Synthesis of high-mannose oligosaccharide analogues through click chemistry: True functional mimics of their natural counterparts against lectins?

Article abstract of DOI:10.1002/chem.201405481

Terminal "high-mannose oligosaccharides" are involved in a broad range of biological and pathological processes, from sperm-egg fusion to influenza and human immunodeficiency virus infections. In spite of many efforts, their synthesis continues to be very

Full text of DOI:10.1002/chem.201405481

DOI: 10.1002/chem.201405481
Full Paper
&
Oligosaccharides
Synthesis of High-Mannose Oligosaccharide Analogues through
Click Chemistry: True Functional Mimics of Their Natural
Counterparts Against Lectins?
[
a]
[b]
[a]
[c]
Marc FranÅois-Heude, Alejandro Mꢀndez-Ardoy, Virginie Cendret, Pierre Lafite,
[c]
[b]
[d]
[a]
Richard Daniellou, Carmen Ortiz Mellet, Josꢀ M. Garcꢁa Fernꢂndez, Vincent Moreau,*
and Florence Djeda ¨ı ni-Pilard*
[a]
Abstract: Terminal “high-mannose oligosaccharides” are in-
volved in a broad range of biological and pathological pro-
cesses, from sperm-egg fusion to influenza and human im-
munodeficiency virus infections. In spite of many efforts,
their synthesis continues to be very challenging and actually
represents a major bottleneck in the field. Whereas multiva-
lent presentation of mannopyranosyl motifs onto a variety
of scaffolds has proven to be a successful way to interfere in
recognition processes involving high-mannose oligosacchar-
ides, such constructs fail at reproducing the subtle differen-
ces in affinity towards the variety of protein receptors (lec-
tins) and antibodies susceptible to binding to the natural li-
gands. Here we report a family of functional high-mannose
oligosaccharide mimics that reproduce not only the terminal
mannopyranosyl display, but also the core structure and the
branching pattern, by replacing some inner mannopyranosyl
units with triazole rings. Such molecular design can be im-
plemented by exploiting “click” ligation strategies, resulting
in a substantial reduction of synthetic cost. The binding af-
finities of the new “click” high-mannose oligosaccharide
mimics towards two mannose specific lectins, namely the
plant lectin concanavalin A (ConA) and the human macro-
phage mannose receptor (rhMMR), have been studied by
enzyme-linked lectin assays and found to follow identical
trends to those observed for the natural oligosaccharide
counterparts. Calorimetric determinations against ConA, and
X-ray structural data support the conclusion that these com-
pounds are not just another family of multivalent manno-
sides, but real “structural mimics” of the high-mannose
oligosaccharides.
Introduction
pathogenic microorganisms and are implicated in bacteria and
[1b]
virus adhesion to host-cells, being a target of the immune
system. All high-mannose glycans share the common penta-
“
High-mannose” type N-glycans play a pivotal role in the quali-
ty control of N-glycoprotein biosynthesis as well as in their in-
saccharide
core
Mana-(1!3)[Mana-(1!6)]Manb-(1!
[
1]
tracellular transportation. Moreover, they are involved in
many biological recognition phenomena at the surface of the
cell-membrane. For instance, they cover the surface of many
4)GlcNAcb-(1!4)GlcNAc (Man GlcNAc ), bearing up to six addi-
3
2
tional mannose residues (Figure 1).
Fragments of the mannooligosaccharide moiety, Man3 to
Man , behave as the recognition motifs (glycotopes) towards
9
[
a] Dr. M. FranÅois-Heude, Dr. V. Cendret, Dr. V. Moreau, Prof. F. Djeda ¨ı ni-Pilard
LG-2A FRE-CNRS 3517, Institut de Chimie de Picardie
Universitꢀ de Picardie Jules Verne, 33 Rue Saint-Leu
different receptor partners. Given their biological significance,
high-mannose oligosaccharides have inspired numerous works
focused on their partial or total synthesis and on their applica-
tions in carbohydrate-based vaccines or glyco-targeted drug
delivery systems, among others. The preparation of such mole-
cules remains very challenging, however, and the design of
more readily accessible artificial mimics that are able to effi-
ciently emulate Man8–9 in molecular recognition events is
highly sought-after in the field of glycobiology.
8
0039 Amiens Cedex 1 (France)
E-mail: vincent.moreau@u-picardie.fr
florence.pilard@u-picardie.fr
[
b] Dr. A. Mꢀndez-Ardoy, Prof. C. Ortiz Mellet
Departamento de Quꢁmica Orgꢂnica, Facultad de Quꢁmica
Universidad de Sevilla, Profesor Garcꢁa Gonzꢃlez 1
4
1012 Sevilla (Spain)
[c] Dr. P. Lafite, Prof. R. Daniellou
[2]
ICOA, UMR CNRS 7311, Universitꢀ d’Orlꢀans
rue de Chartres, BP6759, 45067 Orlꢀans cedex 2 (France)
Inspired by the work of Crich and Yang and of Dondoni
[3]
and co-workers, which suggested that replacing internal
monosaccharide units by 1,2,3-triazole rings preserves the
overall shape of their natural oligosaccharide analogues, we re-
cently reported the preparation of a “click” Man8 mimic
[
d] Prof. J. M. Garcꢁa Fernꢃndez
Instituto de Investigaciones Quımicas, CSIC-Universidad de Sevilla
Americo Vespucio 49, Isla de la Cartuja
4
1092 Sevilla (Spain)
(
pseudo-Man ) by exploiting the copper(I)-catalyzed azide-
8
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201405481.
[4]
alkyne cycloaddition (CuAAC). Encouraged by the strong af-
Chem. Eur. J. 2014, 20, 1 – 15
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assays (ELLA) and isothermal titration calorimetry (ITC) meas-
urements. Both lectins displayed comparable affinity for Mann
and pseudo-Mann homologues, which strongly supports the
validity of the approach. The crystal structure of a ConA:pseu-
do-Man complex at 2.0 ꢄ resolution provided further evidence
9
to support this conclusion. The pseudo-Man8–9 prototypes 4
and 5 keep the three peripheral units H, F, D or I, F, D and the
disaccharide AB or the trisaccharide ABG in relative positions
that are homotopic to those encountered in the natural Man8
and Man9 oligosaccharides (Figure 1). We reasoned that the
inner branched mannopyranosyl trisaccharide moiety in the
latter could act as a scaffold that preserves the overall orienta-
tion of the D1, D2, and D3 arms. Pseudo-Man 1 and pseudo-
3
Man 2 mimic the D1 arm, whereas pseudo-Man 3 is con-
4
5
ceived to imitate the pentasaccharide Man entangling the D2
5
and D3 arms. A simultaneous multiple “click-coupling” ap-
[5]
proach involving the known a-d-mannopyranosyl azide 6 or
the dimannosyl azide derivative 7 and the alkynyl partners 8–
1
1 was implemented to generate triazole connectors at the
position of the inner carbohydrate units G, E, C, and H
Figure 3).
(
Results and Discussion
Synthesis of “high-mannose” oligosaccharide mimics 1–5
The required O-alkynyl derivatives 8–11 were prepared from
known phenyl 2-O-benzyl-4,6-O-benzylidene-1-thio-a-d-man-
Figure 1. Structure of high-mannoses glycans Man5–9
.
[6]
[4]
nopyranoside (12), and mannopyranosyl donors 13 and 14
[
7]
(
Figure 3). Preparation of 8 and 9 first required reductive
[8]
finity displayed by this compound towards the mannose-spe-
cific lectin concanavalin A (ConA), we have now accomplished
ring-opening of the 4,6-O-benzylidene group of 12 to give
[9]
diol 15 (85% yield), which was subsequently acetylated at O-
6 and O-3 (!16) and engaged in glycosylation of N,N-
dibenzylaminoethanol, affording 17. It is worth mentioning
that insertion of the aminoethyl arm proved to be problematic
when performed at a later stage in the reaction sequence, for
example, when propargyl or triazole moieties were already
present in the precursor, and thus had to be carefully planned.
The use of N,N-dibenzylethanolamine as the aglyconic moiety
was motivated by the fact that carbamate protection of the
amino group led to N-alkylation during the O-propargylation
step. De-O-acetylation of 17 afforded the pivotal intermediate
the synthesis of a series of “click” Man mimics family including
n
pseudo-Man3–5 and pseudo-Man8–9 (1–5; Figure 2).
The final compounds are equipped with an aminoethyl
spacer arm at the reducing end to highlight their suitability as
building blocks for the construction of multivalent systems
after clustering onto appropriately functionalized scaffolds. A
study of their binding capabilities towards ConA and the bio-
medically relevant human macrophage mannose receptor
(rhMMR) in comparison with the natural mannooligosacchar-
ides has been conducted by undertaking enzyme-linked lectin
Figure 2. Structure of targeted “High-Mannose” type oligosaccharides mimics 1–5.
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then protected as the corresponding levulinic ester, giving 23,
which was used as glycosyl donor to incorporate the N,N-di-
benzylethanolamine aglycone (!24; 67% yield). The levulinyl
group was selectively removed by treatment with hydrazine to
[4]
afford alcohol 25, which was glycosylated with donor 13, fol-
[10]
lowing the trichloroacetimidate method. The reaction led ex-
clusively to a(1-6)-disaccharide 26 in high yield (73%), in
agreement with the a-directing character of acyl substituents
[11]
at O-3 and O-6 in mannopyranosylation reactions. Base-cata-
lyzed methanolysis of the disaccharide 26 (!27) followed by
treatment with propargyl bromide gave disaccharidic tripro-
pargyl ether scaffold 10 (Scheme 2).
Compound 12 was also used as the starting material in the
route towards the triply-clickable trisaccharide 11 (Scheme 3).
Glycosylation of 12 with known trichloroacetimidate donor
[7]
14
led to a(1-3)-disaccharide 28 in good yield (73%). Subse-
quent reductive opening of the benzylidene acetal afforded ac-
Figure 3. Synthetic strategy and key intermediates.
18 (49% overall yield from 12). Treatment of 18 with propargyl
bromide in the presence of sodium hydride gave the doubly-
clickable precursor 8, whereas regioselective tert-butyldime-
thylsilylation at O-6 (!19), propargylation of the remaining
hydroxyl OH-3 (!20), and final tetrabutylammonium fluoride-
promoted desilylation provided the mono-clickable derivative
9
(81% yield from 17; Scheme 1).
Synthesis of disaccharide 10 starts with benzoylation of 12
Scheme 2. Synthesis of propargylated disaccharide 10. Reagents and condi-
tions: a) BzCl, pyridine, 08C to RT, 2 h, 91%; b) BH
h, 94%; c) Levulinic acid, DMAP, DCC, CH Cl , RT, 6 h, 90%; d) N,N-dibenzy-
lethanolamine, NIS, TfOH, CH Cl , 08C, 1 h, 67%; e) NH NH , AcOH, pyridine,
RT, 20 min., 80%; f) donor 13, TMSOTf, CH Cl
, ꢀ408C, 2 h, inverse proc.
3%; g) MeONa, MeOH, RT, 12 h, 84%; h) propargyl bromide, NaH, DMF, 08C
to RT, 2 h, 91%.
3 2 2 2
·THF, Bu BOTf·CH Cl , RT,
(
!21) followed by the reductive ring-opening of the 4,6-O-
1
2
2
benzylidene group to afford 22. The primary hydroxyl was
2
2
2
2
2
2
7
Scheme 1. Synthesis of 8 and 9. Reagents and conditions: a) BH
Bu BOTf·CH Cl , 08C to RT, 1 h, 85%; b) Ac O, pyridine, 08C to RT, 3 h, 81%;
c) N,N-dibenzylethanolamine, NIS, TfOH, CH Cl , 08C, 1 h, 79%; d) NH
MeOH, RT, 2 d, 90%; e) propargyl bromide, NaH, DMF, 08C to RT, 2 h, 80%;
f) TBDMSCl, pyridine, RT, 12 h, 97%; g) propargyl bromide, NaH, DMF, 08C to
RT, 2 h, 91%; h) TBAF (1m in THF), 08C to RT, 12 h, 92%.
3
·THF,
Scheme 3. Synthesis of propargylated trisaccharide 11. Reagents and condi-
tions: a) TMSOTf, CH Cl ·THF, Bu BOTf·CH Cl , 08C
, ꢀ408C, 1 h, 73%; b) BH
to RT, 1 h, 94%; c) donor 13, TMSOTf, CH Cl
, ꢀ408C, 1 h, inverse proc.,
70%; d) N,N-dibenzylethanolamine, NIS, TfOH, CH Cl , 08C, 1 h, 76%;
e) MeONa, MeOH, RT, 6 h, 85%; f) propargylbromide, NaH, DMF, 08C to RT,
2 h, 60%.
2
2
2
2
2
2
3
2
2
2
2
2
3
/
2
2
2
2
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ceptor 29, which was glycosylated with donor 13, using the in-
mimics by transfer hydrogenolysis using ammonium formate
and palladium on charcoal. In our hands, this procedure afford-
ed rather modest yields of the desired fully unprotected
pseudo-Man derivatives under classical heating. However, we
were delighted to see that the use of microwave heating in
the presence of resin-supported ammonium formate led clean-
ly to the target fully unprotected pseudo “high-mannose” type
oligosaccharides 1–5, which were isolated in 77, 92, 80, 81,
and 74% yield, respectively, after HPLC purification.
[
12]
verse procedure to ensure a good yield (!30, 70%). Installa-
tion of the spacer (!31), followed by base-catalyzed metha-
nolysis (!32) and treatment with propargyl bromide provided
11.
Disaccharide azide 7, which is an intermediate required for
the synthesis of pseudo-Man 2, was obtained in 66% yield by
4
glycosylation reaction between the known 2,3,4,6-tetra-O-
[
13]
acetyl-a-d-mannopyranosyl trichloroacetimidate (33)
and
[
14]
azide 36 {prepared in two steps from the 1,2-orthoester de-
All Man mimics 1–5 were fully characterized by NMR spec-
n
[
15a]
rivative 34,
using FeCl as catalyst in the azido glycosylation
troscopy. In the case of pseudo-Man 5, a mass spectrometry
3
9
[
15b]
reaction }, thereby completing the whole set of building
blocks to undertake the preparation of the target click high-
mannose oligosaccharide mimics (Scheme 4).
fragmentation study was also performed to confirm the linking
pattern. The MS/MS spectrum (negative mode) obtained is pre-
ꢀ
sented in Figure 4. The [MꢀH] ion of 5 (m/z 1275.53) gener-
ates ions coming from the successive loss of the peripheral
mannosidic units D (m/z 1113.45), F (m/z 951.38), and I (m/z
789.34). The loss of triazole rings is monitored by the presence
of the fragments of m/z 870.36, 708.30, 627.26, and 546.24.
The latter fragment is characteristic of the trisaccharidic scaf-
fold ABG, from which the disaccharidic (m/z 384.18) and the
monosaccharidic (m/z 222.10) fragments are finally generated.
Scheme 4. Synthesis of azide 7. Reagents and conditions: a) TMSN
CH Cl , RT, 15 h, 63%; b) MeONa, MeOH, RT, 2 h, 80%; c) 33, TMSOTf, CH
408C, 1 h, 66%.
3 3
, FeCl ,
2
2
2 2
Cl ,
Evaluation of the lectin binding affinities of “high-mannose”
oligosaccharide mimics 1–5
ꢀ
Evaluation of the binding affinity of the oligomannoside
mimics towards ConA and rhMMR lectins was first carried out
by conducting enzyme-linked lectin assays (ELLA). In this tech-
nique, inhibition of the binding between yeast mannan immo-
bilized in a microtiter plate and the corresponding lectin in so-
lution by increasing concentrations of the ligands is deter-
The CuAAC reactions between 8–11 and azide 6 were car-
ried out under microwave heating, using sodium ascorbate
I
and copper(II) sulfate to generate the Cu catalyst in situ in
[16]
a 4:1 mixture of N,N-dimethylformamide (DMF) and water.
The protected pseudo-Man 37 (76%), pseudo-Man 38 (75%),
3
5
[19]
pseudo-Man 39 (72%), and pseudo-Man 40 (75%) were thus
mined. The resulting IC50 values are then taken to be propor-
tional to the corresponding binding constants. Data for the
new click mimics were compared with those obtained for their
natural partners. Interestingly, affinities of the same order of
magnitude were obtained for pseudo-Man /Man pairs in the
8
9
obtained. The CuAAC reaction of 7 and 9 (!41), followed by
base-catalyzed methanolysis, afforded pentabenzylated
pseudo-Man 42 (71%, 2 steps; Scheme 5).
4
[
4]
[17]
We and others have already observed that the final de-
O-benzylation step of derivatives containing triazole rings
failed under catalytic hydrogenation conditions. Gin and co-
n
n
case of the higher homologues against both lectins, which
strongly supports their functional equivalency (Table 1).
ELLA results indicate that the lower mimics 1 and 2 are not
[
18]
workers previously described the simultaneous de-O-benzy-
lation of 18 benzyl ether groups in cyclooligosaccharide
recognized well by the lectins, with pseudo-Man 2 exhibiting
4
Scheme 5. Partially benzylated pseudo-Man3–9 37–40 and 42 and final deprotection: a) resin-supported ammonium formate, Pd/C, MW, 74–92%.
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Figure 4. ESI-MS/MS spectrum of pseudo-Man
9
5, each gray pentagon and gray circle refer to the loss of, respectively, one triazole and one mannosidic unit.
Table 1. IC50 [mm] values for the inhibition of ConA-HRP (ConA) and
rhMMR binding to yeast mannan (378C, pH 7.3)
Ligand
IC50 vs.
ConA
Rel.
potency
IC50 vs.
rhMMR
Rel.
potency
[
b]
MeaMan (I)
triMan (II)
875
81
1
11
38
33
2850
296
62
52.5
36
1
10
[
a]
[
[
[
b]
b]
b]
Man
Man
Man
Man
Man
5
6
7
8
9
23
26
17
5.6
3.9
45.9
54.3
79.2
395
715
1.1
2.12
30.6
157
518
3
51
[
[
b]
156
224
0.87
3.8
20.8
82.5
123
6.7
7.2
4
b]
Ps.Man
Ps.Man
Ps.Man
Ps.Man
Ps.Man
III
3
4
5
8
9
(1)
(2)
(3)
(4)
(5)
1000
230
42
10.6
7.1
2600
1340
93
18.1
5.5
Figure 5. ELLA plots (logarithm scale) for the inhibition of rhMMR binding to
yeast mannan by increasing concentrations of the Pseudo-Man 4 and
Pseudo-Man 5 in comparison with the natural high-mannose glycans Man
and Man
8
9
8
144
950
9
.
[
(
a] triMan (II) : 3,6-di-O-(a-d-mannopyranosyl)-a-d-mannopyranosyl azide
see the Supporting Information). [b] Work previously reported.
[
4]
Man derivative 4, which lacks this moiety, removes a significant
8
[4]
contribution to the binding energy.
In principle, the inner disaccharide AB in pseudo-Man 4 and
8
only 4- and 2-fold higher affinities towards ConA and rhMMR,
the trisaccharide moiety ABG in pseudo-Man9 5 (Figure 2)
might act as simple scaffolds, bearing three peripheral manno-
sidic ligands, with the observed lectin affinity enhancements
being simply the result of a particularly favorable multivalent
respectively, compared with methyl a-d-mannopyranoside
(MeaMan) I, which was used as reference. Binding capabilities
of mimics 3–5 were much more noticeable and were very simi-
lar to those shown by their natural counterparts. For both lec-
[20]
effect of the triad-type presentations. To investigate this pos-
sibility, trivalent glycodendron III (Figure 6) was prepared (see
the Supporting Information) and evaluated. The much weaker
lectin affinity displayed by III as compared with 4 and 5 (by 52
and 172-fold, respectively), in spite of bearing an identical
number of external mannosyl-triazole units, strongly suggests
tins, pseudo-Man 3 is a strong ligand, being only less than 2-
5
fold weaker than the natural Man . Pseudo-Man 4 is a stronger
5
8
ligand than Man , but was found to be around 2-fold weaker
7
than Man ; pseudo-Man 5 was established to be the strongest
8
9
ligand of the series (Figure 5). Although the presence of the
aminoethyl aglycone in the “click” mimics might be involved in
lectin binding to some extent, for example, by establishing salt
bridges with amino acid residues in the protein, the ConA af-
finity data obtained previously for an analogue of the pseudo-
that the latter behave as real Man -type complementary struc-
n
tural motifs, providing an optimal overall shape and presenta-
tion or even participating in the binding process, as occurs in
the natural Man and Man counterparts.
8
9
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ꢀ
DH8, ꢀTDS, and K values for ConA-binding pseudo-Man
a
3–5
1
–3 are also indicative of weaker affinities compared with
those of II, which is consistent with the ELLA measurements.
In contrast, ITC experiments provided evidence that pseudo-
Man8–9 4 and 5 are high affinity ligands for ConA, with Ka
values similar to those of II but with higher ꢀDH8 values (17.2
ꢀ
1
and 16.2 kcalmol , respectively). Surprisingly the ꢀTDS values
are much more negative, with 4 and 5 exhibiting 18- and 2.5-
fold higher ꢀTDS values relative to I and II, respectively. This
entropy penalty is consistent with cross-linking processes. Fi-
nally, ITC data also indicated that the number of binding sites
(N) per monomer of ConA binding to I, II, and pseudo-Man3–5
1–3 is close to 1.0, in agreement with reported data, which
confirms the monovalency of these ligands for ConA. In con-
trast, 4 and 5 exhibit N values of 0.23 and 0.25, respectively, in-
dicating that 4 and 5 are probably involved in multivalent
binding with ConA.
Figure 6. Glycodendrimer III used as trivalent mannosyl-triazole control in
ELLA.
The thermodynamic signature (ꢀDH8, ꢀTDS, and K values)
a
for the binding of MeaMan I, 3,6-di-O-(a-d-mannopyranosyl)-
a-d-mannopyranosyl azide II, pseudo-Man3–5, and pseudo-
Man8–9 1–5 to ConA was further studied by isothermal titration
calorimetry (ITC), which is a technique used widely to investi-
[
21]
gate carbohydrate–lectin interactions. After integrating the
heat signal as a function of the ratio between the carbohy-
drate and the lectin, the isotherm was fitted to the one-site
binding model (see the Supporting Information). The average
values for the thermodynamic parameters are given in Table 2.
Trisaccharide II, for which ConA possesses an extended binding
Crystal structure of ConA:pseudo-Man 5 complex was de-
9
termined at 2.0 ꢄ resolution (See Table 5 in the Supporting In-
formation). Only one terminal mannosyl residue (D, F, or I) and
the triazolyl aglycone could be modeled in the binding site of
each subunit of dimeric ConA (Figure 7A). However, structural
alignment of the complex with a previously reported ConA:-
ꢀ
1
site, exhibited a ꢀDH8 value of 11.4 kcalmol and a K value
a
4
ꢀ1
[22,23]
[24]
of 47.6ꢅ10 m , which are consistent with reported data,
Mana(1-6)[Mana(1-3)]Man trimannoside complex gave simi-
considering the slightly different experimental conditions, and
lar interactions between the mannosyl moiety and the binding
site residues (Figure 7B).
are much more favorable compared with those for I. The
[25]
As reported for other ConA:mannoside structures, Arg228,
Asn14, Asp208, and Leu99 interact with the terminal mannosyl
residue through a network of hydrogen bonds. Tyr12 and
Tyr100 complete the binding site and link the B or G unit at O-
6 or O-3 (B) or O-2 (G) through hydrogen bonds involving
a water molecule. Moreover, Tyr12, Leu99, and Tyr100 form
a hydrophobic pocket in which the triazole ring can be stabi-
lized and act as a good mimic of mannose.
Table 2. Thermodynamic parameters for ConA (258C, pH 7.2)
[
a]
Ligand
N
ꢀDH8
ꢀTDS
K
a
ꢀ
1
ꢀ1
ꢀ1
ꢀ4
[
kcalmol
]
[kcalmol
]
[m ꢅ10 ]
MeaMan (I)
triMan (II)
1
1
1
1
0.8
0.23
0.28
6.1
11.4
6.1
7.7
6.4
1.7
12.3
5.3
3.7
1.3
0.97
47.6
0.19
8.62
3.35
Ps.Man
Ps.Man
Ps.Man
Ps.Man
Ps.Man
3
4
5
8
9
(1)
(2)
(3)
(4)
(5)
Another striking aspect of the ConA:5 structure is the forma-
tion of an unexpected oligomer, which led to crystallization of
17.2
16.2
32.6
29.9
37.03
30.30
the complex in the trigonal H3 space group, instead of the re-
2
ported monoclinic P21 groups for ConA:Mana(1-6)[Mana(1-
[a] Number of sites per monomer.
3)]Man trimannoside that crystallized under the same condi-
Figure 7. A) X-ray structure of the ConA:5 complex (binding site region). 5 (colored green) is shown with the corresponding 2Fo-Fc omit map density con-
toured at 1.5s. Residues interacting with the ligand are indicated. B) Overlay of the ConA:5 complex (green) with the ConA:Mana(1-6)[Mana(1-3)]Man triman-
noside complex (cyan) (PDB code 3D4K).
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6
ꢃ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
Figure 8. Modeling of ConA cross-linking by pseudoMan
9
5. A) Supramolecular assembly visible in crystal structure of ConA:5 complex. In each of the four in-
teracting dimers, Chain A (resp. B) is colored in purple (resp. orange). Mannoside binding site in each chain are in red (Chain A) and cyan (Chain B). B) Detailed
view of the modeled full length 5 bridging two ‘B’ binding sites. Mannoside moieties are labeled according to Figure 2.
[
24]
tions.
dimers (Figure 8A). This geometry allows two sites of Chain A
and two sites of Chain B) from different dimers to be in close
ConA:pseudo-Man9 exists as an assembly of four
the conclusion that the “click” high-mannose oligosaccharide
analogues reported here behave as true functional mimics of
the parent oligosaccharides.
(
contact. Using the terminal mannosyl residue as visible anchor
point in the X-ray structure, a model of 5 was docked into
each pair of binding sites (Figure 8B).
Experimental Section
This molecular modeling confirmed the possible bridging of
General Methods
two binding sites of ConA by the pseudo-Man molecule. Simi-
9
[
25d]
lar results were obtained by Dimick et al.
with a short di-
Optical rotations were measured with a JASCO DIP-370 digital po-
larimeter, using a sodium lamp (l=589 nm) at 208C. NMR experi-
ments were performed at 300.13 and 600.13 MHz with a Bruker
AVANCE DPX300 or AVANCE III IPSO600 spectrometers equipped
mannoside analogue that could crosslink two ConA binding
sites. Thus, 5 interacts with ConA lectin through interactions
that are similar to those reported for oligomannoside com-
pounds and change the oligomeric state of the protein, possi-
bly by crosslinking ConA dimers.
with a Z-gradient unit for pulsed-field gradient spectroscopy. Me Si
4
was used as an external standard and calibration was performed
by using the signal of the residual protons or of the carbon of the
solvents as a secondary reference. Measurements were performed
at 300 K with careful temperature regulation. The length of the 908
pulse was approximately 7 ms. 1D NMR data spectra were collected
as 16 K data points. 2D experiments were run using 1 K data
points and 512 time increments. The phase-sensitive (TTPI) se-
quence was used and processing resulted in a 1 Kꢅ1 K (real-real)
matrix. Details concerning experimental conditions are given in the
Figure captions. Low-resolution electrospray mass spectra were ob-
tained with a hybrid quadrupole/time-of-flight (Q-TOF) instrument,
equipped with a pneumatically assisted electrospray (Z-spray) ion
source (Micromass). High-resolution mass spectra were recorded in
positive mode with a ZabSpec TOF (Micromass, UK) tandem hybrid
mass spectrometer with EBETOF geometry. The compounds were
Conclusion
A new family of high-mannose oligosaccharide mimics that re-
produce the peripheral presentation of mannopyranosyl glyco-
topes as well as the core structure and branching pattern has
been developed. Replacement of some of the inner monosac-
charide residues by triazole rings preserves the overall architec-
ture of the natural compounds and allows click chemistry liga-
tion strategies to be implemented to assemble the mimetics,
which represents a very significant simplification compared
with classical glycosylation methods. The new pseudo-Man de-
rivatives displayed binding affinities towards the mannose-spe-
cific lectins ConA and the human macrophage mannose recep-
tor that reproduced the trends encountered for the natural
counterparts in enzyme-linked lectin assays, with binding po-
tencies that also rivaled those of their respective natural part-
ners. Isothermal titration calorimetry also produced data that
was consistent with the ELLA measurements and indicate that
Pseudo-Man (4) and Pseudo-Man (5) are involved in crosslink-
individually dissolved in a water–CH CN mixture (1:1) at a concen-
tration of 10 mgcm and then infused into the electrospray ion
3
ꢀ
3
3
ꢀ1
source at a flow rate of 10 mm mn at 608C. The mass spectrom-
eter was operated at 4 kV while scanning the magnet at a typical
range of 4000–100 Da. The mass spectra were collected as continu-
um profile data. Accurate mass measurement was achieved by
using polyethylene glycol as internal reference masses with a re-
solving power set to a minimum of 10000 (10% valley). HPLC anal-
yses were performed with a Water PrepLC400 System with a PL-
ELS 1000 detector (Waters and Polymer laboratories) detector
8
9
ing processes with ConA. Most interestingly, the X-ray structure
of the complex of ConA with the highest member of the
(
(
Method 1) or Waters 2545 and SQ mass detector (Waters)
Method 2). Analytical chromatography was performed with an
series, pseudo-Man , provided evidence for contacts beyond
9
Apollo C18 5 mm column (length: 250 mm; Ø: 4.6 mm) at
the terminal mannopyranosyl residue and crosslinking abilities
analogous to those observed in the corresponding complex
ꢀ1
1
mLmin rate flow. Preparative chromatography was performed
with a VisionHT C18 HL 5 mm column (length: 250 mm; Ø: 22 mm)
ꢀ
1
with the natural Man . The ensemble of data strongly support
at 23 mLmin rate flow. Analytical TLC were performed with Silica
9
Chem. Eur. J. 2014, 20, 1 – 15
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7
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Gel 60 F254 plates (Merck, Germany). Eluents used were mixtures of
dichloromethane and methanol or cyclohexane and ethyl acetate;
ratios are specified in each case. Products were visualized under
UV light (l=254 nm) followed by charring with a mixture of vanil-
lin in a 5% solution of H SO in ethanol. For “manual” flash chro-
matography, a glass column filled with silica gel (GERUDAN SI 60,
grading 0.040–0.062 mm MERCK) was used. For “automatic” flash
chromatography a Reveleris iES Flash System device supplied by
Grace was used as silica Grace Flash Cartridges (normal phase) or
C-18 Grace Flash Cartridges (inverse phase). The resin-supported
ammonium formate used in de-O-benzylation reactions of partially
benzylated pseudo-Man3–9 37–40 and 42 was prepared from
Dowex marathon MSA resin (Sigma Aldrich) by treatment with
formic acid (99%) for 30 min. The resin was washed with water
The plates were washed three times with PBST as described above
and 100 mL of a solution of biotinylated anti-human MMR antibody
ꢀ
1
(0.2 mgmL , R&D Systems) in PBS was added in each well, and
the plates were further incubated for 1 h at 378C. The complex
NeutrAvidin-biotinylated HRP was preformed separately by succes-
sively adding to Tris buffer (9.6 mL, 50 mm, pH 7.6) a solution of
2
4
ꢀ
1
NeutrAvidin (100 mgmL in Tris buffer, 1.2 mL, Thermo Scientific)
ꢀ1
and a solution of biotin-conjugated HRP (25 mgmL in Tris buffer,
1.2 mL, Thermo Scientific). The mixture was shaken for 30 min at
RT and the solution was immediately transferred into the plates
(60 mL/well). After 1 h at 378C, these plates were washed twice
ꢀ1
with Tris (250 mL/well) and ABTS (0.25 mgmL , 50 mL/well) in cit-
rate buffer (0.2 m, pH 4.0 with 0.015% H O ) was added. After
2
2
5 min at RT, the optical density was measured at 415 nm. Control
wells were processed with anti-human MMR antibody as well as
NeutrAvidin-biotinylated HRP. The concentration of rhMMR that
displayed an absorbance between 0.8 and 1.0 was used for inhibi-
tion experiments. For the competitive lectin-binding inhibition ex-
periment, MeaMan (I) or the click high-mannose mimics 1–5
mixed in a serial of 2-fold dilutions (60 mL per well) in HEPES
buffer (20 mm, pH 7.4) with 60 mL of the appropriate rhMMR con-
centration in PBS buffer on Nunclon (Delta) microtitre plates and
incubated for 1 h at 378C. The above solutions (100 mL) were then
transferred to the mannan-coated titer plates, which were incubat-
ed for 1 h at 378C. The plates were washed and the solution of
biotinylated anti-human MMR antibody in PBS (100 mL) was added
in each well, and the plates were further incubated for 1 h at 378C.
The NeutrAvidin solution was then transferred into the plates
(
twice) and methanol (twice), and finally dried at 308C under
vacuum for 4 h.
Enzyme-linked lectin assay (ELLA)
Horseradish peroxidase-labeled concanavalin A (HRP-Con A;
Sigma), mannan from Saccharomyces cerevisiae (Sigma) recombi-
nant human macrophage mannose receptor (rhMMR; R&D Sys-
tems), biotinylated anti-human MMR antibody (R&D Systems), Neu-
trAvidin (Thermo Scientific), biotin-conjugated HRP (Thermo Scien-
tific), high-mannose oligosaccharides (Man ; Ludger) and all other
n
common reagents and materials were purchased from commercial
sources and used without further purification unless otherwise
stated. All aqueous solutions for lectin-binding assays were pre-
pared from deionized water filtered with a Milli-Q purification
system.
(60 mL/well). After 1 h at 378C, these plates were washed twice
with Tris (250 mL/well) and ABTS was added (50 mL/well). Optical
density at 415 nm was determined after 5 min. The percent of in-
hibition was calculated as follows: %Inhibition=100ꢅ
Nunc-Inmuno plates (MaxiSorp) were coated overnight with yeast
ꢀ
1
mannan at 100 mL/well diluted from a stock solution of 10 mgmL
in 10 mm phosphate buffer saline (PBS, pH 7.3 containing 0.1 mm
(
Ano inibithorꢀA inhibitor)/Ano inibithor
.
with
2
+
2+
Results in triplicate were used to plot the inhibition curves for
each individual ELLA experiment. Typically, the IC50 values (concen-
tration required for 50% inhibition of the ConA-yeast mannan or
the rhMMR-yeast mannan association) obtained from several inde-
pendently performed tests were in the range of ꢁ12%. Neverthe-
less, the relative inhibition values calculated from independent
series of data were highly reproducible.
Ca and 0.1 mm Mn ) at RT. The wells were then washed three
times with 300 mL washing buffer (PBST, containing 0.05% (v/v)
Tween 20). The washing procedure was repeated after each of the
incubations throughout the assay. The wells were then blocked
with 150 mL/well of 1% BSA/PBS for 1 h at 378C.
For concanavalin A affinity evaluations, after washing, the wells
were filled with 100 mL of serial dilutions of horseradish peroxidase
ꢀ
1
labeled concanavalin A (ConA-HRP, Sigma) from 10
to
mgmL in PBS, and incubated at 378C for 1 h. The plates
were washed and 50 mL/well of 2,2’-azinobis-(3-ethylbenzothiazo-
ꢀ4
ꢀ1
Isothermal calorimetric measurements
1
0
Concanavalin A (ConA) was purchased from Sigma–Aldrich
ꢀ1
line-6-sulfonic acid) diammonium salt (ABTS) (0.25 mgmL ) in cit-
(C2010–250MG, Sigma). Protein concentration was determined
rate buffer (0.2m, pH 4.0 with 0.015% H O ) was added. The reac-
2
2
spectrophotometrically at 280 nm by using the Beer–Lambert law
tion was stopped after 20 min by adding 50 mL/well of 1m H SO4
ꢀ1 ꢀ1
2
with a molar extinction coefficient of 32430 cm
m
and is ex-
and the absorbances were measured at 415 nm. Control wells con-
tained citrate-phosphate buffer. The concentration of lectin-
enzyme conjugate that displayed an absorbance between 0.8 and
[26]
pressed in terms of the monomer. The protein purity (94%) was
checked at three concentrations (0.095, 0.047, 0.025 mm) and plot-
ted versus absorbance to monitor linearity. ITC was performed
with a Microcal ITC200 system (GE Healthcare). All experiments
were performed at 258C in 0.1m Hepes buffer containing NaCl
1
.0 was used for inhibition experiments.
To carry out the competitive lectin-binding inhibition experiments,
each compound was added in a serial of 2-fold dilutions (60 mL per
well) in PBS to 60 mL of the appropriate ConA-HRP concentration
in PBS buffer on Nunclon (Delta) microlitre plates and incubated
for 1 h at 378C. The above solutions (100 mL) were then transferred
to the mannan-coated microplates, which were incubated for 1 h
at 378C. The plates were washed and the ABTS substrate was
added (50 mL per well). Color development was stopped after
(
0.9m), CaCl (0.1 mm), and MnCl (0.1 mm) at pH 7.2. Briefly, 1 mL
2
2
of saccharide solution was injected successively by using a comput-
er-controlled 40 mL syringe at an interval of 180 s into the ConA so-
lution (Cell Volume 204.6 mL). For the Man8 and Man9 mimics, the
first four injections were of 0.5 mL. Additional parameters were as
follows: reference power 8 mV, initial delay 60 s, rotation speed
1
000 rpm. Control experiments were performed by the dilution of
2
0 min and the absorbances were measured.
each saccharide solution in buffer and showed insignificant heats
of dilution. These experiments were then subtracted from each ti-
tration to remove heats of dilution of ligand. The experimental
data were then fitted to a theoretical titration curve by using
Origin 7.0-Microcal Software with the single-site model. During this
For determination of recombinant human MMR (rhMMR) binding
affinities, the wells were filled with 100 mL of serial dilutions of
ꢀ1
rhMMR from a 10 mgmL stock solution in PBS (pH 7.3 containing
2
+
2+
0
.1 mm Ca and 0.1 mm Mn ), and incubated at 378C for 1 h.
&
&
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8
ꢃ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
fitting, enthalpy (DH), stoichiometry (N) and association constants
Ka) were adjustable parameters. All experiments were performed
in EtOAc. The organic layer was washed with saturated aq. KHSO4,
NaHCO , brine, dried over Na SO , filtered, and concentrated in
(
3
2
4
with C values 1<C<90 in the present study. The linear trigalactose
Galb(1–4)Galb(1–4)Gala(1-OMe), used as negative control, did not
shown any binding to the lectin (see Table 4 and Figure S5 in Sup-
porting Information). In the case of MeaMan and PseudoMan3
vacuo. The crude product was purified by “automatic” flash chro-
matography (50 min, cyclohexane/EtOAc 1:0 to 8:2 (v/v)) to give
20
1
21 (9.24 g, 91%). [a]D +48.4 (c=1 in CHCl ); H NMR (300 MHz,
3
CDCl ): d=8.13–8.03 (m, 2H; ArH), 7.65–7.08 (m, 18H, ArH), 5.65 (s,
3
(
C<5), the data were fitted with N fixed and K and DH as floating
1H; H-7), 5.60 (d, J(1,2)=1.4 Hz, 1H; H-1), 5.56 (dd, J(2,3)=3.4 Hz,
a
parameters.
J(3,4)=9.0 Hz, 1H; H-3), 4.67 (d, J=11.9 Hz, 1H; OCH Ph), 4.56 (d,
2
J=11.9 Hz, 1H; OCH Ph), 4.53–4.39 (m, 2H; H-4 and H-5), 4.38 (dd,
2
J(1,2)=1.4 Hz, J(1,2)=3.4 Hz, 1H; H-2), 4.28 (dd, J(5,6a)=4.4 Hz,
J(6a,6b)=10.3 Hz, 1H; H-6a), 3.93 ppm (t, J(5,6b)=10.3 Hz, 1H; H-
Crystallographic experiments
13
ConA was purified by affinity chromatography on a Superdex 200
GL column (GE Healthcare), using glucose for elution. Protein was
6b); C NMR (75 MHz, CDCl ): d=165.9 (PhC(O)O), 137.4–126.3
3
(Ar), 101.9 (C-7), 86.7 (C-1), 77.9 (C-2), 76.5 (C-4), 73.3 (OCH Ph),
2
ꢀ
1
concentrated to 20 mgmL in 20 mm Tris buffer pH 7, 50 mm
NaCl, 1 mm MnCl , 1 mm CaCl , in the presence of 5 mm pseudo-
71.3 (C-3), 68.7 (C-6), 65.5 ppm (C-5); HRMS (ESI): m/z calcd for
+
2
2
C H O SNa: 577.1661 [M+Na] ; found: 577.1681.
33 30
6
Man . Crystals suitable for diffraction experiments were obtained
by sitting-drop vapor diffusion in 7 mL drops containing 2 mL Con-
A:pseudo-Man9 solution and 5 mL of the precipitation solution
9
Phenyl
3-O-benzoyl-2,4-di-O-benzyl-1-thio-a-d-mannopyrano-
side (22): Compound 21 (8.4 g, 15.1 mmol) was treated with BH3
(1m in THF, 48.5 mL, 48.5 mmol, 3.2 equiv) and Bu BOTf solution
2
(
10% polyethylene glycol 6000, and 0.1m Sodium Cacodylate
(1m in CH Cl , 16.1 mL, 16.1 mmol, 1.06 equiv) at 08C under an
[24]
2
2
pH 6.8). After equilibration of the drops against the crystalliza-
tion solution, crystals were cryoprotected in 20% polyethylene
glycol 400, before freezing in liquid nitrogen. Data were collected
at the beamline Proxima-1 at the Soleil Synchrotron Facility, re-
inert atmosphere. The reaction mixture was stirred for 2 h at 08C
then the reaction was quenched by the dropwise addition of
MeOH (until gas release ceased) and Et N (until pH reached 10).
The solvent was evaporated under reduced pressure and the crude
product was purified by “automatic” flash chromatography
3
[27]
duced with the X-Ray Detector Software XDS, and scaled with
[
28]
[29]
SCALA by using the CCP4 program suite. ConA:trimannoside
(40 min, cyclohexane/EtOAc 1:0 to 8:2 (v/v)) to afford 22 (7.90 g,
[24]
crystal structure (pdb 3D4K) was then used as a template for
molecular replacement in the PHASER program. The model was
completed by iterative cycles of manual model building and real
space refinement using the program Coot and crystallographic
refinement using Phenix refine. Structure validation with MOL-
PROBITY was integrated as part of the iterative rebuild and re-
finement procedure. Model and structure factors have been depos-
ited at the Protein Data Bank (PDB code 4PF5). Pymol software
was used to generate all images. NAMD software was used to
perform all molecular dynamics simulations of pseudo-Man9 in
complex with ConA. The topology and parameters files for the
20
1
9
8
1
4
4
4
2
4%). [a]D +43.3 (c=1 in CHCl ); H NMR (300 MHz, CDCl ): d=
[30]
3
3
.11–8.07 (m, 2H; ArH), 7.52–7.19 (m, 18H; ArH), 5.60 (d, J(1,2)=
.8 Hz, 1H; H-1), 5.54 (dd, J(2,3)=3.0 Hz, J(3,4)=9.3 Hz, 1H; H-3),
.83 (d, J=10.8 Hz, 1H; OCH Ph), 4.72 (d, J=10.8 Hz, 1H; OCH Ph),
.68 (d, J=12.6 Hz, 1H; OCH Ph), 4.55 (d, J=12.6 Hz, 1H; OCH Ph),
.40–4.24 (m, 3H; H-2, H-4 and H-5), 3.89 (s, 2H; H-6a and H-6b),
[
31]
[32]
2
2
[
33]
2
2
13
.04 ppm (br s, 1H; OH); C NMR (75 MHz, CDCl ): d=165.7
3
(PhC(O)O), 137.9–124.7 (Ar), 85.9 (C-1), 77.4 (C-2), 75.1 (OCH Ph),
2
[34]
[35]
7
4.6 (C-3), 73.3, 73.2 (C-4 and C-5), 72.7 (OCH Ph), 62.0 ppm (C-6);
2
+
HRMS (ESI): m/z calcd for C H O SNa: 579.1817 [M+Na] ; found:
33
32
6
5
79.1804.
[36]
ligand were obtained by using the Antechamber program and
AM1-BCC charges
[37]
[38]
Phenyl 3-O-benzoyl-2,4-di-O-benzyl-6-O-levulinoyl-1-thio-a-d-
in the Ambertools 13 program.
Pseudo-
mannopyranoside (23): DCC (1.01 g, 4.92 mmol, 1.6 equiv) was
added to a solution of 22 (1.7 g, 3.07 mmol), DMAP (64 mg,
Man was originally placed and aligned so that the terminal man-
nose would fit the ligand density in the ligand structure. Two initial
9
0
1
.52 mmol, 0.17 equiv), and levulinic acid (0.57 g, 4.92 mmol,
.6 equiv) in anhydrous dichloromethane (40 mL) under an inert at-
models of pseudo-Man were thus generating, either bridging the
9
ConA between the terminal mannose D and E (model named
pMan9-DE) or D and F (pMan9-DF). Each model was equilibrated
by several cycles, alternating minimization (10000 steps, steepest
descent), and molecular dynamics (200 ps, 200 K). Long simulations
were then run (1 ns), keeping the backbone of ConA restrained to
its position in the X-ray structure.
mosphere at RT. The mixture was stirred for 24 h at RT then filtered
over Celite, washed with dichloromethane, and concentrated. The
residue was dissolved in ethyl acetate and the organic layer was
successively washed with a saturated solution of aq. KHSO4,
NaHCO , and brine. The organic phase was dried over Na SO , fil-
tered, and concentrated to dryness. The crude product was puri-
3
2
4
fied by “manual” flash chromatography (cyclohexane/EtOAc 1:0 to
Synthesis of pseudo-Man8-9 4 and 5
20
7
:3 (v/v)) to afford 23 (1.81 g, 90%). [a]D +14.1 (c=1 in CHCl3);
1
H NMR (300 MHz, CDCl ): d=8.16–8.02 (m, 2H; ArH), 7.69–7.08 (m,
Syntheses of mimics 1–3 and azide 7 are fully described in the
Supporting Information. A, B, C letters refer to each pyranosidic
rings and are used to indicate their associated protons and car-
bons in the NMR data. Arabic numerals 1–6 refer to each proton
and carbon of the pyranosidic rings according to the nomenclature
of carbohydrates. Arabic numerals higher than 6 refer to protons
and carbons of the side chains. For spectra see the Supporting
Information.
3
1
8H; Ar), 5.64 (d, J(1,2)=1.5 Hz, 1H; H-1), 5.52 (dd, J(2,3)=3 Hz,
J(3,4)=9.3 Hz, 1H; H-3), 4.81 (d, J=10.8 Hz, 1H; OCH Ph), 4.75–
2
4
.60 (m, 2H; OCH Ph), 4.54 (d, J=12.3 Hz, 1H; OCH Ph), 4.51–4.31
2 2
(m, 3H; H-5, H-6a and H-6b), 4.29 (dd, 1H; H-2), 4.22 (t, J(4,5)=
9
.3 Hz, 1H; H-4), 2.80–2.67 (m, 2H; H-8 or H-9), 2.66–2.57 (m, 2H;
13
H-8 or H-9), 2.16 ppm (s, 3H; H-11); C NMR (75 MHz, CDCl ): d=
3
2
8
06.5 (C-10), 172.5, 165.6 (OC(O)Ph, OC(O)CH ), 137.6–127.7 (Ar),
2
5.5 (C-1), 77.2 (C-2), 75.1 (OCH Ph), 74.6 (C-3), 73.6 (C-4), 72.5
2
Phenyl
mannopyranoside (21): Benzoyl chloride (4.26 mL, 36.62 mmol,
equiv) was added to a solution of 12 (8.5 g, 18.35 mmol) in anhy-
drous pyridine (200 mL) at 08C. The mixture was stirred at RT for
h, then the reaction was quenched with MeOH (10 mL). Pyridine
was removed under reduce pressure and the residue was diluted
3-O-benzoyl-2-O-benzyl-4,6-O-benzylidene-1-thio-a-d-
(OCH Ph), 70.9 (C-5), 63.5 (C-6), 37.9 (C-8 or C-9), 29.9 (C-11),
2
2
6
8.0 ppm (C-8 or C-9); HRMS (ESI): m/z calcd for C H O SNa:
38 38 8
77.2185 [M+Na] ; found: 677.2174.
2
+
4
(N,N-Dibenzyl-2-aminoethyl)
3-O-benzoyl-2,4-di-O-benzyl-6-O-
levulinoyl-a-d-mannopyranoside (24): N-Iodosuccinimide (0.755 g,
Chem. Eur. J. 2014, 20, 1 – 15
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9
ꢃ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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3
1
.36 mmol, 1.3 equiv) followed by TfOH (0.32 mL, 3.61 mmol,
.4 equiv) were added to a solution of 23 (1.69 g, 2.58 mmol), N,N-
4.82 (d, J=11.1 Hz, 1H; OCH Ph), 4.75 (d, J(1,2)=1.7 Hz, 1H; H-1A),
2
4.69 (d, J=10.8 Hz, 1H; OCH Ph), 4.67 (d, J=13.1 Hz, 1H; OCH Ph),
2
2
dibenzyl amino ethanol (0.685 g, 2.84 mmol, 1.1 equiv), and molec-
ular sieves (1 g) in anhydrous CH Cl (50 mL) under an inert atmos-
4.58–4.41 (m, 4H; OCH Ph), 4.35 (dd, J(5,6a)=2.3 Hz, J(6a,6b)=
2
11.8 Hz, 1H; H-6Ba), 4.32 (d, J=11.9 Hz, 1H; OCH Ph), 4.26 (dd,
2
2
2
phere at 08C. The mixture was stirred at 08C for 2 h, quenched
with triethylamine, and concentrated to dryness. The residue was
diluted in EtOAc, washed with saturated aq. NaHCO and Na S O ,
J(5,6b)=1.7 Hz, J(6a,6b)=11.8 Hz, 1H; H-6Bb), 4.25 (t, J(3,4)=
J(4,5)=9.7 Hz, 1H; H-4A), 4.11 (t, J(3,4)=J(4,5)=9.3 Hz, 1H; H-4B),
4.04 (dd, J(1,2)=1.7 Hz, J(2,3)=3.3 Hz, 1H; H-2B), 3.99 (m, 1H; H-
5B), 3.94 (dd, J(1,2)=1.7 Hz, J(2,3)=3.3 Hz, 1H; H-2A), 3.91–3.71
(m, 4H; H-5A, H-6Aa, H-6Ab and H-8a), 3.67 (d, J=13.9 Hz, 2H;
NCH Ph), 3.60 (d, J=13.9 Hz, 2H; NCH Ph), 3.48 (m, 1H; H-8b), 2.70
3
2
2
3
dried over Na SO , filtered, and concentrated in vacuo. The crude
2
4
product was purified by “automatic” flash chromatography
(
45 min, cyclohexane/EtOAc 1:0 to 7:3 (v/v) with 1% Et N) to afford
3
4 as the a anomer (1.37 g, 67%). [a]D +14.1 (c=1 in CHCl3);
2
2
20
13
2
(t, J=4.6 Hz, 2H; H-7), 2.04 ppm (s, 3H; OC(O)CH3); C NMR
(75 MHz, CDCl ): d=170.8 (OC(O)CH ), 165.6, 165.1 (OC(O)Ph),
1
H NMR (600 MHz, CDCl ) d=8.07 (m, 2H; ArH), 7.62–7.14 (m, 23H;
3
3
3
ArH), 5.55 (dd, J(2,3)=3.6 Hz, J(3,4)=9.6 Hz, 1H; H-3), 5.85 (d,
139.7, 138.3, 138.1, 137.9, 137.7, 133.2, 133.1 (Ar), 130.4–126.7 (Ar),
J(1,2)=1.2 Hz, 1H; H-1), 4.74 (dd, J=10.8 Hz, 1H; OCH Ph), 4.63–
98.2 (C-1B), 98.0 (C-1A), 76.7 (C-2B), 76.5 (C-2A), 75.1 (OCH Ph), 74.9
2
2
4
1
4
.52 (m, 3H; OCH Ph), 4.35 (dd, J(5,6a)=4.8 Hz, J(6a,6b)=12 Hz,
H; H-6a), 4.28 (dd, J(5,6b)=1.8 Hz, J(6a,6b)=12 Hz, 1H; H-6b),
.11 (t, J(3,4)=J(4,5)=9.6 Hz, 1H; H-4), 3.97 (dd, J(1,2)=1.2 Hz,
(C-3A), 74.5 (C-3B), 73.5, 73.4 (C-4A, C-4B, and OCH Ph), 72.8
2
2
(OCH Ph), 72.0 (C-5A), 70.1 (C-5B), 66.0, 65.8 (C-6A and C-8), 63.5
2
(C-6B), 59.0 (2ꢅNCH Ph), 52.8 (C-7), 21.0 ppm (OC(O)CH ); HRMS
2
3
+
J(2,3)=3.6 Hz, 1H; H-2), 3.91 (m, 1H; H-5), 3.78 (m, 1H; H-13a),
(ESI): m/z calcd for C H O N: 1176.5110 [M+H] ; found:
72 74 14
3
3
2
1
2
6
2
.67 (d, J=13.8 Hz, 2H; NCH Ph), 3.64 (d, J=13.8 Hz, 2H; NCH Ph),
1176.5062.
2
2
.51 (m, 1H; H-13b), 2.76–2.53 (m, 6H; H-8, H-9 and H-12),
(N,N-Dibenzyl-2-aminoethyl) 6-O-(2,4-di-O-benzyl-a-d-mannopyr-
13
.13 ppm (s, 3H; H-11); C NMR (150 MHz, CDCl ): d=206.5 (C-10),
3
anosyl)-2,4-di-O-benzyl-a-d-mannopyranoside (27): A solution of
6 (1.96 g, 1.67 mmol) in methanol (15 mL) was added to a freshly
72.6, 165.6 (OC(O)Ph and C-7), 139.4–127.1 (Ar), 97.1 (C-1), 76.2 (C-
), 75.0 (OCH Ph), 74.6 (C-3), 73.3 (C-4), 73.2 (OCH Ph), 69.9 (C-5),
2
2
2
prepared solution of sodium methoxide (0.6m in MeOH, 15 mL).
The mixture was stirred 20 h at RT, then neutralized with acetic
acid and concentrated in vacuo. The crude product was purified by
6.0 (C-13), 63.6 (C-6), 59.0 (NCH Ph), 52.7 (C-12), 38.0 (C-8 or C-9),
2
9.9 (C-11), 28.0 ppm (C-8 or C-9); HRMS (ESI): m/z calcd for
+
C H O N: 786.3642 [M+H] ; found: 786.3633.
4
8
52
9
“
automatic” flash chromatography (40 min, cyclohexane/EtOAc 1:0
2
0
(N,N-Dibenzyl-2-aminoethyl)
3-O-benzoyl-2,4-di-O-benzyl-a-d-
to 8:2 (v/v) with 1% Et N) to afford 27 (1.32 g, 86%). [a] +39.7
3
D
(c=1.2 in CHCl ); H NMR (300 MHz, CDCl ): d=7.5–7.15 (m, 30H;
3 3
ArH), 5.03 (d, J(1,2)=1.5 Hz, 1H; H-1B), 4.96 (d, J=11.2 Hz, 1H;
1
mannopyranoside (25): A solution of 24 (1.32 g, 1.68 mmol) in
pyridine (5 mL) was treated for 10 min at RT with a solution of pyri-
dine/AcOH/hydrazine hydrate (12:8:1, 20 mL). The reaction
medium was concentrated, then the residue was dissolved in
CH Cl and the organic layer was successively washed with a satu-
OCH Ph), 4.81 (d, J(1,2)=1.3 Hz, 1H; H-1A), 4.72–4.57 (m, 4H;
2
OCH Ph), 4.54 (d, J=11.8 Hz, 1H; OCH Ph), 4.41 (d, J=11.7 Hz, 1H;
2
2
OCH Ph), 3.87 (dd, J(5,6a)=4.1 Hz, J(6a,6b)=11.2 Hz, 1H; H-6Aa),
2
2
2
rated solution of aq. NaOAc, NaHCO , and brine. The organic phase
4.01–3.53 (m, 16H; NCH Ph, H-2A, H-2B, H-6Ab, H-6Ba, H-6Bb, H-
3
2
was dried over Na SO , filtered, and concentrated to dryness. The
5A, H-5B, H-8a, H-3A, H-3B, H-4A and H-4B), 3.46 (m, 1H; H-8b),
2.66 (app. t, J=5.8 Hz, 2H; H-7), 2.29 ppm (br. s, 2H, OH); C NMR
2
4
1
3
crude product was purified by “manual” flash chromatography (cy-
2
D
0
clohexane/EtOAc 1:0 to 7:3 (v/v)) to afford 25 (0.924 g, 80%). [a]
(75 MHz, CDCl ): d=139.7–127.1 (Ar), 97.4 (C-1B), 96.9 (C-1A), 78.8,
3
1
+
6.5 (c=0.63 in CHCl3); H NMR (300 MHz, CDCl ): d=8.13–7.99
78.6 (C-2A and C-2B), 76.6–76.5 (C-3A or C-3B or C-4A or C-4B),
3
(
m, 2H; ArH), 7.64–7.07 (m, 23H; ArH), 5.55 (dd, J(2,3)=3.3 Hz,
75.1, 74.7 (OCH Ph), 73.1, 73.0 (OCH Ph), 72.1, 71.9 (C-3A or C-3B or
2
2
J(3,4)=9.9 Hz, 1H; H-3), 4.81 (d, J(1,2)=1.8 Hz, 1H; H-1), 4.76 (d,
J=10.8 Hz, 1H; OCH Ph), 4.65 (d, J=11.1 Hz, 1H; OCH Ph), 4.60–
C-4A or C-4B), 71.5, 70.9 (C-5A and C-5B), 66.3 (C-6A), 65.9 (C-8),
62.4 (C-6B), 59.1 (2ꢅNCH Ph), 52.9 ppm (C-7); HRMS (ESI): m/z
2
2
2
+
4
.56 (m, 2H; OCH Ph), 4.19 (t, J(3,4)=J(4,5)=9.9 Hz, 1H; H-4), 3.97
calcd for C H O N: 926.4479 [M+H] ; found: 926.4471.
2
56 64 11
(
dd, J(1,2)=1.8 Hz, J(2,3)=3.3 Hz, 1H; H-2), 3.84–3.69 (m, 4H; H-5,
(
N,N-Dibenzyl-2-aminoethyl)
6-O-(3,6-di-O-propargyl-2,4-di-O-
H-6a, H-6b and H-8a), 3.69–3.61 (m, 4H; NCH Ph), 3.48 (m, 1H; H-
8
OH); C NMR (75 MHz, CDCl ): d=165.7 (OC(O)Ph), 139.7–127.0
2
benzyl-a-d-mannopyranosyl)-3-O-propargyl-2,4-di-O-benzyl-a-d-
mannopyranoside (10): NaH (60% in mineral oil, 400 mg,
9
1
b), 2.71 (t, J=6 Hz, 2H; H-7), 1.94 ppm (app. t, J=12.6 Hz, 1H;
1
3
3
.98 mmol, 7 equiv) and propargyl bromide (80% in toluene,
.4 mL, 12.83 mmol, 9 equiv) were added to a solution of 27
(
Ar), 98.3 (C-1), 76.4 (C-2), 75.1 (OCH Ph), 74.7 (C-3), 73.4 (C-4 and
2
OCH Ph), 72.1 (C-5), 66.1 (C-8), 62.2 (C-6), 59.2 (2ꢅNCH Ph),
5
2
2
(
1.32 g, 1.43 mmol) in anhydrous DMF (20 mL) cooled to 08C
under an inert atmosphere. The reaction mixture was stirred for
h at 08C and then slowly warmed to RT for 2 h. The reaction mix-
2.8 ppm (C-7); HRMS (ESI): m/z calcd for C H O N: 688.3275
43 46 7
+
[M+Na] ; found: 688.3253.
1
(N,N-Dibenzyl-2-aminoethyl) 6-O-(6-O-acetyl-3-O-benzoyl-2,4-di-
ture was neutralized with MeOH and diluted with H O (50 mL) and
2
O-benzyl-a-d-mannopyranosyl)-3-O-benzoyl-2,4-di-O-benzyl-a-d-
mannopyranoside (26): A solution of 13 (0.94 g, 1.44 mmol,
aqueous phase was extracted with EtOAc (3ꢅ50 mL). The com-
bined organic layers were washed with water and brine, dried over
Na SO , filtered, and concentrated in vacuo. The crude product
[4]
1
.1 equiv) in CH Cl (25 mL) was added dropwise to a solution of
2 2
2
4
2
5 (0.9 g, 1.31 mmol) and TMSOTf (0.33 mL, 1.83 mmol, 1.4 equiv)
was purified by “automatic” flash chromatography (65 min, cyclo-
in anhydrous CH Cl (25 mL) under an inert atmosphere at ꢀ408C.
hexane/EtOAc 1:0 to 7:3 (v/v) with 1% Et N) to afford 10 (1.35 g,
91%); [a]D +23.6 (c=0.8 in CHCl ); H NMR (300 MHz, CDCl ): d=
3 3
2
2
3
20
1
The mixture was stirred at ꢀ408C for 2 h, then the reaction was
quenched with triethylamine and concentrated to dryness. The
crude product was purified by “automatic” flash chromatography
7.45–7.13 (m, 30H; ArH), 5.05 (d, J(1,2)=1.8 Hz, 1H; H-1B), 4.93 (d,
J=11 Hz, 1H; OCH Ph), 4.88 (d, J=11 Hz, 1H; OCH Ph), 4.73 (d, J=
2
2
(
2
70 min, cyclohexane/EtOAc 1:0 to 7:3 (v/v) with 1% Et N) to afford
12.3 Hz, 1H; OCH Ph), 4.73 (d, J(1,2)=1.7 Hz, 1H; H-1A), 4.69–4.55
3
2
2
0
1
6 (1.13 g, 73%). [a]D +12.1 (c=1 in CHCl ); H NMR (300 MHz,
(m, 5H; OCH Ph), 4.30–4.06 (m, 6H; OCH CCH), 3.99 (dd, J(2,3)=
3
2
2
CDCl ): d=8.13–7.99 (m, 4H; ArH), 7.65–6.95 (m, 36H; ArH), 5.62
2.9 Hz, J(3,4)=9.2 Hz, 1H; H-3B), 3.96–3.84 (m, 5H; H-2B, H-3A, H-
5A, H-6Ba, and H-5B), 3.83–3.75 (m, 3H; H-2A, H-6Aa and H-4A or
H-4B), 3.75–3.65 (m, 4H; H-6Ab, H-6Bb, H-8a and H-4A or H-4B),
3
(
dd, J(2,3)=3.3 Hz, J(3,4)=9.3 Hz, 1H; H-3B), 5.55 (dd, J(2,3)=
.3 Hz, J(3,4)=9.7 Hz, 1H; H-3A), 5.17 (d, J(1,2)=1.7 Hz, 1H; H-1B),
3
&
&
Chem. Eur. J. 2014, 20, 1 – 15
www.chemeurj.org
10
ꢃ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
3
3
.64 (d, J=13.7 Hz, 2H; NCH Ph), 3.58 (d, J=13.7 Hz, 2H; NCH Ph),
.49–3.35 (m, 1H; H-8b), 2.63 (app. t, J=5.7 Hz, 2H; H-7), 2.36 (t,
syl)-2,4-di-O-benzyl-1-thio-a-d-mannopyranoside (30): Com-
pound 13 (3.13 g, 4.82 mmol, 1.6 equiv) in anhydrous CH Cl
2 2
2
2
[4]
J=2.4 Hz, 1H; OCH CCH), 2.32 (t, J=2.3 Hz, 1H; OCH CCH),
(75 mL) was added dropwise to a solution of 29 (2.79 g,
3.01 mmol) and TMSOTf (0.24 mL, 2.41 mmol, 0.5 equiv) in anhy-
2
2
13
2
.31 ppm (t, J=2.3 Hz, 1H; OCH CCH); C NMR (75 MHz, CDCl3):
2
d=139.7–127.2 (ArH), 98.5 (C-1B), 98.0 (C-1A), 80.2 (C-3A), 80.1
drous CH Cl2 (75 mL) under an inert atmosphere at ꢀ408C. The
2
(
OCH CCH), 80.1 (C-3B), 75.8 (C-2B), 75.5 (C-2A), 75.1 (OCH Ph),
mixture was stirred at ꢀ408C for 1 h, quenched with triethylamine,
and concentrated to dryness. The crude product was purified by
“automatic” flash chromatography (70 min, cyclohexane/EtOAc 1:0
2
2
7
4.8–74.5 (OCH CCH, C-5A and C-5B), 73.1, 72.6 (OCH Ph), 71.8,
2 2
7
1.7 (C-4A and C-4B), 68.8 (C-6A), 66.3 (C-6B), 65.9 (C-8), 59.1 (2ꢅ
20
NCH Ph), 58.6, 57.8, 57.6 (OCH CCH), 52.7 ppm (C-7); HRMS (ESI):
to 0:1 (v/v)) to afford 30 (2.95 g, 70%): [a] +54.3 (c=1 in CHCl3);
2
2
D
+
1
m/z calcd for C H O N: 1040.4949 [M+H] ; found: 1040.5001.
H NMR (600 MHz, CDCl ): d=8.07 (d, J=7.4 Hz, 2H; ArH), 7.63–
6
5
70 11
3
6
3
.98 (m, 43H; ArH), 5.59 (dd, J(2,3)=3.3 Hz, J(3,4)=9.5 Hz, 1H; H-
C), 5.51 (m, 1H; H-2A), 5.46 (s, 1H; H-1A), 5.25 (s, 1H; H-1B), 5.10
Phenyl 3-O-(2-O-acetyl-3,4,6-tri-O-benzyl-a-d-mannopyranosyl)-
-O-benzyl-4,6-O-benzylidene-1-thio-a-d-mannopyranoside (28):
TMSOTf (0.45 mL, 4.52 mmol, 0.5 equiv) was added to a solution of
2
(d, J(1,2)=1.4 Hz, 1H; H-1C), 4.90 (d, J=10.4 Hz, 1H; OCH Ph), 4.87
2
[
7]
(d, J=10.8 Hz, 1H; OCH Ph), 4.75–4.42 (m, 11H; OCH Ph), 4.33 (d,
2 2
J=12.1 Hz, 1H; OCH Ph), 4.31 (d, J(5,6)=3.3 Hz, 2H; H-6C), 4.24
1
4
(5.76 g, 9.04 mmol) and 12 (4.08 g, 9.04 mmol, 1 equiv) in an-
2
hydrous CH Cl (125 mL) under inert atmosphere at ꢀ408C. The
2
2
(dd, J(2,3)=4.1 Hz, J(3,4)=9.2 Hz, 1H; H-3B), 4.13–4.05 (m, 4H; H-
mixture was stirred at ꢀ408C for 1 h, quenched with triethylamine,
2
1
B, H-4B, H-4C and H-5A), 4.03 (dd, J(1,2)=1.4 Hz, J(2,3)=3.3 Hz,
H; H-2C), 4.01 (dd, J(2,3)=3.3 Hz, J(3,4)=9.2 Hz, 1H; H-3A), 3.96–
and concentrated to dryness. The crude product was purified by
“
automatic” flash chromatography (55 min, cyclohexane/EtOAc 1:0
20
3.90 (m, 2H; H-5C and H-6Aa), 3.89–3.85 (m, 1H; H-5B), 3.84–3.75
(m, 2H; H-4A and H-6Ab), 6.69–6.62 (m, 2H; H-6Ba and H-6Bb),
2
to 7:3 (v/v)) to afford 28 (6.65 g, 79%). [a] +83.9 (c=1 in CHCl3);
D
1
H NMR (300 MHz, CDCl ): d=7.52–7.12 (m, 30H; ArH), 5.65 (s, 1H;
3
1
3
.10 (s, 3H; OC(O)CH ), 2.04 ppm (s, 3H; OC(O)CH ); C NMR
3 3
H-7), 5.63 (dd, J(1,2)=1.8 Hz, J(2,3)=3.3 Hz, 1H; H-2B), 5.51 (d,
J(1,2)=1.4 Hz, 1H; H-1A), 5.35 (d, J(1,2)=1.8 Hz, 1H; H-1B), 4.89 (d,
J=10.9 Hz, 1H; OCH Ph), 4.76–4.62 (m, 4H; OCH Ph), 4.57–4.44 (m,
(150 MHz, CDCl ): d=171.0, 170.2 (OC(O)CH ), 165.5 (OC(O)Ph),
3
3
1
38.8–127.5 (Ar), 100.0 (C-1B), 98.2 (C-1C), 85.6 (C-1A), 79.2 (C-2B or
2
2
C-4C or C-4B or C-5A), 78.1 (C-3A), 77.4 (C-2B or C-4C or C-4B or C-
5
7
3
H; OCH Ph), 4.35–4.28 (m, 3H; H-3A, H-5A and H-5B), 4.24 (dd,
2
A), 76.6 (C-2C), 75.5 (OCH Ph), 75.2 (C-2B or C-4C or C-4B or C-5A),
2
J(5,6a)=3.6 Hz, J(6a,6b)=10.3 Hz, 1H; H-6Ba), 4.07 (br. s, 1H; H-
A), 3.98 (dd, J(2,3)=3.3 Hz, J(3,4)=8.9 Hz, 1H; H-3B), 3.93–3.78
m, 3H; H-4A, H-4B and H-6Bb), 3.77–3.69 (m, 2H; H-6Aa and H-
5.1, 75.0 (OCH Ph), 74.5, 74.4 (C-4A and C-3C), 73.6 (OCH Ph), 73.5
2
2
2
(C-2B or C-4C or C-4B or C-5A), 73.0 (OCH Ph and C-3B), 72.5 (C-
2
(
1
3
5B), 72.1, 71.8 (OCH Ph), 70.0 (C-5C), 69.3 (C-6B), 69.0 (C-2A), 66.2
2
6
1
1
7
Ab), 2.11 ppm (s, 3H; OC(O)CH3); C NMR (75 MHz, CDCl ): d=
3
(C-6A), 63.4 (C-6C), 21.1, 21.0 ppm (OC(O)CH ); HRMS (ESI): m/z
3
70.1 (OC(O)CH ), 138.7–128.1 (Ar), 101.4 (C-7), 99.1 (C-1B), 87.1 (C-
A), 79.3 (C-2A), 79.2 (C-3A), 78.1 (C-3B), 75.2 (OCH Ph), 74.5 (C-4B),
3.6 (OCH Ph), 73.3 (C-5A or C-5B), 73.2 (OCH Ph), 72.4 (C-4A), 71.8
3
+
calcd for C H O SNa: 1437.5432 [M+Na] ; found: 1437.5372.
84
86 18
2
2
2
(N,N-Dibenzyl-2-aminoethyl) 3-O-(2-O-acetyl-3,4,6-tri-O-benzyl-a-
(
OCH Ph), 69.2 (C-6A), 68.5 (C-6B), 68.3 (C-2B), 65.5 (C-5A or C-5B),
2
d-mannopyranosyl)-6-O-(6-O-acetyl-3-O-benzoyl-2,4-di-O-benzyl-
a-d-mannopyranosyl)-2,4-di-O-benzyl-a-d-mannopyranoside
2
9
1.2 ppm (OC(O)CH ); HRMS (ESI): m/z calcd for C H O SNa:
3 55 56 11
47.3441 [M+Na] ; found: 947.3467.
+
(
31): N-Iodosuccinimide (0.207 g, 0.92 mmol, 1.3 equiv) followed by
TfOH (0.10 mL, 1.13 mmol, 1.6 equiv) were added to a solution of
0 (1.0 g, 0.71 mmol), dibenzylaminoethanol (0.188 g, 0.78 mmol,
.1 equiv) and molecular sieves (1 g) in anhydrous CH Cl (50 mL)
Phenyl 3-O-(2-O-acetyl-3,4,6-tri-O-benzyl-a-d-mannopyranosyl)-
,4-di-O-benzyl-1-thio-a-d-mannopyranoside (29): Compound 28
6.6 g, 7.1 mmol) was treated with BH3 (1m in THF, 22.8 mL,
2
(
3
1
2
2
2
2.8 mmol, 3.2 equiv) and Bu BOTf (1m in CH Cl , 7.6 mL,
2 2 2
under an inert atmosphere at 08C. The mixture was stirred at 08C
for 2 h, then the reaction was quenched with triethylamine and
concentrated to dryness. The residue was diluted in EtOAc, washed
with saturated aq. NaHCO and Na S O , dried over Na SO , filtered,
and concentrated in vacuo. The crude product was purified by “au-
tomatic” flash chromatography (55 min, cyclohexane/EtOAc 1:0 to
7
.6 mmol, 1.06 equiv) at 08C under an inert atmosphere. The reac-
tion mixture was stirred for 3 h at 08C then the reaction was
quenched by the addition of MeOH dropwise (until gas release
3
2
2
3
2
4
ceased) and Et N (until pH reached 10). The solvent was evaporat-
3
ed under reduced pressure and the crude product was purified by
“
automatic” flash chromatography (45 min, cyclohexane/EtOAc 1:0
20
7
:3 (v/v) with 1% Et N) to afford 31 (0.827 g, 76%). [a] +26.4 (c=
3
D
20
to 1:1 (v/v)) to afford 29 (6.29 g, 94%). [a] +81.2 (c=1 in CHCl3);
1
D
3.2 in CHCl ); H NMR (600 MHz, CDCl ): d=8.08 (d, J=7.4 Hz, 2H;
3 3
ArH), 7.67–7.01 (m, 48H; ArH), 5.62 (dd, J(2,3)=3.2 Hz, J(3,4)=
9
2
H-1C), 4.91 (d, J=11.6 Hz, 1H; OCH Ph), 4.89 (d, J=11.4 Hz, 1H;
OCH Ph), 4.74 (d, J(1,2)=1.2 Hz, 1H; H-1A), 4.73–4.62 (m, 4H;
OCH Ph), 4.60–4.44 (m, 6H; OCH Ph), 4.41–4.32 (m, 3H; OCH Ph
1
H NMR (300 MHz, CDCl ): d=7.42–7.16 (m, 30H, ArH), 5.50 (dd,
3
J(1,2)=1,8 Hz, J(2,3)=3.3 Hz, 1H; H-2B), 5.49 (d, J(1,2)=1.5 Hz, 1H;
H-1A), 5.25 (d, J(1,2)=1,8 Hz, 1H; H-1B), 4.89 (d, J=11.1 Hz, 1H;
OCH Ph), 4.82 (d, J=10.8 Hz, 1H; OCH Ph), 4.72–4.45 (m, 9H;
.5 Hz, 1H; H-3C), 5.55 (dd, J(1,2)=1.7 Hz, J(2,3)=3.2 Hz, 1H; H-
B), 5.24 (d, J(1,2)=1.7 Hz, 1H; H-1B), 5.15 (d, J(1,2)=1.7 Hz, 1H;
2
2
2
OCH Ph), 4.19–4.04 (m, 4H; H-2A, H-3A, H-4A and H-5A), 4.00 (dd,
2
2
J(2,3)=3.3 Hz, J(3,4)=9 Hz, 1H; H-3B), 3.95–3.87 (m, 1H; H-5B),
2
2
2
3
6
1
.85 (t, J(3,4)=J(4,5)=9 Hz, 1H; H-4B), 3.79 (m, 2H; H-6Aa and H-
and H-6Ca), 4.31 (dd, J(5,6b)=4.2 Hz, J(6a,6b)=11.6 Hz, 1H; H-
Cb), 4.18 (dd, J(2,3)=2.8 Hz, J(3,4)=9.5 Hz, 1H; H-3A), 4.13 (t,
J(3,4)=J(4,5)=9.5 Hz, 1H; H-4C), 4.09–3.98 (m, 4H; H-3B, H-2C, H-
A and H-5C), 3.92–3.84 (m, 3H; H-4B, H-5A and H-6Aa), 3.80–3.66
m, 4H; H-2A, H-5B, H-6Ab and H-8a), 3.66–3.52 (m, 6H; NCH Ph,
Ab), 3.74–3.65 (m, 2H; H-6Ba and H-6Bb), 2.12 (s, 3H; OC(O)CH ),
3
6
13
.85 ppm (br. s, 1H; OH); C NMR (75 MHz, CDCl ): d=170.2
3
(
OC(O)CH ), 138.7–127.7 (Ar), 99.9 (C-1B), 85.5 (C-1A), 79.2, 78.9 (C-
3
4
(
2
A and/or C-3A and/or C-4A and/or C-5A), 78.0 (C-3B), 75.4, 75.1
2
(
(
(
(
[
OCH Ph), 75.0 (C-2A or C-3A or C-4A or C-5A), 74.6 (C-4B), 73.7
2
H-6Ba and H-6Bb), 3.48–3.42 (m, 1H; H-8b), 2.71–2.60 (m, 2H; H-7),
2
OCH Ph), 73.4 (C-2A or C-3A or C-4A or C-5A), 72.5 (C-5B), 72.0
13
2
.11 (s, 3H; OC(O)CH ), 2.06 ppm (s, 3H; OC(O)CH ); C NMR
3 3
OCH Ph), 69.3 (C-6B), 69.0 (C-2B), 62.1 (C-6A), 21.1 ppm
2
(75 MHz, CDCl ): d=171.0, 170.1 (OC(O)CH ), 165.5 (OC(O)Ph),
3 3
OC(O)CH ); HRMS (ESI): m/z calcd for C H O SNa: 949.3597
3
55 58 11
1
7
39.8–126.9 (Ar), 99.8 (C-1B), 98.1 (C-1C), 97.4 (C-1A), 78.6 (C-3A),
+
M+Na] ; found: 949.3643.
8.1 (C-3B), 77.8 (C-2A), 76.7 (C-2C), 75.2 (OCH Ph and C-4A), 75.0,
2
Phenyl 3-O-(2-O-acetyl-3,4,6-tri-O-benzyl-a-d-mannopyranosyl)-
-O-(6-O-acetyl-3-O-benzoyl-2,4-di-O-benzyl-a-d-mannopyrano-
74.9 (OCH Ph), 74.5 (C-3C), 74.4 (C-4B), 73.5 (OCH Ph and C-4C),
2 2
6
72.8, 72.6 (OCH Ph), 72.4 (C-5B), 72.2 (C-5A), 72.0 (OCH Ph), 70.0 (C-
2
2
Chem. Eur. J. 2014, 20, 1 – 15
www.chemeurj.org
11
ꢃ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
5
C), 69.1 (C-6B), 69.0 (C-2B), 65.9 (C-6A), 65.7 (C-8), 63.5 (C-6C), 58.9
H-5B or H-5C), 3.63–3.54 (m, 7H; NCH Ph, H-6Ba, H-6Bb and H-
2
(
2 ꢅ NCH Ph), 52.8 (C-7), 21.1, 21.0 ppm (OC(O)CH ); HRMS (ESI):
6Ab), 3.39 (m, 1H; H-8b), 2.60 (m, 2H; H-7), 2.35 (t, J=2.4 Hz, 1H;
OCH CCH), 2.26 (t, J=2.4 Hz, 1H; OCH CCH), 2.16 ppm (t, J=2.3
2
3
+
m/z calcd for C H O N: 1546.6884 [M+H] ; found: 1546.6853.
9
4
100 19
2
2
13
Hz, 1H; OCH CCH); C NMR (150 MHz, CDCl ): d=140.0–138.0 (Ar),
2
3
(
N,N-Dibenzyl-2-aminoethyl) 3-O-(3,4,6-tri-O-benzyl-a-d-manno-
1
7
3
28.9–127.0 (Ar), 100.2 (C-1B), 98.5 (C-1C), 97.2 (C-1A), 80.2, 80.1,
pyranosyl)-6-O-(2,4-di-O-benzyl-a-d-mannopyranosyl)-2,4-di-O-
benzyl-a-d-mannopyranoside (32): A solution of 31 (0.136 g,
0
solution of sodium methoxide (0.8m in MeOH, 20 mL). The mixture
was stirred for 20 h at RT and then neutralized with acetic acid
until pH 7, and concentrated in vacuo. The crude product was puri-
fied by “automatic” reverse phase chromatography (50 min, H O/
acetonitrile 1:1 to 0:1 (v/v)) to afford 32 (102 mg, 85%): [a]
+
9.9 (OCH CCH), 79.8 (C-3B or C-3C), 79.5 (C-3A), 79.3 (C-3B or C-
2
C), 78.0 (C-2A), 75.7 (C-2C), 75.3–74.7 (C-4A, C-4B, C-4C, OCH CCH,
2
.09 mmol) in methanol (20 mL) was added to a freshly prepared
OCH Ph), 74.6 (OCH Ph), 74.3 (C-2B), 73.5 (OCH Ph), 72.7 (C-5A or
2
2
2
C-5B or C-5C), 72.6, 72.5, 72.4 (OCH Ph), 71.7, 71.6 (C-5A and/or C-
2
5
5
B and/or C-5C), 69.4 (C-6B), 68.7 (C-6C), 66.3 (C-6A), 65.9 (C-8),
9.0 (2ꢅNCH Ph), 58.6, 58.1, 57.6 (OCH CCH), 52.7 ppm (C-7);
2
2
2
+
HRMS (ESI): m/z calcd for C H O N: 1472.6880 [M+H] ; found:
20
92 98 16
D
1
472.6849.
1
39.1 (c=0.5 in CHCl3); H NMR (600 MHz, CDCl ): d=7.42–7.21
3
(
1
1
m, 45H; ArH), 5.29 (d, J(1,2)=1.2 Hz, 1H; H-1B), 5.14 (d, J(1,2)=
.2 Hz, 1H; H-1C), 4.96 (d, J=11.4 Hz, 1H; OCH Ph), 4.89 (d, J=
(N,N-Dibenzyl-2-aminoethyl) 6-O-(3,6-di-O-[1-(a-d-mannopyrano-
syl)-1,2,3-triazol-4-yl]methyl-2,4-di-O-benzyl-a-d-mannopyrano-
syl)-3-O-[1-(a-d-mannopyranosyl)-1,2,3-triazol-4-yl]methyl-2,4-di-
O-benzyl-a-d-mannopyranoside (39): A freshly prepared solution
of copper sulfate (16 mg, 0.07 mmol, 0.6 equiv) and sodium ascor-
bate (43 mg, 0.13 mmol, 1.2 equiv) in water (0.5 mL) was added to
a vigorously stirred solution of 10 (114 mg, 0.11 mmol) and azide
2
1.4 Hz, 1H; OCH Ph), 4.80 (d, J=11.4 Hz, 1H; OCH Ph), 4.77 (d,
2
2
J(1,2)=1.2 Hz, 1H; H-1A), 4.72–4.63 (m, 9H; OCH Ph), 4.44 (d, J=
1
J(2,3)=3 Hz, J(3,4)=9.6 Hz, 1H; H-3A), 4.06 (dd, J(2,3)=3.6 Hz,
J(3,4)=9.6 Hz, 1H; H-3C), 4.03 (dd, J(1,2)=1.2 Hz, J(2,3)=3 Hz, 1H;
H-2B), 3.98 (t, J(3,4)=J(4,5)=9.6 Hz, 1H; H-4A), 3.95 (dd, J(2,3)=3
Hz, J(3,4)=9 Hz, 1H; H-3B), 3.94–3.87 (m, 3H; H-4B, H-5C and H-
6
(
5
3
2
2.6 Hz, 1H; OCH Ph), 4.39 (d, J=11.4 Hz, 1H; OCH Ph), 4.17 (dd,
2 2
[5]
6
(90 mg, 0.44 mmol, 4 equiv) in anhydrous DMF (3 mL). The re-
action mixture was placed under microwave irradiation at 1008C
for 30 min then concentrated. The crude product was purified by
“automatic” reverse phase chromatography (50 min, water/acetoni-
Aa), 3.86 (dd, J(1,2)=1.2 Hz, J(2,3)=9.6 Hz, 1H; H-2A), 3.84–3.96
m, 3H; H-2C, H-6Ca and H-6Cb), 3.75–3.69 (m, 4H; H-4C, H-5A, H-
20
B and H-8a), 3.70–3.59 (m, 7H; H-6Ab, H-6Ba, H-6Bb and NCH Ph),
trile, 7:3 to 0:1 (v/v)) to afford 39 (130 mg, 72%). [a] +55.1 (c=
2
D
1
.46 (m, 1H; H-8b), 2.69 (dt, J=1.2, 6 Hz, 2H; H-7), 2.42 (d,
0.83 in MeOH); H NMR (600 MHz, MeOD): d=8.01, 7.97, 7.86 (3 ꢅ
J(2,OH)=2.4 Hz, 1H; OH), 2.31 (d, J(3,OH)=9 Hz, 1H; OH),
s, 3H; H-5 triazole), 7.40–7.12 (m, 30H, ArH), 5.95 (d, J(1,2)=2.2 Hz,
1H; H-1C or D or E), 5.88 (d, J(1,2)=2.5 Hz, 1H; H-1C or D or E),
5.87 (d, J(1,2)=2.4 Hz, 1H; H-1C or D or E), 4.91 (d, J(1,2)=1.5 Hz,
13
1
.97 ppm (m, 1H; OH); C NMR (75 MHz, CDCl ): d=139.6–137
3
(
Ar), 129.5–126.2 (Ar), 101.5 (C-1B), 97.6 (C-1A and C-1C), 80.1 (C-
3
B), 78.8 (C-2C and C-3A), 77.8 (C-2A), 76.6 (C-4C or C-4B or C-5A),
1H; H-1B), 4.89 (d, J=12 Hz, 1H; OCH Ph), 4.88 (d, J(1,2)<1 Hz,
2
7
5.1 and 74.9 (OCH Ph), 74.4 (C-5B or C-5C), 73.5, 72.8, 72.5 and
1H; H-1A), 4.78–4.73 (2d, 2H; OCH Ph and OCH (CCHN )), 4.72–
2
2
2
3
7
2.2 (OCH Ph), 72.1 (C-4C or C-4B or C-5A), 72.0 (C-5B or C-5C),
4.65 (2d, 2H; OCH Ph and OCH (CCHN )), 4.64–4.58 (m, 5H; H-2C
2 2 3
2
7
1.9 (C-3C), 71.5 (C-4C or C-4B or C-5A), 69.2 (C-6B), 68.8 (C-2B),
to E, OCH Ph and OCH (CCHN )), 4.57–4.53 (m, 3H; OCH Ph), 4.53–
2 2 3 2
6
7
6.2 (C-6A), 65.9 (C-8), 62.3 (C-6C), 59.0 (2 ꢅ NCH Ph), 52.7 ppm (C-
4.49 (m, 2H; OCH (CCHN )), 4.48 (d, J=11 Hz, 1H; OCH Ph), 4.43 (d,
2 3 2
J=12.4 Hz, 1H; OCH (CCHN )), 4.16 (dd, J(2,3)=3.5 Hz, J(3,4)=
2 3
2
+
); HRMS (ESI): m/z calcd for C H O N: 1358.6416 [M+H] ;
83
92 16
found: 1358.6428.
8.8 Hz, 1H; H-3C or D or E), 4.08 (dd, J(2,3)=2 Hz, J(3,4)=8.6 Hz,
1
3
H; H-3C or D or E), 4.07 (dd, J(2,3)=1.9 Hz, J(3,4)=8.6 Hz, 1H; H-
C or D or E), 3.96–3.46 (m, 27H; H-2A, H-2B, H-3A, H-3B, H-4A to
(
N,N-Dibenzyl-2-aminoethyl) 3-O-(3,4,6-tri-O-benzyl-2-O-proparg-
yl-a-d-mannopyranosyl)-6-O-(2,4-di-O-benzyl-3,6-di-O-propargyl-
a-d-mannopyranosyl)-2,4-di-O-benzyl-a-d-mannopyranoside
(
propargyl bromide (80% in toluene, 0.3 mL, 2.6 mmol, 9 equiv)
were added to a solution of 32 (0.392 g, 0.29 mmol) in anhydrous
DMF (20 mL) and cooled to 08C under an inert atmosphere. The
mixture was stirred for 1 h at 08C and then slowly warmed to RT
for 1 h. The reaction mixture was neutralized with MeOH, diluted
with H O (50 mL) and the aqueous phase was extracted with
EtOAc (3ꢅ50 mL). The combined organic layers were washed with
water and brine, dried over Na SO , filtered, and concentrated in
vacuo. The crude product was purified by “automatic” flash chro-
matography (60 min, cyclohexane/EtOAc 1:0 to 7:3 (v/v) with 1%
E, H-5A, H-5B, H-6Aa to Ea, H-6Ab to Eb, H-8 and NCH Ph), 3.30–
2
13
3
.21 (m, 3H; H-5C to E), 2.65 ppm (m, 2H; H-7); C NMR (75 MHz,
11): NaH (60% in mineral oil, 81 mg, 2.02 mmol, 7 equiv) and
MeOD): d=146.4, 146.3, 146.0 (3ꢅC-4 triazole), 140.8, 140.0, 139.8,
1
9
3
7
7
7
6
39.7 (Ar), 130.0–128.1 (Ar), 125.1, 125.0, 124.9 (3ꢅC-5 triazole),
9.5 (C-1B), 99.2 (C-1A), 88.3, 88.2 (C-1C to E), 82.0 (C-3A), 81.1 (C-
B), 78.4, 78.3, 78.2 (C-5C to E), 76.6, 76.3 (C-2B and C-2A), 75.9,
5.8 (OCH Ph), 75.7, 75.6 (C-4A and C-4B), 74.3, 73.7 (OCH Ph), 73.0,
2.6 (C-5A and C-5B), 72.6, 72.5 (C-3C to E), 70.5 (C-6A or C-6B),
0.2, 70.1 (C-2C to E), 68.5, 68.4, 68.3 (C-4C to E), 67.6 (C-6A or C-
B), 66.6 (C-8), 65.2, 64.1, 63.7 (3 ꢅ OCH (CCHN )), 62.5, 62.4, 62.3
2
2
2
2
3
2
4
(C-6C to E), 60.0 (2ꢅNCH Ph), 54 ppm (C-7); HRMS (ESI): m/z calcd
2
+
for C H O N : 1655.7045 [M+H] ; found: 1655.7061.
83
103 26 10
2
D
0
Et N) to afford 11 (253 mg, 60%). [a] +54.9 (c=0.88 in CHCl3);
(N,N-Dibenzyl-2-aminoethyl) 3-O-(2-O-[1-(a-d-mannopyranosyl)-
1,2,3-triazol-4-yl]methyl-3,4,6-tri-O-benzyl-a-d-mannopyranosyl)-
6-O-(3,6-di-O-[1-(a-d-mannopyranosyl)-1,2,3-triazol-4-yl]methyl-
2,4-di-O-benzyl-a-d-mannopyranosyl)-2,4-di-O-benzyl-a-d-man-
nopyranoside (40): A freshly prepared solution of copper sulfate
(22 mg, 0.09 mmol, 0.6 equiv) and sodium ascorbate (58 mg,
0.18 mmol, 1.2 equiv) in water (0.5 mL) was added dropwise to
a vigorously stirred solution of 11 (216 mg, 0.15 mmol) and azide
3
1
H NMR (600 MHz, CDCl ): d=7.39–7.12 (m, 45H; ArH), 5.22 (d,
3
J(1,2)=1.2 Hz, 1H; H-1B), 5.01 (d, J(1,2)=1.7 Hz, 1H; H-1C), 4.88,
.87 (2d, J=10.8, 12.2 Hz, 2H; OCH Ph), 4.79 (d, J=11.5 Hz, 1H;
4
2
OCH Ph), 4.71 (d, J(1,2)=1.4 Hz, 1H; H-1A), 4.69 (d, J=11.6 Hz, 1H;
OCH Ph), 4.66–4.44 (m, 9H; OCH Ph), 4.37 (d, J=12.1 Hz, 1H;
OCH Ph), 4.28 (dd, J(CH ,CH)=2.3 Hz, J=16.3 Hz, 1H; OCH CCH),
4
5
1
5
2
2
2
2
2
2
.23 (dd, J(CH ,CH)=2.4 Hz, J=16 Hz, 1H; OCH CCH), 4.17–4.09 (m,
2
2
[5]
H; OCH CCH and H-3A), 4.07 (dd, J(1,2)=1.2 Hz, J(2,3)=2.8 Hz,
6 (105 mg, 0.51 mmol, 3.5 equiv) in DMF (5 mL). The reaction mix-
2
H; H-2B), 3.99–3.85 (m, 6H; H-3B, H-3C, H-4A, H-4B, H-4C and H-
A or H-5B or H-5C), 3.86–3.83 (m, 2H; H-2C and H-6Aa), 3.82 (dd,
ture was placed under microwave irradiation at 1008C for 30 min
then concentrated. The crude product was purified by “automatic”
reverse phase chromatography (50 min, water/acetonitrile 1:1 to
J(1,2)=1.4 Hz, J2,3 =3 Hz, 1H; H-2A), 3.81–3.74 (m, 2H; H-6Ca and
H-5A or H-5B or H-5C), 3.70–3.64 (m, 3H; H-8a, H-6Cb and H-5A or
20
0:1 (v/v)) to afford 40 (229 mg, 75%). [a]D +57.3 (c=0.53 in
&
&
Chem. Eur. J. 2014, 20, 1 – 15
www.chemeurj.org
12
ꢃ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
1
20
MeOH); H NMR (600 MHz, MeOD): d=8.0, 7.95, 7.87 (3 s, 3ꢅ1H;
by HPLC (Method 2) to afford 5 as a white solid (67 mg; 74%). [a]D
1
H-5 triazole), 7.36–7.03 (m, 45H; ArH), 5.90 (d, J(1,2)=2.5 Hz, 1H;
H-1D or H-1E or H-1F), 5.87 (d, J(1,2)=2.5 Hz, 2H; H-1D and/or H-
+63.5 (c=0.32, H O); H NMR (600 MHz, D O): d=8.44 (s, 1H;
2
2
HC(O)O), 8.26, 8.25, 8.24 (3s, 3ꢅ1H; H-5 triazole), 8.15 (d, J(1,2)=
2.3 Hz, 1H; H-1D or H-1E or H-1F), 8.14 (d, J(1,2)=2.2 Hz, 1H; H-1D
or H-1E or H-1F), 8.13 (d, J(1,2)=2.2 Hz, 1H; H-1D or H-1E or H-1F),
5.19 (d, J(1,2) <1 Hz, 1H; H-1B), 4.92 (d, J(1,2)=1.6 Hz, 1H; H-1C),
4.89 (d, J(1,2)=1.4 Hz, 1H; H-1A), 4.89–4.72 (m, 9H; H-2D to F and
OCH (CCHN )), 4.19 (dd, J(1,2)=1.6 Hz, J(2,3)=2.6 Hz, 1H; H-2C),
1
1
3
E and/or H-1F), 5.19 (d, J(1,2)=1.1 Hz, 1H; H-1B), 4.94 (d, J(1,2)=
.3 Hz, 1H; H-1C), 4.83 (d, J(1,2)=1.1 Hz, 1H; H-1A), 4.79–4.74 (m,
H; OCH Ph), 4.71–4.41 (m, 18H; OCH Ph, OCH (CCHN ) and H-2D
2
2
2
3
to F), 4.37 (d, J=11.9 Hz, 1H; OCH Ph), 4.30 (d, J=11.9 Hz, 1H;
OCH Ph), 4.15 (d, J(2,3)=2.8 Hz, J(3,4)=9.6 Hz, 1H; H-3A), 4.14–
2
2
2
3
4.07 (m, 3H; H-3D to F), 3.97 (t, J(3,4)=J(4,5)=9.6 Hz, 1H; H-4A),
3.94–3.65 (m, 22H; H-2A to C, H-3B, H-3C, H-4B to F, H-5A to C, H-
6Aa, H-6Ca, H-6Da to Fa, H-6Db to Fb and H-8a), 3.64–3.49 (m, 8H;
4.17–4.12 (m, 4H; H-2A and H-3D to F), 4.02 (dd, J(1,2)<1 Hz,
J(2,3)=3.2 Hz, 1H; H-2B), 4.02–3.69 (m, 25H; H-3A to C, H-4B to F,
H-5A to C, H-6Aa to Fa, H-6Ab to Fb and H-8), 3.63 (t, J(3,4)=
H-6Ab, H-6Ba, H-6Bb, H-6Cb and NCH Ph), 3.45 (m, 1H; H-8b),
J(4,5)=9.8 Hz, 1H; H-4A), 3.37–3.25 ppm (m, 5H; H-5D to F and H-
2
13
13
3
.33–3.25 (m, 3H; H-5D to F), 2.57 ppm (m, 2H; H-7); C NMR
7); C NMR (150 MHz, D O): d=170.8 (HC(O)O), 145.2, 144.9, 144.8
2
(
(
(
75 MHz, MeOD): d=146.5, 146.4, 146.3 (C-4 triazole), 140.8–139.5
Ar), 130.2–127.6 (Ar), 124.9, 124.7 (C-5 triazole), 101.5 (C-1B), 99.6
C-1C), 98.6 (C-1A), 88.3 (C-1D to F), 81.7 (C-3B or C-3C), 80.5 (C-3B
(C-4 triazole), 125.5 125.4, 125.4 (C-5 triazole), 100.7 (C-1A), 100.6
(C-1B), 100.1 (C-1C), 87.3 (C-1D to F), 79.0, 78.9 (C-3A and C-3C),
78.3 (C-2B), 76.5, 76.4 (C-5D to F), 73.7 (C-3B), 71.7, 71.4, 70.4 (C-4B
and/or C-4C and/or C-5A and/or C-5B and/or C-5C), 70.8 (C-3D to
F), 69.8 (C-2A), 69.3 (C-6C), 68.5 (C-2D to F), 67.4 (C-4A), 66.9 (C-2C),
66.6 (C-4D to E), 66.8, 65.9 (C-4B and/or C-4C and/or C-5A and/or
C-5B and/or C-5C), 65.7 (C-6A), 63.8 (C-8), 63.7, 63.5, 62.0
(OCH (CCHN )), 61.3 (C-6B), 60.8 (C-6D to F), 39.4 ppm (C-7); HRMS
or C-3C), 80.1 (C-3A), 79.3 (C-2A), 78.3 (C-5D to F), 77.8 (C-2B), 76.5,
7
7
6
6.4, 76.1, 75.8, 73.9, 73.0, 72.9 (C-2C, C-4A to C, C-5A to C); 75.9,
5.7, 75.6, 74.3, 73.7, 73.6, 73.1 (OCH Ph), 72.6 (C-3D to F); 70.5 (C-
2
B and C-6C), 70.1 (C-2D to F), 68.5 (C-4D to F), 67.6 (C-6A), 66.5
(
C-8), 65.2, 64.3 (3ꢅOCH (CCHN )), 62.4 (C-6D to F), 59.8 (2ꢅ
2 3
2
3
+
NCH Ph), 59.8 ppm (C-7); HRMS (ESI): m/z calcd for C110H131O N :
2
(ESI, amine form): m/z calcd for C H O N : 1277.4756 [M+H] ;
2
31 10
47 77 31 10
+
087.8922 [M+H] ; found: 2087.8989.
found: 1277.4742.
2
-Aminoethyl 6-O-(3,6-di-O-[1-(a-d-mannopyranosyl)-1,2,3-tria-
zol-4-yl]methyl-a-d-mannopyranosyl)-3-O-[1-(a-d-mannopyrano-
syl)-1,2,3-triazol-4-yl]methyl-a-d-mannopyranoside hydrofor-
Acknowledgements
mate (4): Palladium on charcoal (10%, 30 mg, 0.28 mmol) and
resin with ammonium formate (1.3 g) was added to a solution of
This work was supported by the Conseil Rꢀgional de Picardie,
the Ministꢆre de l’Enseignement et de la Recherche, the Span-
ish Ministerio de Innovaciꢇn y Ciencia (MICINN, contract num-
bers SAF2013-44021-R, and CTQ2010-15848), the Junta de An-
dalucꢁa and the European Union (FEDER and FSE). We also
thank the CITIUS for technical support. The authors acknowl-
edge Dr. David Lesur (FRE 3517) and Dr. Serge Pilard (Plate-
forme Analytique, UPJV, France) for MS and HPLC. We are
grateful to the staff of the Soleil Proxima 1 beamline (Gif/
Yvette, France) for their help in the collection of diffraction
data.
3
9 (93 mg, 0.056 mmol) in an isopropanol/H O mixture (5:1 (v/v),
2
1
2 mL). The reaction mixture was placed under microwave irradia-
tion at 808C for 40 min, filtered over Celite and concentrated in
vacuo. The crude product was then purified by HPLC (Method 2)
20
to afford 4 as a white solid (51 mg; 81%). [a] + 60.5 (c=0.43 in
D
1
H O); H NMR (600 MHz, D O): d=8.46 (s, 1H; OC(O)H), 8.26, 8.24,
2
2
8
.23 (3s, 3H, H-5 triazole), 6.16 (d, J(1,2)=2.4 Hz, 1H; H-1C or H-1D
or H-1E), 6.15 (d, J(1,2)=1.8 Hz, 1H; H-1C or H-1D or H-1E), 6.14 (d,
J(1,2)=2.4 Hz, 1H; H-1C or H-1D or H-1E), 4.94 (d, J(1,2)=1.8 Hz,
1
1
4
H; H-1B), 4.91 (d, J(1,2)=1.8 Hz, 1H; H-1A), 4.89 (d, J=12.1 Hz,
H; OCH (CCHN )), 4.86 (d, J=13.2 Hz, 1H; OCH (CCHN )), 4.83–
2
3
2
3
.71 (m, 7H; H-2C to E and OCH (CCHN )), 4.21 (dd, J(1,2)=1.8 Hz,
2
3
J(2,3)=3 Hz, 1H; H-2A), 4.18 (dd, J(1,2)=1.8 Hz, J(2,3)=2.4 Hz, 1H;
H-2B), 4.83–4.72 (m, 3H; H-3C to E), 4.0–3.94 (m, 2H; H-6Aa and H-
Keywords: carbohydrates · click chemistry · glycoconjugates ·
oligosaccharides · structure–activity relationships
8
a), 3.90–3.69 (m, 19H; H-3A, H-3B, H-4A to E, H-5A, H-5B, H-6Ab,
H-6Ba to Ea, H-6Bb to Eb and H-8b), 3.37–3.30 (m, 3H; H-5C to E),
13
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.0–3.22 ppm (m, 2H; H-7); C NMR (150 MHz, D O): d=171.0
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(
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Published online on && &&, 0000
&
&
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ÝÝ These are not the final page numbers!

Full Paper
FULL PAPER
Simplifying the complex: “Click” emula-
tors of “high-mannose oligosaccharides”
containing triazole rings in the core act
as functional mimics of the natural
sugars towards the mannose specific
lectins concanavalin A and the human
macrophage mannose receptor. This
strategy may prove of general use for
the design of high affinity ligands of
biologically relevant targets binding
complex carbohydrates.
&
Oligosaccharides
M. FranÅois-Heude, A. Mꢀndez-Ardoy,
V. Cendret, P. Lafite, R. Daniellou,
C. Ortiz Mellet, J. M. Garcꢁa Fernꢃndez,
V. Moreau,* F. Djeda ¨ı ni-Pilard*
&
& – &&
Synthesis of High-Mannose
Oligosaccharide Analogues through
Click Chemistry: True Functional
Mimics of Their Natural Counterparts
Against Lectins?
Chem. Eur. J. 2014, 20, 1 – 15
www.chemeurj.org
15
ꢃ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ