC–F = 175 Hz, C-6ꢀꢀꢀ),
74.9, 70.8, 70.1, 70.0, 69.8, 69.7, 69.6, 69.5, 69.4, 68.5, 68.3, 67.8,
67.1, 66.2, 65.8, 62.5, 62.2, 62.1, 61.9, 50.7, 20.9, 20.8, 20.8, 20.7,
20.7, 20.6; ESI-MS: m/z: calcd for C52H72FN3O33: 1286.1; found:
1286.4 [M + H]+.
2-(2-Azidoethoxy)ethyl a-D-mannopyranosyl-(1 → 2)-a-D-man-
nopyranosyl-(1 → 2)-a-D-mannopyranosyl-(1 → 3)-a-D-manno-
pyranoside (14). A solution of 12 (170 mg, 0.0872 mmol) in
aqueous acetic acid (80%, 10 mL) was stirred at 60 ◦C. After
10 h, TLC indicated the completion of de-benzylidenation. The
reaction mixture was concentrated under reduced pressure. The
residue was dissolved in MeOH (10 mL), to which a solution of
MeONa in MeOH (0.5 M, 0.5 mL) was added. The mixture was
stirred at rt overnight. After neutralized with Dowex 50W (H+),
the mixture was filtered and the filtrate was concentrated. The
residue was then dissolved into pyridine–Ac2O (10 mL, 1 : 1) and
resulting mixture was stirred overnight. The reaction mixture was
concentrated under reduced pressure and the residue was purified
by flash chromatography to give the corresponding O-acetylated
derivative (90 mg, 78%) as a white solid. dH (500 MHz, CDCl3):
5.44 (dd, 1 H, J = 3.5, 10.0 Hz, H-3ꢀꢀꢀ), 5.39–5.28 (m, 7 H), 5.19
(dd, 1 H, J = 3.0, 9.5 Hz, H-3ꢀꢀ), 5.17 (d, 1 H, J = 1.8 Hz, H-1ꢀꢀꢀ),
5.04 (d, 1 H, J = 2.0 Hz, H-1ꢀꢀ), 4.99 (dd, 1 H, J = 1.8 Hz, H-1),
4.93 (dd, 1 H, J = 1.8 Hz, H-1ꢀ), 4.34–4.10 (m, 12 H), 4.08–4.02 (m,
2 H), 3.96 (t, J = 2.5 Hz, 1 H), 3.90–3.84 (m, 1 H), 3.76–3.68 (m,
5 H), 3.42 (t, 2 H, J = 5.5 Hz, CH2N3), 2.42–2.06 (13 s, 39 H, 13
CH3CO); dC (CDCl3, 125 MHz): 170.9, 170.8, 170.7, 170.5, 170.4,
170.0, 169.9, 169.8, 169.7, 169.7, 169.5, 169.5, 169.4, 100.2, 99.5,
99.3, 97.5, 74.2, 70.7, 70.1, 70.0, 69.9, 69.7, 69.6, 69.5, 69.4, 68.4,
68.3, 68.0, 67.0, 66.5, 66.1, 65.7, 62.6, 62.4, 62.1, 62.0, 50.7, 20.9,
To
a solution of the O-acetylated compound (70 mg,
0.0544 mmol) in anhydrous MeOH (5 mL) was added a solution
of MeONa in MeOH (0.5 M, 0.2 mL). The mixture was stirred
at rt for 2 h, and then neutralized with Dowex 50W (H+). The
reaction mixture was filtered and the filtrate was concentrated to
give 15 (41 mg, 95%) as a white solid. dH (500 MHz, CD3OD):
5.40 (d, 1 H, J = 1.5 Hz, H-1ꢀꢀꢀ), 5.25 (d, 1 H, J = 1.5 Hz, H-1ꢀꢀ),
5.05 (d, 1 H, J = 2.0 Hz, H-1), 4.82 (d, 1 H, J = 1.5 Hz, H-1ꢀ),
4.72–4.60 (m, 2 H, H-6ꢀꢀꢀ), 4.09–4.01 (m, 4 H), 3.98–3.59 (m, 24 H),
3.42 (t, 2 H, J = 5.0 Hz, CH2N3); dC (CD3OD, 125 MHz): 102.8,
101.0, 100.8, 100.5, 82.1 (d, JC–F = 175 Hz, C-6ꢀꢀꢀ), 79.1, 78.8, 78.8,
73.7, 73.6, 73.5, 72.3, 72.1, 71.1, 70.7, 70.6, 70.4, 70.0, 69.9, 67.8,
67.7, 66.6, 66.3, 66.0, 61.8, 61.6, 61.4, 50.4; ESI-MS: m/z: calcd
for C28H48FN3O21: 779.7; found: 781.5 [M + H]+, 804.2 [M + Na]+.
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2-(2-Aminoethoxy)ethyl a-D-mannopyranosyl-(1
mannopyranosyl-(1 2)-a-D-mannopyranosyl-(1
→
→
2)-a-D-
3)-a-D-
selectively remove the Dde protecting groups. The reaction mixture
was neutralized with acetic acid to pH 7.0, diluted with 40 mL
water, and lyophilized. The crude de-protected cyclic peptide was
purified by RP-HPLC to give the compound 19 (15 mg, 84%).
ESI-MS calcd. for C60H108N16O14: M = 1277.60; found: 1278.03
(M + H)+, 639.40 (M + 2H)2+, 589.55 (M − Boc + 2H)2+, 539.39
(M − 2Boc + 2H)2+, 359.93 (M − 2Boc + 3H)3+.
→
mannopyranoside (16). A mixture of 14 (20 mg, 0.0257 mmol)
and 10% Pd/C (20 mg) in MeOH (100 mL) was stirred under H2
at 40 PSI for 2 h. The mixture was filtered through a pad of celite,
and the filtrate was concentrated under vacuum to give 16 (18 mg,
98%) as a white solid. dH (500 MHz, CD3OD): 5.29 (s, 1 H, H-1ꢀꢀꢀ),
5.24 (d, 1 H, J = 1.5 Hz, H-1ꢀꢀ), 4.98 (d, 1 H J = 1.5 Hz, H-1), 4.81
(d, 1 H J = 1.2 Hz, H-1ꢀ), 4.04–4.00 (m, 4 H), 3.94–3.56 (m, 28
H); ESI-MS: m/z: calcd for C28H51NO22: 753.7; found: 754.4 [M
+ H]+.
c[Lys(-propynoic amide)-Lys-Lys (-propynoic amide)-Pro-Gly-
Lys(-propynoic amide)-Lys-Lys (propynoic amide)-Pro-Gly] (20).
To a solution of cyclic peptide 19 (26 mg, 0.02 mmol) in
DMF (5 mL), were added propynoic acid (11.2 mg, 0.16 mmol)
and dicyclohexylcarbodiimide (DCC) (32 mg, 0.16 mmol). The
resulting mixture was shaken at rt for 30 min. After dilution
with water (50 mL), the suspension was filtered and the filtrate
was lyophilized. The crude acylated cyclic peptide was subject
to preparative RP-HPLC to give the corresponding acylated
cyclic decapeptide (23 mg, 77%). ESI-MS calcd for C72H108N16O18:
1484.80; found: 1485.67 [M + H]+, 743.52 [M + 2H]2+, 693.35 [M −
Boc + 2H]2+, 642.73 [M − 2Boc + 2H]2+, 428.62 [M − 2Boc + 3H]3+.
To selectively remove the Boc protective groups, the above
acylated cyclic peptide (23 mg, 0.015 mmol) was treated with 90%
TFA in DCM at rt for 10 min, then the solution was diluted with
water and lyophilized. The product was purified by preparative
RP-HPLC to give the alkynylized cyclic decapeptide 20 (18 mg,
93%). ESI-MS calcd. for C62H92N16O14: 1284.70; found: 1285.64
[M + H]+, 643.32 [M + 2H]2+.
2-(2-(8-Azido-3,6-dioxaoctanamido)ethoxy)ethyl-a-D-mannopy-
ranosyl-(1
ranosyl-(1
→
2)-a-D-mannopyranosyl-(1
3)-a-D-mannopyranoside (18). Compound 16
→
2)-a-D-mannopy-
→
(5.6 mg, 0.0076 mmol) was dissolved in aqueous NaHCO3
(50 mM, 2 mL) containing MeCN (1 mL) and MeOH (0.1 mL).
To the solution was added a solution of 17 (5 mg, 0.0175 mmol)
in MeCN (1 mL). The mixture was stirred at rt for 3 h and then
lyophilized. The residue was loaded onto a column of Sephadex
G-10 and eluted with 0.1 M HOAc. The fractions containing the
product were pooled and lyophilized to give 18 (5.2 mg, 74%)
as a white foam. dH (500 MHz, CD3OD): 5.29 (d, 1 H, J =
1.5 Hz, H-1ꢀꢀꢀ), 5.24 (d, 1 H, J = 1.5 Hz, H-1ꢀꢀ), 4.98 (d, 1 H, J =
1.5 Hz, H-1), 4.81 (d, 1 H, J = 1.5 Hz, H-1ꢀ), 4.05–3.97 (m, 6
H), 3.94–3.56 (m, 32 H), 3.46 (t, 2 H, J = 5.0 Hz,), 3.41 (t, 2 H,
J = 5.0 Hz, CH2N3); ESI-MS: m/z: calcd for C34H60N4O25: 924.8;
found: 925.6 [M + H]+.
Synthesis of glycopeptides 21–24. General procedures. To a
solution of the individual azido-tagged oligosaccharide (2, 14,
15, or 18) (5.0 mol. equiv), the alkynyl-substituted cyclic peptide
20 (1.0 mg, 0.78 lmol, 1 mol. equiv.) in an aqueous t-BuOH
(50%, 0.95 mL) were added CuSO4 (10 mM in 1 : 1 t-BuOH–H2O,
15 ll, 0.15 lmol, 0.2 mol. equiv.) and sodium ascorbate (20 mM
in 1 : 1 t-BuOH–H2O, 15 ll, 0.30 lmol, 0.4 mol. equiv.). The
reaction mixture was stirred at 60 ◦C. The reaction was monitored
by RP-HPLC. After 6–12 h, HPLC indicated the completion of
the 1,3-dipolar cycloaddition to give a single product. The product
was easily purified by preparative RP-HPLC to give the template-
assembled oligosaccharide cluster (21–24).
The orthogonally protected linear decapeptide. Peptide synthe-
sis was performed on an automatic peptide synthesizer using
distinctly protected fluorenylmethyloxycarbonyl (Fmoc)-amino
acids as building blocks, HATU as the coupling reagent, and
Fmoc-Lys(Boc)-NovaSyn TGT acidic sensitive resin as the solid
support. The linear peptide was synthesized on a 0.4 mmole scale
and cleaved from resin by treatment with 50% acetic acid in
DCM for 2 h at rt. The crude peptide was precipitated by cold
ethyl ether and purified by preparative RP-HPLC. The column
was eluted with 30% MeCN containing 0.1% TFA in 40 min to
give the linear decapeptide (810 mg, 95%). ESI-MS calcd. for
C112H182N16O23: M = 2120.74; found: 2121.83 (M + H)1+, 1061.36
(M + 2H)2+, 707.88 (M + 3H)3+, 874.49 (M − Boc + 3H)3+, 641.17
(M − 2Boc + 3H)3+.
The fully protected cyclic decapeptide. The linear peptide
obtained above (30 mg, 14 lmol) was dissolved in 40 mL
acetonitrile. To the solution was added 0.5 M 2-(1H-benzotriazole-
1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU)
(28 lmol) in DMF and 1.0 M diisopropylethylamine (DIPEA).
The mixture was gently shaken at rt for 2 h. The reaction
mixture was diluted with 30 mL water and lyophilized. The crude
cyclic peptide was subjected to purification by preparative RP-
HPLC. The peptide was eluted with 30% MeCN containing 0.1%
TFA in 40 min to give the fully protected cyclic decapeptide (26 mg,
88%). ESI-MS calcd. for C112H180N16O22: M = 2102.72; found:
2103.59 (M + H)1+, 1052.32 (M + 2H)2+, 668.47 (M − Boc +
3H)3+, 635.15 (M − 2Boc + 3H)3+.
Glycopeptide 21. dH (500 MHz, D2O): 8.48 (s, 4 H, H-triazole),
4.94 (s, 4 H, 4 H-1); ESI-MS: m/z: calcd for C102H168N28O42:
2457.62; found: 1230.3 [M + 2H]2+, 841.5 [M + 3Na]3+, 820.7 [M +
3H]3+, 766.6 [M + 3H-Man]3+, 712.6 [M + 3H-2Man]3+, 658.6 [M +
3H-3Man]3+.
Glycopeptide 22. dH (500 MHz, D2O): 8.48 (s, 4 H, H-triazole),
5.45 (s, 4 H, 4 H-1ꢀꢀꢀ), 5.36 (s, 4 H, 4 H-1ꢀꢀ), 5.05 (s, 4 H, 4 H-1), 4.83
(s, 4 H, 4 H-1ꢀ); ESI-MS: m/z: calcd for C174H288N28O102: 4403.5;
found: 1469.2 [M + 3H]3+, 1115.6 [M + 2H + 2Na]4+.
Glycopeptide 23. dH (500 MHz, D2O): 8.48 (s, 4 H, H-triazole),
5.41 (s, 4 H, 4 H-1ꢀꢀꢀ), 5.27 (s, 4 H, 4 H-1ꢀꢀ), 5.07 (s, 4 H, 4 H-1), 4.83
(s, 4 H, 4 H-1ꢀ); ESI-MS: m/z: calcd for C174H284N28O98F4: 4411.5;
found: 1471.9 [M + 3H]3+, 1118.0 [M + H + 3Na]4+, 1113.8 [M +
2H + 2Na]4+, 1109.8 [M + 3H + Na]4+.
The selectively deprotected cyclic decapeptide 19. The above-
obtained cyclic peptide (29 mg, 0.014 mmol) was dissolved in
10 mL 2% hydrazine in DMF, and gently shaken at rt for 20 min to
Glycopeptide 24. dH (500 MHz, D2O): 8.48 (s, 4 H, H-triazole),
5.41 (s, 4 H, 4 H-1ꢀꢀꢀ), 5.27 (s, 4 H, 4 H-1ꢀꢀ), 5.07 (s, 4 H, 4 H-1), 4.83
(s, 4 H, 4 H-1ꢀ); ESI-MS: m/z: calcd for C198H332N32O114: 4984.1;
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found: 1662.9 [M + 3H]3+, 1260.8 [M + H + 3Na]4+, 1257.0 [M +
2H + 2Na]4+, 1253.6 [M + 3H + Na]4+.
short pulse of 3.5 M MgCl2 with a flow rate of 100 lL min−1 for
30 seconds.
Glycopeptide 25. Compound 23 (2.0 mg, 0.45 lmol) was
dissolved in 2 mL 0.05 M NaHCO3, 1 mL CH3CN and 0.1 mL
methanol. To this solution, compound 17 (5 mg, 0.0175 mmol) in
1 mL CH3CN was added, and the reaction mixture was stirred at
room temperature for 1 h. The mixture was purified by RP-HPLC
and lyophilized to give compound 25 as a white foam (1.9 mg,
90%). ESI-MS: m/z: calcd for C186H302N34O104F4: 4753.5; found:
1586 [M + 3H]3+, 1423.5 [M + 3H-FMan3]3+, 1369.2 [M + 3H-
FMan4]3+, 1260.3 [M + 3H-2FMan3]3+, 1206.0 [M + 3H-FMan3-
FMan4]3+, 1098.0 [M + 3H-3FMan3]3+, 1039.6 [M + 3H-2FMan3-
FMan4]3+.
Acknowledgements
The human monoclonal antibody 2G12 was provided by the
National Institutes of Health (NIH)’s AIDS Research and Ref-
erence Reagent Program. This work was supported in part by the
National Institutes of Health (AI054354).
References
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Glycopeptide 27. To a solution of compound 26 (1.0 mg,
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in aqueous t-BuOH (50%, 0.25 mL) were added CuSO4 (10 mM
in 1 : 1 t-BuOH–H2O, 2 ll, 0.02 lmol, 0.2 mol. equiv.) and sodium
ascorbate (20 mM in 1 : 1 t-BuOH–H2O, 2 ll, 0.40 lmol, 0.4 mol.
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Binding studies
Surface plasmon resonance (SPR) technology was used for
analyzing the binding of the synthetic oligomannoses and their
clusters to monoclonal antibody (mAb) 2G12. The experiments
were carried out with a biacore 3000 system in a HBS-P buffer
(10 mM Hepes, 150 mM NaCl, 0.005% surfactant P20, pH 7.4)
at 25 ◦C. For coupling of mAb 2G12 to CM5 sensor chip, the
chip surface was activated by injection of EDC–NHS for 7 min
at 5 lL min−1, followed by injection of 20 lg mL−1 mAb 2G12
solution in an acetate buffer (10 mM pH 5.5) until the target level
of 10 000 response units (RU) was reached. The reaction was then
quenched by injection of ethanolamine HCl (1 M, pH 8.5) for
7 min at 5 lL min−1. A reference surface was prepared by a similar
procedure, but without the injection of antibody solution. Each
synthetic compound was injected to reference and mAb 2G12
channels, and a binding profile was obtained by subtraction of the
reference surface signal from the mAb 2G12 surface signal. For
a kinetic study, three analytes, compounds 22–24, at six different
concentrations were injected at 30 lL min−1 for 4 min, followed
by 5 min dissociation. The censor surface was regenerated with a
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