New branched amino acids for high affinity dendrimeric DC-SIGN ligands

Article abstract of DOI:10.1016/j.bmc.2017.12.036

A branched amino acid was synthesized from methyl glucopyranoside; this amino acid presents three amino groups protected by Fmoc and one acid group and can be used in classic peptide synthesis. In parallel, similar azido terminated blocks were synthesized

Full text of DOI:10.1016/j.bmc.2017.12.036

Accepted Manuscript
New branched amino acids for high affinity dendrimeric DC-SIGN ligands
Laurent Cattiaux, Vanessa Porkolab, Franck Fieschi, Jean-Maurice Mallet
PII:
S0968-0896(17)31683-8
https://doi.org/10.1016/j.bmc.2017.12.036
BMC 14141
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
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22 December 2017
24 December 2017
Please cite this article as: Cattiaux, L., Porkolab, V., Fieschi, F., Mallet, J-M., New branched amino acids for high
affinity dendrimeric DC-SIGN ligands, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/
j.bmc.2017.12.036
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New branched amino acids for high affinity
dendrimeric DC-SIGN ligands
L Cattiaux, V Porkolab, F Fieschi, J-M Mallet
École Normale Supérieure-PSL Research University, LBM UMR 7203 ; and Univ. Grenoble Alpes, IBS,

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
New branched amino acids for high affinity dendrimeric DC-SIGN ligands
Laurent Cattiaux, a Vanessa Porkolab,b Franck Fieschi,b Jean-Maurice Mallet*a
a Laboratoire des Biomolécules, Département de chimie, École normale supérieure, PSL ResearchUniversity, Sorbonne Universités, UPMC Univ. Paris 06,
CNRS, 24 rue Lhomond, 75005 Paris,France.
b Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, Avenue des Martyrs, F-38044 Grenoble, France
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A branched amino acid was synthesized from methyl glucopyranoside; this amino acid presents
three amino groups protected by Fmoc and one acid group and can be used in classic peptide
synthesis. In parallel, similar azido terminated blocks were synthesized.
Successive coupling reaction and deprotection afforded dendrimers with up to 27 azido
functional groups. As an example of application, D-mannose and L-fucose residues were linked
through CuAAC coupling and resulting glycodendrimers were evaluated in their interaction with
DC-SIGN using SPR competition assay.
Available online
Keywords:
glycoside
dendrimers
DC- SIGN
SPR
2017 Elsevier Ltd. All rights reserved
.
branched amino acid
Introduction
SIGN, a large bouquet of mannose is required for good
affinity (ca 30 D-mannoses) 5. Our project is the
construction of very active yet simple mannosylated/
fucosylated synthetic ligands for DC-SIGN. Different
approaches were found in the literature mainly with
mannosylated polymers and mannosylated dendrimers
whereas fucosylated conjugates (dendrimer and
DC-SIGN (dendritic cell-specific ICAM-3-grabbing non
integrin) also called CD209 is a type II transmembrane
C-type lectin with a single C-terminal Carbohydrate
Recognition Domain (CRD). This receptor binds to
“self” glycan ligands found on human cells as well as to
“foreign” glycans derived from bacterial or parasitic
6
polymers) are less frequently prepared . Dendrimers
1
pathogens . It facilitates intra cellular DC delivery of
allow a better control both in geometry and in
homogeneity (macroscopic and microscopic) than
polymeric carriers. Various studies have shown the
usefulness of dendritic manosylated ligands in the design
of high affinity DC-SIGN ligands with mannose
derivatives or ꢀ D-Man 1->2 Man terminated branches 7.
high-mannose-type structure and related structure and
promotes their processing towards antigen presentation.
A critically important example of such involvement of
DC-SIGN is its strong binding to highly mannosylated
HIV gp120 promoting virus transposition from mucosal
surface to lymphoid system and thus the transfer of HIV
2
to T-cell . We plan to use this property in a program
Results
aiming the preparation of vaccines:
a
suitable
construction equipped with a DC-SIGN ligand as war
head will be able to deliver antigenic protein intra DC
and thus promoted its presentation by DC to the immuno
competent cells 3.
We want here to present new highly branched
glycodendrimers as potent DC-SIGN ligands. As many
carbohydrates ligands are needed, a less commonly used
3-branched dendrimer is proposed to produce high
valency more rapidly. With a tripler unit, a 27-branches
dendrimer needs a generation 2 and 14 synthetic blocks
(9+3+1), whereas with a doubler unit, a 32-branches
DC-SIGN recognizes both mannosylated and
fucosylated ligands. Although protein-carbohydrate
interactions are essential to many biological processes,
individual interactions usually exhibit weak binding
dendrimer needs
a generation 4 and 31 blocks
(16+8+4+2+1) and the double of coupling steps. Amide
linkage was selected as the dendrimer building function
for its stability, biocompatibility and its potential
biodegradability. Dendrimers made from amino acid are
not new, as dendrimers of lysine are well established;
4
affinities (mM range). Mammen et al. demonstrated
that nature uses multivalency to overcome this problem.
Thus multiple copies of binding sites on the lectin but
also multiple ligands are necessary. In fact, for DC-

however this core offers only two branches and of very
different length.
depicted on scheme 2. A trityl was selected as primary
alcohol protection to give 7 11. T. Lindhorst in her
preparation of octopus carbohydrate 12 compared various
methods for amino alkyl introduction and finally
selected hydroboration of allyl groups as her preferred
route for multiple carbohydrate alcohols substitution.
Thus the three secondary alcohol functions were
allylated. The allyl groups were hydroborated (9-BBN)
and the generated alcohol function transformed into an
azido group. Finally, the trityl group was removed and
replaced with a terbutyloxy carbonymethyl group under
phase transfert catalysis conditions to give the key block
13 (scheme 2). Further transformations involved:
reduction of the azido groups with Ph3P followed by in
situ introduction of Fmoc 13 and tert butyl ester cleavage
with TFA.
We propose here the first member of new amino acids
family originated from carbohydrates (scheme 1). This
glucose amino acid derivative combines a rigid and
chiral core with short flexible arms. This approach can
be extended to various sugars and gives us the
possibility to use solid phase synthesis and an automated
synthesis to build dendrimers. In addition, this offers
also the possibility to add/insert natural amino acid as
spacer (such as glycine) inside the core to expand it or to
introduce charges (Glu, Asp, Arg, Lys) to modulate
solubility and charges.
The "last layer" of the dendrimer will be terminated by
azido groups (for click CuAAC functionalization of
protected sugars such as 4, 5 or 6) using a triazido
The dendrimer construction is straightforward as
depicted in scheme 3, it involved iterative coupling steps
starting from S-trityl cysteamine: amide formation
promoted by DCC, removal of Fmoc (DBU/octyl thiol)
14. This latter method uses a strong base (DBU) in
presence of octyl thiol, a powerful nucleophile that traps
the dibenzofulvene and avoids side reaction of amine.
Thus two versatile key dendrimeric scaffolds were
prepared: IIc (generation 1 with nine azido terminal
groups, from block 1 and three blocks 2) and IIIc
(generation 2 with 27 azido terminal groups, from four
blocks 1 and nine blocks 2. (scheme 3)
8
aminoacid 2 as the terminator group. R. Riguera
already showed the versatility of this strategy for the
preparation of clikable dendrimers. The other end will be
functionalized by a thiol, a cysteamine protected as S-
9
trityl ether 3 . This thioester, once deprotected, will
allow specific and orthogonal linkage in various uses.
Alternatively, the trityl may deserve as an anchor on
surface as described. 10
We used first a glucose scaffold starting from methyl α-
D-glucoside, an inexpensive derivative, presenting one
primary alcohol (to link the acid function) and three
secondary hydroxyl groups of similar reactivity for the
installation of 3-amino propyl group. The synthesis is
Scheme 1: Strategy, general structure and synthetic blocks for glycodendrimers

The azido dendrimers were then coupled with acylated
propargyl glycosides by CuAAC to give the protected
Affinities of all compounds were measured by SPR in the
previously established competition assay that measure the
18
15
16
glycodendrimers. Compounds 4
and 5
were prepared
ability of compounds to inhibit the binding of extracellular
(ECD) DC-SIGN to mannosylated bovine serum albumin
(BSA-Man) surface onto the CM4 sensor chip. A fixed
amount of DC-SIGN ECD and increasing concentrations of
dendrimers were injected over the surface. The natural
monosaccharide D-mannose and L-fucose were used both to
control the surface stability and to estimate the ß factor value
4. This value corresponds to the capability of dendrimers
displaying several D-mannose or L-fucose copies to improve
their inhibitory potency related to the monovalent species.
The inhibition curves and IC50 values are shown in Fig 2 (the
initial sensorgrams can be seen in supplementary Figure S1).
The ß factor values are summarized in Table 1.
according to literature procedure. In addition, a block 6 with
two mannoses was prepared on a glycerol scaffold (it was
prepared from 1,3-benzylidene glycerol via 2-propargyl
17
glycerol
and its mannosylation using pentabenzoyl
mannose) to increase further the numbers of branches.
Figure 2: DC-SIGN inhibition activities of dendrimers
bearing L-fucose or D-mannose ligands. DC-SIGN ECD (20
µM) and compounds were co-injected over a BSA-Man
surface. (a) Comparison of the inhibitory potency of
compounds toward DC-SIGN ECD/ BSA-Man interaction. (b)
IC50 (grey bars) are expressed as dendrimer concentration
(µM).
Scheme 2: Elongating and terminating blocks: (a) allyl
bromide, NaH 60% in oil, DMF, 12h ; (b) 0.5M 9-BBN in
THF, 100°C, 2h ; then H2O 0°C, 3M aq NaOH, 35% H2O2,
12h ; (c) MsCl, Et3N, CH2Cl2, 0°C, 15min ; (d) NaN3, DMF,
65°C, 2h ; (e) PTSA (cat) 1 :1 CH2Cl2/ Methanol, 50°C, 12h ;
(f) tert-butyl 2-bromoacetate, NBu4Br (cat), 50% aq NaOH,
toluene, 80°C, 2h ; (g) Ph3P, THF, H2O, 15h ; (h) FmocOSu,
Et3N, 15min ; (i) TFA/CH2Cl2, RT, 5h
The affinity and the relative potency inhibitory (ß factor) of
dendrimers increase with the number of D-mannose or L-
fucose displaying by dendrimers. A significant ß factor,
superior to 40, was obtained for IIIg bearing 27 copies of D-
mannose. The potency enhancement for DC-SIGN results
from different molecular mechanisms including clustering,
6f,19
statistical rebinding and potentially the chelation effect
.
Compared to others scaffolds already tested with our DC-
SIGN competition assay 7b,20, the multivalent compound IIIg
is able to generate, up to date, one of the best avidity for DC-
SIGN.
After deacylation, the four dendrimers If, Id, Ih, IId (Scheme
3) was purified on Sephadex columns and characterized in
NMR and mass spectrometry (Fig. 1). Although NMR gave
correct spectra integration, like others 7d, we were not able to
obtain clean mass spectra for the IIIg (27 Man dendrimer)
MW 12226
Table 1: Valency, IC50 and avidity values of dendrimers
inhibiting binding of DC-SIGN ECD to immobilized BSA-
Man.
compound valency ligand
IC50 (µM)
β
Fucose
IIh
1
9
Fuc
Fuc
2480 17
9.5 0.7
1
29
Mannose
IIg
IIi
1
9
18
27
Man
Man
Man
Man
3057 66
36.1 1.3
20 0.7
1
9
8
IIIg
2.75 0.1
41
Figure 1: Maldi Mass spectra of IIg

This remarkable affinity may relies on the peculiar feature of
the dendrimer, the presence of a rigid and chiral core with
flexible and short arm that allows large inter group distance
with a limited global flexibility and binding entropy.
range avidity than IIg.
Conclusion
We have described the synthesis of a branched amino acid
from methyl glucopyranoside used in the preparation of key
polyazido dendrimers, easily functionalized by CuAAC with
sugars (mannose or fucose) to give 9 or 27-branch ligands.
Preliminary studies on these glycosylated dendrimers have
shown nice cooperative effect, in DC-SIGN recognition. This
strategy is currently extended to other highly branched amino
acids (with various sugars cores), in solid phase synthesis for
an easy approach to lectin ligands
Moreover, comparison of inhibitory potencies of IIh and IIg
reveals that for an identical valency, monovalent ligands play
a decisive role in DC-SIGN binding. The 9 L-fucose of IIh
with an IC50 of 9.5 µM inhibits three times more than the
corresponding dendrimer with 9 D-mannose IIg. On the other
hand, the ligand presentation of IIi influences negatively the
multivalent interactions for his target leading to the same
B/ substituants
a
b
c
f
d
e
g
h
i
Scheme 3: Structure of the dendrimers (I, II and III)

Scheme 3 part C) Synthetic scheme for dendrimers preparation: reagents and conditions : a) DCC, CH2Cl2; b) DBU, n-octylthiol,
THF; c) click reaction: CuSO4, Na+ ascorbate, dioxane / water; d) NaOH, MeOH
×
-CH2-). 13C
Experimental
, -O-CH3, H4, H2), 1.77 – 1.45 (app.s, 6H, 3
NMR (75 MHz, CDCl3) ꢣ 172.44 (-COOH), 156.76
(NHCOFmoc), 156.60 (NHCOFmoc), 156.55 (NHCOFmoc),
Chemical synthesis: All compounds were homogeneous by
TLC analysis and had spectral properties consistent with their
assigned structures. Compound purity was checked by TLC
on Silica gel 60 F254 (E. Merck) with detection by charring
with sulfuric aciꢁꢂꢃꢄꢅꢆꢇꢈꢉꢃꢊꢋꢌꢅꢈꢍꢎꢅꢏꢌꢍꢐꢋꢑꢃꢒꢍꢓꢃꢐꢔꢌꢕꢅꢌꢈꢔꢁꢃꢅꢉꢃ
ꢖꢗꢆꢗꢊꢍꢃ ꢏꢔꢆꢃ ꢘꢙꢃ ꢚꢛꢂꢃ ꢜꢔꢌꢊꢝꢞꢂꢃ ꢟꢜꢠꢃ ꢓꢐꢔꢊꢎꢌꢍꢃ ꢒꢔꢌꢔꢃ ꢌꢔꢊꢅꢌꢁꢔꢁꢃ ꢒꢗꢎꢋꢃ
ꢡꢌꢢꢝꢔꢌꢃ 300, 400 MHz instruments. CyHex stands for
cyclohexane.
143.98 (6
127.04 (6
×
×
Cq ar), 141.33 (6
Cq ar), 124.98 (6
×
×
Cq ar), 127.67 (6
Cq ar), 119.98 (6
×
×
Cq ar),
Cq ar),
97.31 (C1), 81.43 (C3), 80.03 (C2), 77.65 (C4), 71.54 (-CH2-O-
), 70.73 (-CH2-O-), 70.26 (C6), 70.11 (C5), 68.74 (C7), 68.40
(-CH2-O-), 66.64 (CH2Fmoc), 66.32 (CH2Fmoc), 65.90
(CH2Fmoc), 55.16 (O-CH3), 47.32 – 47.25 (3
× CHFmoc),
39.04 (FmocNH-CH2-), 38.80 (FmocNH-CH2-), 38.62
(FmocNH-CH2-), 30.44 (-CH2-), 30.22 (-CH2-), 29.60 (-CH2-
).
Methyl-2,3,4-tri-O-(3-azidopropyl)-6-O-carboxymethyl -α-
D-glucopyranoside (2) :To a solution of 13 (500 mg, 0.8967
mmol) in dry CH2Cl2 (18 mL) was added TFA (2 mL). The
mixture was stirred for 3 hours at room temperature, washed
with water (10 mL), the resulting organic layer was dried over
MgSO4, and concentrated to give 2 (417mg, 93%) as a yellow
Compound 6: step 1: Synthesis of 1,3-Propanediol, 2-(2-
propyn-1-yloxy) - To a solution of 2-phenyl-1,3-dioxan-5-ol
(500 mg, 2.77 mmol, 1 equiv.) in dry DMF (5mL) were added
propargyl bromide 80 wt. % in toluene (618 µL, 5.55 mmol, 2
equiv.) and sodium hydride 60 wt. % (222 mg, 5.55 mmol, 2
equiv.) portionwise. The mixture was stirred overnight and
the sodium hydride was quenched by addition of MeOH and
the solution was concentrated. The residue was dissolved in
CH2Cl2 (20 mL) and washed with water (2 × 10 mL). The
organic layers were combined, dried over magnesium sulfate
and concentrated. The residue was purified by column
chromatography on silica gel (CyHex 100% then
CyHex/AcOEt 70:30) to get the propargyl derivative (498 mg,
syrup. [α] +62 (c 1, CHCl3). ESI-HRMS m/z [M-H]-
D
calculated for C18H30N9O8: 500.2217, found: 500.2206. 1H
NMR (300 MHz, CDCl3) ꢣ 4.75 (d, J = 3.5 Hz, 1H, H1), 4.19
(d, J = 17.3 Hz, 1H, H7a), 4.13 (d, J = 17.3 Hz, 1H, H7b), 3.87
– 3.73 (m, 4H, 2
H5, H6), 3.51 (t, J = 9.2 Hz, H3), 3.41 – 3.27 (m, 9H, 3
CH2-, O-CH3), 3.26 – 3.19 (m, 2H, H2, H4), 1.90 – 1.66 (m,
6H, 3
-CH2-). 13C NMR (75 MHz, CDCl3) ꢣ 174.16
×
-CH2-O-), 3.73 – 3.55 (m, 5H, -CH2-O-,
×
N3-
×
+
(COOH), 97.62 (C1), 81.36 (C3), 80.48 (C2), 77.49 (C4), 70.31
(-CH2-O), 70.02 (-CH2-O), 69.97 (C5), 69.41 (-CH2-O), 68.47
(C7), 67.76 (C6), 55.36 (OCH3), 48.41 (N3-CH2-), 48.25 (N3-
CH2-), 48.17 (N3-CH2-), 29.75 (-CH2-), 29.57 (-CH2-), 29.43(-
CH2-).
83%). MS ESI-HRMS m/z [M+Na]+ calcd for C13H14NaO3
1
241.0835, found 241.0837. H NMR (300 MHz, CDCl3) ꢣ
7.57 – 7.51 (m, 2H, Har), 7.43 – 7.35 (m, 3H, Har), 5.59 (s, 1H,
H Benzylidene), 4.47 – 4.31 (m, 4H, CH2, CH2 propargyl),
4.11 (dddd, J = 12.8, 1.5 Hz, 2H, CH2), 3.67 (p, J = 1.7 Hz,
1H, CH), 2.47 (t, J = 2.4 Hz, 1H, H alkyne). 13C NMR (75
MHz, CDCl3) ꢣ 138.0 (Cq ar), 128.93 (Car), 128.2 (2 × Cq ar),
126.1 (2 × Cq ar), 101.3 (C Benzylidene), 79.4 (-C≡CH),
74.9(-C≡CH), 68.8 (CH), 68.7 (2 × CH2), 55.6 (CH2
propargyl).
Compound 1 : Compound 14 was dissolved in a mixture of
dry CH2Cl2 (80 mL) and TFA (20 mL). After 5 hours stirring
at room temperature, the solution was concentrated and the
residue was dissolved in the minimum of CH2Cl2 and cold
diethyl ether (300 mL) was added to promote precipitation.
The mixture was kept overnight at 4°C and filtered to give 1
(6.09g, 55% over 3 steps). Rf: 0.57 (CH2Cl2/MeOH 95:5).
Step 2: To a mixture of CH2Cl2, TFA and water (10 mL, 8/1/1
v/v/v) was added the propargyl derivative (200 mg, 0.917
mmol). The mixture was stirred overnight at room
temperature and TFA was carefully neutralized by
triethylamine. The solution was concentrated and the residue
was purified by column chromatography on silica gel (CyHex
100% then CyHex/AcOEt 50:50) to get the 1,3-propanediol,
[α] +27 (c 1, CHCl3). ESI-HRMS m/z [M+H]+ calculated for
D
C63H68N3O14: 1090.4701, found: 1090.4746. 1H NMR (300
MHz, CDCl3) ꢣ 7.81 – 7.05 (m, 24H, Har), 5.50 – 5.13 (m, 3H,
NHFmoc), 4.80 – 4.61 (br s, 1H, H1), 4.46 – 3.89 (m, 11H, 3
1
2-(2-propyn-1-yloxy) (104 mg, 67%). H NMR (300 MHz,
×
CH2Fmoc, 3
×
CHFmoc, H7), 3.89 – 3.33 (m, 10H, 3
× -
CDCl3) ꢣ 4.25 (d, J = 2.3 Hz, 2H, CH2 propargyl), 3.82 – 3.53
CH2-O-, H6, H5, H3), 3.32 – 2.97 (m, 11H, 3
×
FmocNH-CH2-

(m, 5H, 2 × CH2, CH), 3.04 (s, 2H, OH), 2.46 (t, J = 2.3 Hz,
1H, H alkyne). 13C NMR (75 MHz, CDCl3) ꢣ 80.0 (-CꢤCH),
79.3 (CH), 75.0 (-C≡CH), 61.9 (2 × CH2), 57.3 (CH2
propargyl).
glucopyranoside (9) : 8 (6.62 g, 11.90 mmol, 1 equiv.) was
dissolved in 9-BBN 0.5M in THF (142 mL, 71.37 mmol, 2
equiv. / allyl). The solution was stirred under reflux for 2
hours under argon. Then, the solution was cooled at 0°C and
the excess of 9-BBN was quenched with cold water (50 mL).
Then, 3M aq sodium hydroxide (150 mL) and hydrogen
peroxide (35%) (150mL) were added carefully at 0°C. The
mixture was stirred from 0°C to room temperature overnight
and CH2Cl2 (400 mL) was added. The aqueous layer was
discarded and the organic layer was further washed with a
saturated solution of Na2S2O3 (200 mL) and brine. The
organic layer was dried over MgSO4 and concentrated. The
residue was purified by column chromatography on silica gel
(AcOEt/MeOH 95:5) to give 9 (5.23 g, 72%) as a white foam.
Step 3: To a solution of step 2 compound (20 mg, 0.1538
mmol, 1 equiv.) in dry CH2Cl2 (3 mL) were added 1,2,3,4,6-
penta-O-benzoyl-ꢀ,ꢥ-D-mannopyranoside (323 mg, 0.4615
mmol, 3 equiv.) and BF3,Et2O (0.3 mL, 2.30 mmol, 15
equiv.). The mixture was stirred overnight under argon at
room temperature. Then the reaction was diluted with CH2Cl2
(5 mL) and BF3, Et2O was neutralized with aq sat NaHCO3.
The organic layer was dried over MgSO4, and concentrated.
The residue was purified by column chromatography on silica
gel (CyHex/AcOEt 8:2) to give 6 (115 mg, 58%). MS ESI-
+
HRMS m/z [M+Na]+ calcd for C74H62NaO21 1309.3676,
Rf: 0.28 (AcOEt/MeOH 95:5). [α] +67 (c 1, CHCl3). ESI-
D
1
HRMS m/z [M+Na]+ calculated for C35H46NaO9: 633.3040,
found 1309.3656. H NMR (300 MHz, CDCl3) ꢣ 8.31 – 7.17
1
(m, 40H, Har), 6.34 – 6.14 (m, 2H, 2 × H4 Man), 6.12 – 5.93 (m,
2H, 2 × H3 Man), 5.88 – 5.80 (m, 2H, 2 × H2 Man), 5.26 (2d, J =
1.7 Hz, 2 × H1 Man), 4.89 – 4.77 (m, 4H, 4 × H6 Man), 4.74 –
4.55 (m, 6H, 2 × H6 Man, 2 × H5 Man), 4.50 (app. dd, J = 2.3, 0.8
Hz, 2H, CH2 propargyl), 4.30 – 4.17 (m, 1H, CH), 4.16 – 4.01
(m, 2H, CH2), 3.92 – 3.77 (m, 2H, CH ), 2.73 (t, J = 2.4 Hz,
1H, H alkyne). 13C NMR (75 MHz, CDCl3) ꢣ 166.2 – 165.3 (8
× CO Bz), 133.4 – 128.3 (40 × Car, 8 × Cq ar), 98.2 (C1 Man),
97.7 (C1 Man), 79.8 (-CꢤCH), 76.3 (CH), 75.5 (-C≡CH), 70.4
(C2 Man), 70.4 (C2 Man), 70.13 (2 × C3 Man), 69.2 (C5 Man), 69.1
(C5 Man), 67.8 (CH2), 67.3 (CH2), 66.8 (C4 Man), 66.76 (C4 Man),
62.8 (C6 Man), 62.8 (C6 Man), 58.4 (CH2 propargyl).
found: 633.3055. H NMR (300 MHz, CDCl3) ꢣ 7.41 (d, J =
7.1 Hz, 6H, Hortho Trt), 7.30 – 7.09 (m, 9H, Har Trt), 4.90 (d, J
= 3.3 Hz, 1H, H1), 3.86 (t, J = 5.7 Hz, 2H, -CH2-O-), 3.81 –
3.60 (m, 7H, 2 × HO-CH2-, -CHaHb-O-, -CH2-O-), 3.58 (m,
1H, H5), 3.47 (m, 1H, H3), 3.42 – 3.18 (m, 9H, H2, H4, H6b, O-
CH3, HO-CH2-, -CHaHb-O-), 3.02 (dd, J = 10.1, 4.1 Hz, 1H,
H6a), 2.68 (br s, 3H, OH), 1.87 – 1.68 (m, 4H, 2 × -CH2-),
1.37 (m, 2H, -CH2-). 13C NMR (75 MHz, CDCl3) ꢣ 143.99 (3
× Cq ar Trt), 128.92 (6 × Car Trt), 127.98 (6 × Car Trt), 127.20
(3 × Car Trt), 97.13 (C1), 86.48 (Cq Trt), 81.42 (C3), 80.49
(C2), 78.73 (C4), 72.39 (-CH2-O-), 71.07 (-CH2-O-), 70.40
(C5), 69.26 (-CH2-O-), 62.26 (C6), 61.30 (HO-CH2-), 61.12
(HO-CH2-), 60.85 (HO-CH2-), 55.12 (O-CH3), 32.77(-CH2-),
32.61 (-CH2-), 32.37(-CH2-).
Methyl
2,3,4-tri-O-allyl-6-O-trityl-α-D-glucopyranoside
(8): To a solution of methyl 6-O-trityl-α-D-glucopyranoside 7
(9.55 g, 21.88 mmol, 1 equiv.) in dry DMF (200 mL) were
added portionwise under argon allyl bromide (8.52 mL, 98.46
mmol, 4.5 equiv.) and sodium hydride (3.28 g, 82.04 mmol,
3.75 equiv.). Then, the mixture was heated at 60°C for 1 hour.
The excess of sodium hydride was quenched with methanol
(100 mL) and the solution was concentrated. The residue was
dissolved in CH2Cl2 (300 mL) and washed with water (3 ×
100 mL). The aqueous layers were combined and washed
with CH2Cl2. The organic layers were combined, dried over
magnesium sulfate and concentrated. The residue was purified
by column chromatography on silica gel (CyHex/AcOEt 95:5
then 90:10) to give 8 (8.13g, 67%), as a colourless oil. Rf:
Methyl
2,3,4-tri-O-(3-azidopropyl)-6-O-trityl-α-D-
glucopyranoside (11): To a solution of 9 (8.52 g, 13.95
mmol, 1 equiv.) in dry CH2Cl2 (100 mL) at 0°C were added
under argon triethylamine (7.9 mL, 55.81 mmol, 4 equiv.) and
slowly mesyl chloride (4.1 mL, 50.22 mmol, 3.6 equiv.). The
solution was stirred at 0°C for 1 hour, diluted with CH2Cl2
(100 mL) and washed with water (50 mL). The organic layer
was dried over MgSO4 and concentrated. The residue 10 was
dissolved in dry DMF (80 mL) in the presence of sodium
azide (9.07 g, 139.5 mmol, 10 equiv.) and the solution was
heated at 65°C overnight. The mixture was diluted in water
(300 mL), then extracted with CH2Cl2 (3×100 mL). The
organic layer was dried over MgSO4 and concentrated. The
residue was purified by column chromatography on silica gel
(CyHex/AcOEt 90:10 then 80:20) to give 11 (6.65g, 69%) as
0.89 (CyHex/AcOEt 80:20). [α] +62 (c 1, CHCl3). ESI-
D
HRMS m/z [M+Na]+ calculated for C35H40NaO6: 579.2723,
found: 579.2737. 1H NMR (300 MHz, CDCl3) ꢣ 7.64 – 7.50
(m, 6H, Hortho Trt), 7.42 – 7.20 (m, 9H, Har Trt), 6.13 – 5.92
(m, 2H, H All), 5.72 – 5.52 (m, 1H, H All), 5.45 – 5.28 (m,
4H, H All), 5.28 – 5.17 (m, 2H, H All), 5.11 – 4.99 (m, 2H, H
All), 4.94 (d, J = 3.6 Hz, 1H, H1), 4.47 – 4.14 (m, 5H, 2 ×
CH2-O-, -CHaHbO-), 3.90 – 3.80 (m, 1H, -CHaHbO-), 3.80 –
3.72 (m, 2H, H3, H5), 3.62 – 3.43 (m, 6H, OCH3, H2, H4, H6a),
3.19 (dd, J = 10.0, 4.4 Hz, 1H, H6b). 13C NMR (75 MHz,
a white syrup. Rf: 0.16 (CyHex/AcOEt 90:10). [α] +51 (c 1,
D
CHCl3). ESI-HRMS m/z [M+Na]+ calculated for
C35H43N9NaO6: 708.3234, found: 708.3261. 1H NMR (300
MHz, CDCl3) ꢣ 7.40 (dt, J = 8.6, 2.0 Hz, 6H, Har), 7.28 – 7.09
(m, 9H, Har), 4.83 (d, J = 3.5 Hz, 1H, H1), 3.77 (dt, J = 9.7,
6.2 Hz, 1H, -CHaHb-O-), 3.71 – 3.52 (m, 5H, -CHaHb-OR, -
CHaHb-O-, -CHaHb-O-, H5), 3.45 (t, J = 9.2, 1H, H3), 3.41 –
3.20 (m, 10H, H2, H4, H6a, O-CH3, 2 × N3-CHaHb-), 3.12 (ddd,
J = 9.2, 7.1, 5.3 Hz, 1H, -CHaHb-OR), 3.05 – 2.91(m, 2H, H6b,
N3-CHaHb-), 2.82 (m, 1H, N3-CHaHb-), 1.87 – 1.71 (m, 4H, 2
× -CH2-), 1.43 (m, 2H, -CH2-).13C NMR (75 MHz, CDCl3) ꢣ
143.91 (3 × Cq ar Trt), 128.80 (6 × Car Trt), 127.81 (6 × Car
Trt), 127.05 (3 × Car Trt), 97.42 (C1), 86.34 (Cq Trt), 81.67
(C3), 80.85 (C4 or C2), 78.41 (C4 or C2), 70.18 (C5), 70.11 (-
CH2-O-), 69.41 (-CH2-O-), 67.72(-CH2-O-), 62.30 (C6), 54.94
(O-CH3), 48.48 (N3-CH2-), 48.30 (N3-CH2-), 48.27 (N3-CH2-),
29.83 (-CH2-), 29.57 (-CH2-), 29.50 (-CH2-).
CDCl3) ꢣ 144.05 (3 × Cq
Trt), 135.34 (H2C=CH),
ar
135.03(H2C=CH), 134.77 (H2C=CH), 128.83 (6 × Car Trt),
127.80 (6 × Car Trt), 126.97 (3 × Car Trt), 117.52 (H2C=CH),
116.90 (H2C=CH), 116.88 (H2C=CH), 98.08 (C1), 86.30 (Cq
Trt), 81.77 (C3), 79.77 (C2), 77.99 (C4), 74.55(-CH2-O-),
73.81 (-CH2-O-), 72.63 (-CH2-O-), 70.20 (C5), 62.57 (C6),
54.88 (OCH3).
Methyl
2,3,4-tri-O-(3-hydroxypropyl)-6-O-trityl-α-D-

Hz, 1H, NH), 5.64 (br t, J = 5.7 Hz, 1H, NH), 5.55 (m, 1H,
NH), 4.85 (d, J = 3.4 Hz, 1H, H1), 4.53 – 4.32 (m, 6H, 3 ×
CH2Fmoc), 4.27 – 4.13 (m, 4H, 3 × CHFmoc, H7a), 4.06 (d, J
= 16.6 Hz, 1H, H7b), 3.96 (dd, J = 10.6, 3.3 Hz, 1H, H6a), 3.91
– 3.79 (m, 4H, 2 × -CH2-O-), 3.75 – 3.51 (m, 5H, -CH2-O-,
H3, H5, H6b), 3.49 – 3.18 (m, 11H, 3 × FmocNH-CH2-, O-
CH3, H2, H4), 1.94 – 1.63 (m, 6H, 3 × -CH2-), 1.50 (s, 9H,
CH3 tBu). 13C NMR (75 MHz, CDCl3) ꢣ 169.61 (CO ester),
156.61 (2 × NHCOFmoc), 156.56 (NHCOFmoc), 144.07 –
119.97 (Car), 97.30 (C1), 81.79 (Cq tBu), 81.56 (C3), 80.11
(C2), 77.61 (C4), 71.61 (-CH2-O-), 70.64 (-CH2-O-), 70.23
(C5), 69.95 (C6), 69.24 (C7), 68.43 (-CH2-O-), 66.57
(CH2Fmoc), 66.39 (CH2Fmoc), 66.30 (CH2Fmoc), 55.08 (O-
CH3), 47.35 (CHFmoc), 47.30 (2 × CHFmoc), 39.10
(FmocNH-CH2-), 38.83(FmocNH-CH2-), 38.61 (FmocNH-
CH2-), 30.44 (-CH2-), 30.27(-CH2-), 30.21 (-CH2-), 28.17 (3
× CH3 tBu).
Methyl 2,3,4-tri-O-(3-azidopropyl)-α-D-glucopyranoside
(12) : To a solution of 11 (18.95 g, 27.64 mmol, 1 equiv.) in a
mixture CH2Cl2/MeOH 1:1 (200 mL) was added TsOH (1.57
g, 8.292 mmol, 0.3 equiv.). The solution was stirred for 18
hours and neutralized with triethylamine (1.54 mL, 11.06
mmol, 0.4 equiv.). The solution was concentrated and the
residue was purified by column chromatography on silica gel
(CyHex 100% then CyHex/AcOEt 60:40) to give 12 (9.83g,
80%) as an oil. Rf: 0.08 (CyHex/AcOEt 70: 30). [α] +78 (c 1,
D
CHCl3). ESI-HRMS m/z [M+Na]+ calculated for
C16H29N9NaO6: 466.2138, found: 466.2144. 1H NMR (300
MHz, CDCl3) ꢣ 4.72 (d, J = 3.5 Hz, 1H, H1), 3.91 – 3.53 (m,
8H, 3 × -CH2-O-, H6), 3.53 – 3.43 (m, 2H, H3, H5), 3.43 –
3.27 (m, 9H, OCH3, 3 × N3-CH2- ), 3.26 – 3.13 (m, 2H, H2,
H4), 2.04 (br s, 1H, OH), 1.89 – 1.69 (m, 6H, 3 × -CH2-).13C
NMR (75 MHz, CDCl3) ꢣ 97.54 (C1), 81.33 (C3), 80.75 (C2),
77.72 (C4), 70.69 (C5), 69.95 (-CH2-O-), 69.45 (-CH2-O-),
67.70 (-CH2-O-), 61.59 (C6), 55.15 (OCH3), 48.43 (N3-CH2-),
48.30 (N3-CH2-), 48.18 (N3-CH2-), 29.78 (-CH2-), 29.61 (-
CH2-), 29.46 (-CH2-).
Dendrimer assembly
Methyl-2,3,4-tri-O-(3-azidopropyl)-6-O-
Dendrimer Ia : To a solution of 1 (300mg, 0.2752 mmol, 1
equiv.) and 3 (105mg, 0.3303 mmol, 1.2 equiv.) in dry
CH2Cl2 (5 mL) was added DMAP (cat.). The solution was
cooled at 0°C and DCC (113mg, 0.550 mmol, 2 equiv.) was
added. The reaction was stirred for 3 hours from 0°C to room
temperature, filtered and concentrated. The residue was
purified by column chromatography on silica gel (DCM/EtOH
98 : 2) to give Ia (349mg, 91%) as a white foam. Rf: 0.42
(CH2Cl2 /EtOH 95 : 5). [α] (c = 1, CHCl3). ESI-HRMS m/z
[M+Na]+ calculated for C84H86N4NaO13S : 1413.5810,
tertbutyloxycarbonylmethyl-α-D-glucopyranoside (13) :
To a solution of 12 in toluene (120 mL) were added tert-butyl
bromoacetate (14.4 mL, 97.92 mmol,
5
equiv.),
tetrabutylammonium bromide (631 mg, 1.958 mmol, 0.1
equiv.) and an aqueous 12.5M sodium hydroxide (62 mL, 783
mmol, 40 equiv.). The reaction was stirred vigorously for 2
hours at 80°C, then diluted with AcOEt (200 mL). The
organic layer was washed with an aq 1M HCl (50 mL) and sat
aq NaHCO3 (100 mL). The organic layer was dried over
MgSO4, concentrated and the residue was purified by column
chromatography on silica gel (CyHex 100% then
CyHex/AcOEt 85:15) to give 13 (9.17g, 84%) as an oil. Rf:
1
found : 1413.5817. H NMR (300 MHz, CDCl3) ꢣ 7.74 – 7.02
(m, 39H, Har), 6.96 – 6.83 (br s, 1H, NHCO), 5.38 – 5.27 (br
s, 1H, NHFmoc), 5.27 – 5.19 (br s, 1H, NHFmoc), 5.14 – 5.00
(br s, 1H, NHFmoc), 4.65 (d, J = 3.2 Hz, 1H, H1), 4.42 – 4.21
(m, 6H, 3 × CH2Fmoc), 4.17 – 4.01 (m, 3H, 3 × CHFmoc),
3.89 (s, 2H, H7), 3.79 – 3.30 (m, 10H, 3 × -CH2-O-, H3, H5,
H6), 3.30 – 2.98 (m, 13H, 3 × FmocNH-CH2-, -OCH3, H2, H4,
H8), 2.33 (t, J = 6.3 Hz, 2H, H9), 1.77 – 1.49 (m, 6H, -CH2-).
13C NMR (75 MHz, CDCl3) ꢣ 169.51 (NHCO), 156.54 (2 ×
NHCO Fmoc), 156.47 (NHCO Fmoc), 144.58 (Cq ar), 144.02
(Cq ar), 143.98 (Cq ar), 141.32 (Cq ar), 129.55 – 119.98
(Car), 97.15 (C1), 81.33 (C3), 80.21 (C2), 77.97 (C4), 71.51 (-
CH2-O-), 70.99 (-CH2-O-), 70.57 (C7), 70.33 (-CH2-O-),
69.98 (C5), 68.42 (C6), 66.87 (Cq Trt), 66.53 (CH2 Fmoc),
66.42 (CH2 Fmoc), 66.34 (CH2 Fmoc), 55.09 (-OCH3), 47.33
(CH Fmoc), 47.27 (2 × CH Fmoc), 38.92 (FmocNH-CH2-),
38.81 (FmocNH-CH2-), 38.72 (FmocNH-CH2-), 37.67 (C8),
32.18 (C9), 30.47 (-CH2-), 30.35 (-CH2-), 29.69 (-CH2-).
0.47 (CyHex/AcOEt 70:30). [α] +59 (c 1, CHCl3). ESI-
D
HRMS m/z [M+Na]+ calculated for C22H39N9NaO8: 580.2819,
1
found: 580.2825. H NMR (300 MHz, CDCl3) ꢣ 4.80 (d, J =
3.5 Hz, 1H, H1), 4.08 (d, J = 16.5 Hz, 1H, H7a), 4.02 (d, J =
16.5 Hz, 1H, H7b), 3.91 – 3.57 (m, 9H, H5,H6, 3 × -CH2-O-),
3.56 (t, J = 9.6 Hz, 1H, H3), 3.48 – 3.33 (m, 10H, 3 × N3-
CH2-, OCH3, H4), 3.30 (dd, J = 9.6, 3.5 Hz, 1H, H2), 1.93 –
1.79 (m, 6H, 3 × -CH2-), 1.48 (s, 9H, CH3 tBu).13C NMR (75
MHz, CDCl3) ꢣ 169.31 (CO ester), 97.57 (C1), 81.54 (Cq tBu),
81.47 (C3), 80.56 (C2), 77.53 (C4), 70.11 (C5), 69.90 (-CH2-O-
), 69.77 (-CH2-O-), 69.38 (-CH2-O-), 69.16 (C7), 67.69 (C6),
55.19 (OCH3), 48.45(N3-CH2-), 48.38 (N3-CH2-), 48.20 (N3-
CH2-), 29.80 (-CH2-), 29.66 (-CH2-), 29.47 (-CH2-), 28.11 (3
× CH3 tBu).
Compound 14 : To a solution of 13 (5.65 g, 10.14 mmol, 1
equiv.) in THF (75 mL) was added triphenylphosphine (26.5
g, 101.4 mmol, 10 equiv.). The reaction was stirred for 5
hours at room temperature and iminophosphorane was
hydrolyzed by addition of water (37.5 mL). The mixture was
stirred overnight. Then, the reaction mixture was diluted with
THF until solubilization and N(Et)3 (5.63 mL, 40.56 mmol, 4
equiv.) and FmocOSu (12.31 g, 36.50 mmol, 4 equiv.) were
added. The mixture was stirred for 1 hour at room temperature
and concentrated. The residue was purified by column
Dendrimer Ib : To a solution of Ia (349 mg, 0.2509 mmol,
1 equiv.) dissolved in dry THF (20 mL) were added octan-1-
thiol (1.311 mL, 7.527 mmol, 10 equiv. / Fmoc) then DBU (4
µl, 0.02509 mmol, 0.1 equiv.). The reaction was stirred
overnight at room temperature and concentrated. The residue
was diluted in diethyl ether (50 mL) and centrifuged. The
supernatant was kept aside and the pellet was washed with
diethyl ether and centrifuged once again. The ether layers
were combined and washed with water (10 mL). The aqueous
layer is combined with the centrifuged residue then
lyophilized to get the compound Ib (170mg, 93%) as oil. ESI-
HRMS m/z [M+H]+ calculated pour C39H57N4O7S : 725.3948,
chromatography on silica gel (CyHex/AcOEt 50:50). [α] +23
D
(c 1, CHCl3). ESI-HRMS m/z [M+Na]+ calculated for
C67H75N3O14Na: 1168.5147, found: 1168.5095. 1H NMR (300
MHz, CDCl3) ꢣ 7.88 – 7.08 (m, 39H, Har), 5.74 (br t, J = 5.8

1
55.30 (13 × OCH3), 48.43 – 48.19 (27 ×N3-CH2), 37.69 (C8),
36.59 – 36.26 (12 × CONH-CH2-), 32.11 (C9), 29.79 – 29.49
(39 × -CH2-).
found : 725.3955. H NMR (300 MHz, CD3OD) ꢣ 7.49 – 7.37
(m, 6H, H ortho Trt), 7.37 – 7.18 (m, 9H, Har), 4.84 (d, J = 3.5
Hz, 1H, H1), 4.01 (s, 2H, H7), 3.92 – 3.54 (m, 9H, 3 × -CH2-
O-, H5, H6), 3.50 (t, J = 9.2 Hz, 1H, H3), 3.34 (s, 3H, OCH3)
3.32 – 3.23 (m, 2H, H2, H4), 3.20 – 3.12 (m, 2H, H8), 2.88 –
2.64 (m, 6H, 3 × H2N-CH2-), 2.43 (t, J = 6.5 Hz, 2H, H9),
1.93 – 1.53 (m, 6H, 3 × -CH2-).13C NMR (75 MHz, MeOD) ꢣ
170.91 (CO amid), 144.73 (3 × Cq ar Trt), 129.38 (6 × Car
Trt), 127.68 (6 × Car Trt), 126.60 (3 × Car Trt), 97.38 (C1),
81.29 (C3), 80.24 (C2), 77.83 (C4), 71.22 (-CH2-O-), 70.99 (-
CH2-O-), 70.17 (C5), 69.93 (C7), 68.63 (C6), 66.53 (Cq Trt),
54.28 (O-CH3), 38.84 (2 × H2N-CH2-), 38.75 (H2N-CH2)-,
37.30 (C8), 33.08 (-CH2-), 33.01 (-CH2-), 32.47 (-CH2), 31.63
(C9).
Dendrimer IIc:
A solution of Ib (177mg, 0.2451 mmol,
1 equiv.), DMAP (cat.) and 2 (442mg, 0.8824 mmol, 3.6
equiv.) dissolved in dry CH2Cl2 The solution was cooled at
0°C and DCC (303mg, 1.471 mmol, 6 equiv.) was added. The
reaction was stirred 3 hours under argon from 0°C to room
temperature, filtered and concentrated. The residue obtained
was purified by column chromatography on silica gel
(CH2Cl2/MeOH 95:5) to get IIc (312mg, 59%) as a yellow
oil. Rf : 0.57 (DCM/MeOH 95 : 5). [α] + 55 (c = 1, CHCl3).
1H NMR (300 MHz, CDCl3) ꢣ 7.46 – 7.36 (m, 6H, H ortho Trt),
7.36 – 7.18 (m, 9H, H ar Trt), 7.12 – 6.82 (m, 4H, NH), 4.84
(d, J = 3.1 Hz, 3H, 3 × H1), 4.74 (d, J = 3.1 Hz, 1H, H1), 4.15
– 4.01 (br s, 8H, 4 × H7), 4.01 – 3.51 (m, 40H, 12 × -CH2-O-,
4 × H3, 4 × H5, 4 × H6), 3.50 – 3.11 (m, 46H, 4 × OCH3, 9 ×
N3-CH2-, 3 × -NH-CH2, 4 × H2, 4 × H4, H8), 2.49 – 2.37 (t, J
= 6.3 Hz, 2H, H9), 1.99 – 1.68 (m, 24H, 12 × -CH2). 13C
NMR (75 MHz, CDCl3) ꢣ 169.71 – 169.61 (4 × CO amide),
144.59 (3 × Cq), 129.72 – 126.84 (15 × Car), 97.55 (4 × C1),
81.37 (4 × C3), 80.61 (4 × C2), 77.76 (4 × C4), 71.00 – 67.73
(12 × -CH2-O, 4 × C5, 4 × C6, 4 × C7), 66.81 (Cq Trt), 55.32
(4 × OCH3), 48.43 – 48.18 (9 × N3-CH2), 37.71 (C8), 36.67
(CONH-CH2-), 36.42 (CONH-CH2-), 36.31 (CONH-CH2-),
32.09 (C9), 29.79 – 29.49 (12 × -CH2-).
Dendrimer IIa: To a solution of Ib (94 mg, 0.1298 mmol, 1
equiv.) and 1 (467 mg, 0.4285 mmol, 3.3 equiv.) in dry
CH2Cl2 (15 mL) was added DMAP (cat.). The solution was
cooled at 0°C and DCC (161 mg, 0.7788 mmol, 6 equiv.). The
reaction was stirred for 6 hours under argon from 0°C to room
temperature, filtered and concentrated. The residue was
purified by column chromatography on silica gel
(CH2Cl2100% then CH2Cl2/EtOH 95:5) to get IIa (397mg,
78%) as a slightly yellow foam. Rf: 0.39 (DCM/EtOH 95:5).
1H NMR (300 MHz, CDCl3) ꢣ 7.70 – 7.05 (m, 87H, Har), 7.01
– 6.75 (m, 4H, NHCO), 5.54 – 5.17 (m, 9H, NHFmoc), 4.73 –
4.63 (br s, 3H, 3× H1), 4.57 (d, J = 2.8 Hz, 1H, H1), 4.40 –
4.17 (m, 18H, CH2 Fmoc), 4.17 – 3.97 (m, 9H, CH Fmoc),
3.98 – 3.88 (s, 6H, 3 × H7), 3.88 – 3.81 (s, 2H, H7), 3.79 –
3.32 (m, 46H, 3 × -CONHCH2-, 12 × -CH2-O-, 4 × H3, 4 ×
H5, 4 × H6), 3.31 – 2.94 (m, 40H, 4 × OCH3, 9 ×
FmocNHCH2-, 4 × H2, 4 × H4, H8), 2.33 (t, J = 6.3 Hz, 2H,
H9), 1.78 – 1.44 (app.s, 24H,, 12 × -CH2-).
Dendrimer IId: To a solution of IIc (58 mg, 26.66 µmol, 1
equiv.) and 4 (111 mg, 0.288 mmol, 10.8 equiv.) in dioxane
(2.5 mL) was added a solution of CuSO4, 5 H2O (30 mg,
0.120 mmol, 0.5 equiv. / azide) and sodium ascorbate (31 mg,
0.1680 mmol, 0.7 equiv. / azide) in water (0.5 mL). The
mixture was stirred overnight at room temperature and
concentrated. The residue was dissolved in CH2Cl2 (10 mL)
and washed with a aq solution of Na2EDTA 0.05M (2 × 5
mL). The organic layer was dried over MgSO4, concentrated
and the residue was purified by column chromatography on
silica gel (CH2Cl2/AcOEt/MeOH 5:4:1) to get IId (126 mg,
83%) as a white foam. Rf : 0.16 (DCM/AcOEt/MeOH 5:4:1).
Dendrimer IIIc: To a solution of IIa (154 mg, 39 µmol, 1
equiv.) dissolved in dry THF (15 mL) was added octan-1-thiol
(610 µL, 3.517 mmol, 90 equiv.) then DBU (0.6µl, 4 µmol,
0.1 equiv.). The reaction was stirred overnight at room
temperature and concentrated. The residue was taken in
diethyl ether (50 mL). After centrifugation, the supernatant
was removed and the pellet was washed with diethyl ether and
centrifuged once again. The final pellet was dried under
vacuum to get the IIb, which was immediately used in the
following step (65 mg, 86%). MALDI m/z [M+H]+ calculated
for C93H162N13O28S+ : 1941.137, found : 1941.1607). The
compound was dissolved in dry CH2Cl2 in the presence of 2
(166 mg, 0.3315 mmol, 9.9 equiv.) and DMAP (cat.). The
solution was cooled at 0°C and DCC (124 mg, 0.6028 mmol,
18 equiv.) was added. The solution was stirred overnight
under argon from 0°C to room temperature, filtered and
concentrated. The residue obtained was purified by column
chromatography on silica gel (DCM/MeOH/NH3 aq 95: 5:
[α] +71 (c = 1, CHCl3). 1H NMR (300 MHz, CDCl3) ꢣ 7.87 –
D
7.56 (br s, 9H, H Triaz), 7.32 (d, J = 7.4 Hz, 6H, Hortho Trt),
7.18 (m, 9H, H Trt), 7.05 – 6.76 (br s, 4H, NH), 5.35 – 5.10
(m, 27H, 9 × H2 Man, 9 × H3 Man, 9 × H4 Man), 4.92 (s, 9H, 9 ×
H1 Man), 4.86 – 4.71 (m, 13H, 9 × O-CHaHb-Triaz, 4 × H1 Gluc),
4.70 – 4.54 (m, 9H, 9 × O-CHaHb-Triaz), 4.53 – 4.34 (br s,
18H, 9 × Triaz-CH2-C), 4.31 – 4.17 (dd, J = 12.1, 4.0 Hz, 9H,
9 × H6a Man), 4.15 – 3.86 (m, 26H, 9 × H5 Man, 9 × H6b Man, 4 ×
H7), 3.85 – 3.42 (m, 40H, 12 × -CH2-O-, 4 × H3 Gluc, 4 × H5
Gluc, 4 × H6 Gluc), 3.41 – 3.00 (m, 28H, 4 × -OCH3, 3 ×
CONH-CH2-, 4 × H2 Gluc, 4 × H4 Gluc, H8), 2.34 (t, J = 6.3 Hz,
2H, H9), 2.23 – 2.00 (m, 78H, 12 × -CH2-, 9 × CH3 Ac, 9 ×
CH3 Ac), 1.96 (s, 27H, 9 × CH3 Ac), 1.90 (s, 27H, 9 × CH3
Ac). 13C NMR (75 MHz, CDCl3) ꢣ 170.69 – 169.67 (36 × CO
Ac, 4 × CO amid), 144.57, 129.63, 129.50, 127.96, 127.92,
127.86, 127.76, 127.69, 127.17, 126.81, 123.91 – 123.15 (9 ×
CH Triaz), 97.17 (4 × C1 Gluc), 96.90 – 96.86 (9 × C1 Man), 81.41
(4 × C3 Gluc), 80.52 (4 × C2 Gluc), 77.81 (C4 Gluc), 70.69 – 67.06
(4 × C7, 12 × -CH2-O-, 4 × C5 Gluc, 4 × C6 Gluc, 9 × C2 Man, 9
× C3 Man, 9 × C5 Man), 66.82 (Cq Trt), 65.91 (9 × C4 Man), 62.31
(9 × C6 Man), 60.68 (9 × O-CH2-Triaz), 55.26 – 55.14 (4 ×
OCH3), 47.69 – 47.35 (9 × Triaz-CH2-C), 37.65 (C8), 36.62 –
36.32 (3 × CONH-CH2-), 32.04 (C9), 31.07 – 29.67 (12 × -
1
0.5) to get the IIIc as a yellow oil (144mg, 69%). H NMR
(400 MHz, CDCl3) ꢣ 7.40 – 7.31 (d, J = 7.8 Hz, 6H, Har Trt),
7.31 – 7.13 (m, 9H, Har Trt), 7.05 – 6.79 (m, 13H, NHCO),
4.82 – 4.74 (m, 12H, 12 × H1), 4.71 (d, J = 2.7 Hz, 1H, H1),
4.06 – 3.94 (s, 26H, 13 × H7), 3.90 – 3.45 (m, 130H, 39 ×-
CH2-O-, 13 × H3, 13 × H5, 13 × H6), 3.45 – 3.30 (m, 117H,
13 × OCH3, 27 × N3-CH2-, 12 × CONHCH2-), 3.29 – 3.07
(m, 28H, 13 × H2, 13 × H4, H8), 2.38 (t, J = 6.4 Hz, 2H, H9),
2.10 – 1.60 (m, 78H, 39 × -CH2-). 13C NMR (101 MHz,
CDCl3) ꢣ 169.64 (NHCO), 144.62 (Cq ar), 129.54 (C ar Trt),
127.96 (C ar Trt), 126.81 (C ar Trt), 97.55 (13 × C1), 81.37
(13 × C3), 80.64 (13 × C2), 77.77 (13 × C4), 70.98 – 67.71
(13 × -CH2-O-, 13 × C5, 13 × C6, 13 × C7), 66.82 (Cq Trt),

CDCl3) ꢣ 165.0 – 164.2 (72 × CO Bz), 143.8 – 143.6 (9 × Cq
Triaz), 132.4 – 132.0 (Car), 128.9 – 126.9 (Car), 122.1 –
122.0 (9 × CH Triaz), 97.2 (9 × C1 Man), 96.9 (9 × C1 Man), 96.2
(4 × C1 Glc), 80.4 (4 × C3 Glc), 79.5 (4 × C2 Glc), 76.5 (4 × C4
Glc), 76.4 (CH), 69.2 – 66.8 (-CH2-O-, 18 × C3 Man, 4 × C5 Glc, 4
× C6 Glc, 4 × C7), 68.2 (18 × C5 Man), 66.8 (-CH2-O-), 66.0 (-
CH2-O-), 65.5 (18 × C4 Man), 62.9 (18 × C6 Man), 61.6 (9 × O-
CH2-Triaz), 54.2 (4 × OCH3), 46.5 – 46.10 (9 × Triaz-CH2-
C), 30.2 – 28.7 (12 × -CH2-).
CH2-), 20.89 – 20.69 (27 × CH3 Ac).
Dendrimer IIe: To a solution of IIc (32 mg, 14.7 µmol, 1
equiv.) and 5 (87 mg, 0.265 mmol, 18 equiv.) in dioxane (1.0
mL) was added a solution of CuSO4, 5 H2O (16.5 mg, 66
µmol, 0.5 equiv. / azide) and sodium ascorbate (18.4 mg, 92
µmol, 0.7 equiv. / azide) in water (0.5 mL). The reaction was
stirred overnight at room temperature at 40°C and
concentrated. The residue was dissolved in CH2Cl2 (10 mL).
The solution was washed with an aq solution of Na2EDTA
0.05M (2 × 5 mL). The organic layer was dried over MgSO4,
concentrated. The residue was purified by column
chromatography on silica gel (CH2Cl2/MeOH 95: 5) to obtain
Dendrimer IIg: To a solution of IId (20.0 mg, 3.54 µmol, 1
equiv.) in MeOH was added 1M aq sodium hydroxide (1.270
mL) The reaction was stirred for 48 hours at room
temperature, neutralized with aq 1M HCl (1.270 mL) and
concentrated. The residue was purified by column
chromatography on Sephadex G25 to give after lyophilisation
IIg (10.5 mg, 72%). MALDI m/z [M+Na]+ calculated for
C174H269N31NaO82S+: 4159.745, found : 4160.020.
1
IIe (41mg, 55%). [α]- 64 (c 1, CHCl3). H NMR (300 MHz,
CDCl3) ꢣ 7.73 – 7.58 (m, 9H, H Triaz), 7.44 – 7.35 (m, 6H, H
ortho Trt), 7.33 – 7.19 (m, 9H, H Trt), 7.09 – 6.88 (m, 4H,
NH), 5.41 – 5.24 (m, 18H, 9 × H3 Fuc, 9 × H4 Fuc), 5.22 – 5.18
(m, 9H, 9 × H1 Fuc), 5.17 – 5.09 (m, 9H, 9 × H2 Fuc), 4.90 –
4.79 (m, 12H, 9 × -O-CHaHb-Triaz, 3× H1 Gluc), 4.76 (d, J =
3.3 Hz, 1H, 1 × H1 Gluc), 4.71 – 4.59 (m, 9H, 9 × -O-CHaHb ),
4.57 – 4.40 (m, 18H, 9 × -Triaz-CH2-C), 4.30 – 4.15 (m, 9H,
9 × H5 Fuc), 4.07 – 3.99 (app. s, 8H, 4 × H7), 3.98 – 3.48 (m,
40H, 12 × -CH2-O-, 4 × H3 Gluc, 4 × H5 Gluc, 4 × H6 Gluc), 3.43
(app. s, 12H, 4 × OCH3), 3.40 – 3.06 (m, 16H, 3 × CONH-
CH2-, 4 × H2 Gluc, 4 × H4 Gluc, H8), 2.43 (t, J = 6.5 Hz, 2H, H9),
2.28 – 2.10 (app. s, 51H, 12 × -CH2-, 9 × CH3 Ac), 2.05 (s,
27H, 9 × CH3 Ac), 1.99 (s, 9 × CH3 Ac), 1.16 (d, J = 6.5 Hz,
27H, 9 × H6 Fuc). 13C NMR (75 MHz, CDCl3) ꢣ 170.62 –
169.70 (27 × CO Ac, 4 × CO Amid), 144.60 (3 × Cq ar Trt),
144.08 – 143.93 (9 × Cq Triaz), 129.54 (6 × Car Trt), 127.98
(6 × Car Trt), 126.84 (3 × Car Trt), 123.08 – 122.79 (9 × CH
Triaz), 97.23 (4 × C1 Gluc), 95.64 (9 × C1 Fuc), 81.45 (4 × C3
Gluc), 80.57 (4 × C2 Gluc), 77.27 (4 × C4 Gluc), 71.11 (9 × C4 Fuc),
70.79 – 70.09 (4 × C7, -CH2-O-), 69.98 (4 × C5 Gluc), 69.73 (4
× C6 Gluc), 69.10 (-CH2-O-), 68.04 (9 × C Fuc: C3 or C2),
67.90 (9 × C Fuc: C3 or C2), 66.93 (-CH2-O-), 66.80 (Cq Trt),
64.72 (9 × C5 Fuc), 61.21 (9 × O-CH2-Triaz), 55.28 (4 ×
OCH3), 47.39 – 47.07 (Triaz-CH2-C), 37.57 (C8), 36.9 – 36.0
(3 × CONH-CH2-) 32.07 (C9), 31.23 – 30.82 (12 × -CH2-),
20.86 (9 × CH3 Ac), 20.72 (9 × CH3 Ac), 20.68 (9 × CH3
Ac), 15.89 (9 × C6 Fuc).
Dendrimer IIh: To a solution of IIe (21.0 mg, 4.1 µmol, 1
equiv.) in MeOH (1.1 mL) was added an aq 1M sodium
hydroxide (1.100 mL). The reaction was stirred for 48 hours
at room temperature and neutralized with aq 1M HCl (1.100
mL).The residue was purified by column chromatography on
Sephadex G25 to obtain after lyophilisation IIh as white foam
1
(12.1 mg, 74%). H NMR (300 MHz, D2O) ꢣ 7.98 – 7.76 (m,
9H, CH Triaz), 7.13 (app. s, 15H, H Trt), 4.71 (app. s, 9H, 9 ×
H1 Fuc), 4.71 (app. s, 4H, 4 × H1 Gluc), 4.64 – 4.46 (m, 18H, 9 ×
-O-CH2-Triaz), 4.43 – 4.20 (m, 18H, 9 × Triaz-CH2-C), 3.98
– 2.95 (m, 121H, 12 × -CH2-O-, 3 × CONH-CH2-, 4 × OCH3,
9 × H2 Fuc, 9 × H3 Fuc, 9 × H4 Fuc, 9 × H5 Fuc, 4 × H2 Gluc, 4 × H3
Gluc, 4 × H4 Gluc, 4 × H5 Gluc, 4 × H6 Gluc, 4 × H7, H8), 2.13 –
1.87 (br. s, 18H, -CH2-), 1.72 – 1.48 (br. s, 6H, -CH2-), 1.04 –
0.86 (m, 27H, H6 Fuc). 13C NMR (75 MHz, D2O) ꢣ 171.76 –
171.70 (4 × CO amid), 144.33 – 143.96 (Cq ar Trt, 9 × Cq
Triaz), 129.11 (6 × Car Trt), 128.15 (6 × Car Trt), 127.17 (3
× Car Trt), 124.81 – 124.59 (9 × CH Triaz), 98.48 (9 × C1
Fuc), 96.88 (4 × C1 Gluc), 81.07 – 80.91 (4 × C3 Gluc), 79.46 –
79.25 (4 × C2 Gluc), 77.41 – 77.19 (4 × C4 Gluc), 71.66 (9 × C
Fuc : C2 or C3 or C4), 69.94 – 69.54 (4 × C7,12 × -CH2-O),
69.44 (9 × C Fuc: C2 or C3 or C4), 69.29 – 69.19 (4 × C5 Gluc),
67.84 (9 × C Fuc: C2 or C3 or C4), 67.18 – 67.15 (4 × C6 Gluc),
66.66 (C5 Fuc), 60.57 (9 × -O-CH2-Triaz), 54.88 (4 × OCH3),
47.20 – 47.13 (9 × Triaz-CH2-C), 36.02 (C8), 30.84 (C9),
30.19 – 29.83 (9 × -CH2-), 15.21 (9 × C6 Fuc).
Dendrimer IIi: To a solution of IIf (60.0 mg, 4.36 µmol, 1
equiv.) in MeOH (3.14 mL) was added aq 1M sodium
hydroxide (3.14 mL). The reaction mixture was stirred for 48
hours at room temperature and neutralized with aq HCl 1M
(3.14 mL) and concentrated. The residue was purified by
column chromatography on Sephadex G25 to obtain after
lyophilisation IIi as a white foam (11mg, 41%). MS MALDI
m/z [M+Na]+ calcd for C255H413N31NaO145S+ 6284.55 found
6284.92.
Dendrimer IIId: To a solution of IIIc (124 mg, 19.71 µmol,
1 equiv.) and 4 (247 mg, 0.6386 mmol, 32.4 equiv.) in
dioxane (10 mL) was added a solution of CuSO4, 5 H2O (66
mg, 0.2661 mmol, 13.5 equiv.) and sodium ascorbate (69 mg,
0.3725 mmol, 18.9 equiv.) in water (2 mL). The reaction was
stirred overnight at room temperature and concentrated. A
solution of the residue in CH2Cl2 (20 mL) was washed with
0.05M aq Na2EDTA (2× 10 mL), dried over MgSO4, filtered,
and concentrated. the residue was purified by column
chromatography on silica gel (CH2Cl2100% then
CH2Cl2/MeOH 90:10) to give IIId (202mg, 61%) as a slightly
Dendrimer IIf : To a solution of IIc (31 mg, 14.3 µmol, 1
equiv.) and 6 (330 mg, 257 µmol, 18 equiv.) in dioxane (5
mL) were added a solution of CuSO4, 5 H2O (16 mg,
64µmmol, 0.5 equiv. / azide) and sodium ascorbate (18 mg,
90 µmol, 0.7 equiv. / azide) in water (1 mL). The mixture was
stirred overnight at room temperature and concentrated. The
residue was dissolved in CH2Cl2 (10 mL) and washed with aq
Na2EDTA (2 × 5 mL). The organic layer was dried over
MgSO4, concentrated and the residue was purified by column
chromatography on silica gel (CH2Cl2/AcOEt/MeOH 5:4:1) to
get IIf (60 mg, 30%) as a white foam.13C NMR (75 MHz,
1
yellow foam. Rf: 0.50 (CH2Cl2/MeOH 90:10). H NMR (400
MHz, CDCl3) ꢣ 7.79 – 7.60 (br s, 27H, HTriaz), 7.35 (d, J =
7.7 Hz, 6H, Har Trt), 7.30 – 7.14 (m, 9H, Har Trt), 7.09 –
6.79 (m, 13H, 13H × NHCO), 5.35 – 5.14 (m, 81H, 27 × H2
Man, 27 × H3 Man, 27 × H4 Man), 4.95 (s, 27H, 27 × H1 Man), 4.88
– 4.75 (m, 54H, 27 × O-CH2a-Triaz, H1 Glc), 4.73 – 4.58 (m,
27H, O-CH2b-Triaz), 4.57 – 4.35 (br s, 54H, 27 × Triaz-CH2-

C), 4.34 – 4.21 (m, 27H, 27 × H6a Man), 4.20 – 3.91 (m, 80H,
27 × H5 Man, 27 × H6b Man, 13 × H7), 3.90 – 3.48 (m, 104H, 13
× OCH3, 13 × -CH2-O-, 13 × H3 Glc, 13 × H6 Glc), 3.48 – 3.07
(m, 54H, 13 × -CONHCH2-, 13 × H2 Glc, 13 × H4 Glc, H8),
2.32 – 2.05 (m, 242H, H9, 39 × -CH2-, 54 × CH3 Ac,), 2.03 –
2.00 (br s, 81H, 27 × CH3 Ac), 1.96 – 1.94 (br s, 81H, 27×
CH3 Ac). 13C NMR (101 MHz, CDCl3) ꢣ 170.71 – 169.71
(108 × CO Ac, 13 × NHCO), 144,61 (Cq ar Trt), 143.44 (Cq
Triaz), 129.56 – 123.30 (Car), 97.25 (13 × C1 Glc), 96.91 (27
× C1 Man), 81.46 (13 × C3 Glc), 80.58 (13 × C2 Gluc), 77.92 (13
× C4 Glc), 70.81 – 69.74 (39 × O-CH2-, 13 × C5 Glc,13 × C6 Glc,
13 × C7), 69.40 (27× C2 Man), 69.11 (27× C3 Man), 68.73 (27 ×
C5 Man), 66.95 (Cq Trt), 66.02 (27 × C4 Man), 62.38 (27 × C6
Man), 60.93 (27 × O-CH2-Triaz), 55.30 (13× OCH3), 47.52 –
47.28 (27 × Triaz-CH2-C), 37.72 (C8), 36.66 – 36.38 (12 × -
CONHCH2-), 31.17 – 30.78 (39 × -CH2-), 20.92 – 20.74 (108
× CH3 Ac).
values were calculated, from equation 2, using the values of
fitted parameters (Rhi, Rlo, A1 and A2).
Acknowledgments
This work used the platforms of the Grenoble Instruct centre
(ISBG ; UMS 3518 CNRS-CEA-UJF-EMBL), notably the MP3
and SPR platforms, with support from FRISBI (ANR-10-INSB-
05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble
Partnership for Structural Biology (PSB). V. Porkolab was
supported by a grant from la Région Rhône-Alpes. F. Fieschi
acknowledge for support from CM1102 COST Action.
Supplementary Material
Spectral data are available in supplementary document.
References and notes
Dendrimer IIIg: To a solution of IIIc (20.0 mg, 1.20 µmol)
in MeOH (1.30 mL) was added 1M aq sodium hydroxide
(1.30 mL). The reaction was stirred for 48 hours at room
temperature and neutralized with 1M aq HCl (1.30 mL) and
concentrated. The residue was purified by column
chromatography on Sephadex G25 to obtain after
lyophilisation IIIg (7.2 mg, 50%).
1
2
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7
1




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
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R
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