Effect of dendrimer generation and aglyconic linkers on the binding properties of mannosylated dendrimers prepared by a combined convergent and onion peel approach

Article abstract of DOI:10.3390/molecules23081890

An efficient study of carbohydrate-protein interactions was achieved using multivalent glycodendrimer library. Different dendrimers with varied peripheral sugar densities and linkers provided an arsenal of potential novel therapeutic agents that could be

Full text of DOI:10.3390/molecules23081890

molecules
Article
Effect of Dendrimer Generation and Aglyconic
Linkers on the Binding Properties of Mannosylated
Dendrimers Prepared by a Combined Convergent and
Onion Peel Approach
Celia Sehad 1, Tze Chieh Shiao 1, Lamyaa M. Sallam 1, Abdelkrim Azzouz 1 and
ID
René Roy 1,2,
*
ID
1
Department of Chemistry, University of Québec a Montréal, P.O. Box 8888, Succ. Centre-Ville, Montréal,
QC H3C 3P8, Canada; celia_0709@hotmail.fr (C.S.); chichi_shiao@hotmail.com (T.C.S.);
lsallam@yahoo.com (L.M.S.); azzouz.a@uqam.ca (A.A.)
2
Glycovax Pharma Inc., 424 Guy, Suite 202, Montreal, QC H3J 1S6, Canada
*
Correspondence: roy.rene@uqam.ca or rroy@glycovax.com; Tel.: +1-514-987-3000 (ext. 2546)
Academic Editor: Ashok Kakkar
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 5 July 2018; Accepted: 25 July 2018; Published: 28 July 2018
Abstract: An efficient study of carbohydrate-protein interactions was achieved using multivalent
glycodendrimer library. Different dendrimers with varied peripheral sugar densities and linkers
provided an arsenal of potential novel therapeutic agents that could be useful for better specific
action and greater binding affinities against their cognate protein receptors. Highly effective
click chemistry represents the basic method used for the synthesis of mannosylated dendrimers.
To this end, we used propargylated scaffolds of varying sugar densities ranging from 2 to 18 for
the attachment of azido mannopyranoside derivatives using copper catalyzed click cycloaddition.
Mannopyranosides with short and pegylated aglycones were used to evaluate their effects on the
kinetics of binding. The mannosylated dendrons were built using varied scaffolds toward the
accelerated and combined “onion peel” strategy These carbohydrates have been designed to fight
E. coli urinary infections, by inhibiting the formation of bacterial biofilms, thus neutralizing the
adhesion of FimH type 1 lectin present at the tip of their fimbriae against the natural multiantennary
oligomannosides of uroplakin 1a receptors expressed on uroepithelial tissues. Preliminary DLS
studies of the mannosylated dendrimers to cross- link the leguminous lectin Con A used as a model
showed their high potency as candidates to fight the E. coli adhesion and biofilm formation.
Keywords: dendrimers; mannose; click chemistry; E. coli; UTIs; FimH; DLS
1. Introduction
Modern medicine is exploring several alternative strategies to overcome the expending antibiotic
resistance problems. One of these goals is to find new ways to fight bacterial infections. Of particular
interest are the recurrent urinary tract infections (UTIs). Half of women are affected by UTIs at
least once in their lifetime. Hence, UTIs represent an important public health issue because of their
frequent occurrence. UTIs are mostly caused by uropathogenic Escherichia coli strains (UPEC) that
infest urinary epithelium through their type-1 pili (FimH). At the tips of their pili, E. coli possesses
a carbohydrate binding domain called FimH that adhere to mannosylated glycoproteins receptors
on the urinary epithelial cells [1–3]. In addition, through their quorum sensing molecules, E. coli can
infest hosts cell and forms resilient biofilms. Once formed, bacteria become mature and readily acquire
drugs resistance.
Molecules 2018, 23, 1890; doi:10.3390/molecules23081890
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Besides, as the first defense mechanism against this infectious agent, the innate immune system
exploits the structures of mannosides [ ]. On the other hand, fimbriated E. coli receptor-binding site
(FimH) consists of a specific mannose-binding pocket, with a tyrosine gate (Tyr48, Tyr137) [ ] that also
4,5
6–8
recognizes and binds to the mannosides residues of uroplakin-1a, present at the surface of urothelial
cells as a premise for bacterial infections causing cystitis and pyelonephritis. Ever since, considerable
efforts have been devoted toward the development of new synthetic antiadhesive antagonists that
could act as potent competitive inhibitors [
carrying various aglycones have been reported and their binding to FimH have been fully characterized.
Amongst these, heptyl -D-mannopyranoside (HM) as emerged as one of the strongest monovalent
FimH binders described so far [ ]. The studies supported that the hydrophobic HM’s aglycone strongly
9–13]. Since then, synthetic mono-mannopyranosides
α
3
interacted via van der Waals contacts and aromatic stacking with the tyrosine gates residues Tyr148
and Tyr137 [2,8,13].
However, monomeric carbohydrate residues interacting with proteins often occur with
low-binding affinities [14
unequivocally displayed several advantages over monomeric ones, a process commonly used by
nature to control a wide variety of cellular processes [17 19]. Hence, several glycodendrimers
have been designed to address the issue of low affinity carbohydrate-protein interactions [ 20 27].
–16]. Furthermore, multivalent carbohydrate-protein interactions have
–
5
, –
Mannosylated glycodendrimers were found to be excellent ligands against the leguminous lectin
from Canavalia ensiformis (Concanavalin A) and to be excellent ligands toward the fimbriated E. coli
K12 with nanomolar affinities [21,26,28,29]. Likewise, the glycoside cluster effect helps to enhance
avidity, selectivity and affinity of a multivalent glycoside toward a protein that possess multiple
carbohydrate recognition domains (CRDs). This phenomenon was characterized by strong stabilization
of carbohydrate-protein interactions through macroscopic cross-linking effects [17
mannosylated glycodendrimers constitute excellent lead candidates for the treatment of E. coli
infectious agents by the inhibition of bacterial adhesion and biofilm formation on cell surfaces [ 12 30].
,19]. Therefore,
5
,
,
To these aim, a new variety of mannosylated dendrimers of different sugar densities and linker’s
properties have been developed to prevent infections spreading, by inhibiting E. coli adhesion and
biofilm formations. Thus, we report herein a new class of glycodendrimers [5,20–22,25,28,31,32] that are
built on aromatics, cyclotriphosphazene and pentaerythritol cores using high yielding click chemistry
to anchor mannosylated azides onto the above alkyne-functionalized scaffolds. This synthetic strategy
provides easy access to biodegradability and biocompatibility to the glycodendrimers. In addition,
by using different scaffolds at each layer rather than the identical ones commonly used, we are
providing another application of the so called “onion peel” approach. Hence, pegylated tetraethylene
glycol spacers have been introduced as aglycones, because of the easy availability, water-solubility,
biocompatibility and the variable chain length accessible that should enable good interactions
with the multiple FimH binding pili present at the surface of E. coli. The relative affinities of the
pegylated mannodendrimers were studied using diffraction light scattering measurements (DLS) in
the presence of ConA, toward a better understanding of the multivalent binding interactions and
the effect of the linkers. Although ConA is a leguminous lectin, it also binds natural multiantennary
mannopyranosides that can also be advantageously replaced by synthetic monomeric derivatives
harboring hydrophobic aglycones. Hence, it constitutes an appropriate model to evaluate the relative
propensity of mannosylated dendrimers toward E. coli FimH.
2. Results and Discussion
2.1. Scaffold Syntheses
First, the syntheses of different propargylated aromatic scaffolds with valencies of 2 (
were achieved in moderate to good yields. Scaffolds and were obtained through propargylation
(propargyl bromide, K2CO3, DMF) of polyhydroxylated aromatic cores and , respectively.
The trimeric phloroglucinol was commercially available and the dimeric compound was synthesized
7) and 3 (2)
2
7
1
5
1
5

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after protection of the hydroxyl group of 5-hydroxy dimethyl isophthalate
3
with a MOM-protecting
group (MOMCl, NaH, DMF, 85%), followed by reduction of the intermediate esters of
using LiAlH4 in 86% yield (Scheme 1).
4 to afford 5
Scheme 1. Synthesis of tris-propagylated 2 and bis-propagylated 7 aromatic scaffolds.
Pentaerythritol
(propargyl bromide, DMSO/NaOH) (Scheme 2). It was accompanied by smaller quantities
of the fully tetra-propargylated scaffold 10 which was readily separated by flash chromatography.
The reaction mixture gave both and 10 in a 2:1 ratio, respectively. An extended dendron precursor 11
was then obtained from using bis(2-chloroethyl) ether under phase-transfer catalyzed conditions
8 was next chosen as template for the synthesis of the desired tris-propargylated
core
9
9
9
(PTC) (tetrabutylammonium hydrogensulfate (TBAHS), NaOH, r.t.) in 73% yield (Scheme 2).
Scheme 2. Synthesis of a tripropagylated dendron precursor 11 under PTC conditions.

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For our next scaffold, harboring six propargylated functionalities, we choose the previously
described hexachlorocyclotriphosphazene core 14 (N3P3Cl6) [28 33 35]. The choice for this dense
,
–
hexavalent core was based on previous observations by the Majoral’s group who showed that it was
biologically favorable over several other scaffolds and that it can readily form dendrimers of very high
generation [36–40]. Toward this goal, diol 12 was monopropargylated (propargyl bromide, K2CO3,
acetone, r.t., 12 h) to afford 13 (43%) (Scheme 3) which was treated with 14 under basic conditions
◦
(Cs2CO3, THF, 70 C, 17 h) to provide 15 in essentially quantitative yield (98%). The structural integrity
of the fully substituted 15 was compared to its known 1H- and 31P-NMR [28].
Scheme 3. Synthesis of a hexa-propargylated cyclotriphosphazene core 15.
2.2. Sugar Precursors
It is well known that the aglyconic moiety of a given carbohydrate can affect the binding affinity
and selectivity with lectins. This effect is achieved by either providing better accessibility of the sugar
itself into the deep binding pocket of the lectin (linker effect) or by increasing the binding interactions
network between neighboring amino acids (pharmacophore effect). Because of the high number of pili
at the surface of E. coli, we figured that the linker effect would be a dominant aspect. For comparison
purposes, we also built our mannoside residues with a short linker. Thus, known peracetylated
mannose 17 was chosen as a starting point after treatment of commercial D-mannose 16 with acetic
anhydride in pyridine (Scheme 4). Lewis acid treatment of 17 with bromoethanol in DCM (BF3-Et2O,
DCM, 40%) provided known 2-bromoethyl
α
-D-mannopyranoside 18. Nucleophilic substitution of the
-D-mannopyranoside
bromide by an azide group (NaN3, DMF, 85 ◦C, 12 h) afforded 2-azidoethyl
α
19 in 60% yield. Similarly, using monotosylated tetraethylene glycol as the aglycone (20), analogous
glycosidation of 17 gave tosylate 21 (60%), followed by displacement with azide to give extended
mannoside 22 in 90% yield.

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Scheme 4. Synthesis of azido mannoside precursors having a short (19) and a long spacer (22).
2.3. Glycodendron Syntheses
Having the di-( ) and tri-propargylated (11) scaffolds in hands, together with the two
family of short (19) and long (22) azido mannosides, we were set for the convergent syntheses
of our mannosylated dendrons using the efficacious [ ]-dipolar copper-catalyzed azide-alkyne
cycloaddition (CuAAC) (click chemistry) (Scheme 5). We first combined bis-propargylated scaffold
7
1,3
7
with elongated azido mannoside 22 under typical CuAAC conditions to obtain dimer 23 in 75% yield.
Alternatively, the tri-mannosylated dendron was accomplished by treating chloride 11 with the short
mannoside 19 under the above click conditions to provide dendron 25 (82%) having an orthogonal
chloride functionality at the focal point. Displacement of the chloride by an azide as above gave 26
in 90% yield. Final sugar deprotection using the Zemplén transesterification procedure (NaOMe,
MeOH) gave the necessary precursors 24 and 27 in quantitative yields. Completion of the reactions
were evidenced by proton NMR spectroscopy, wherein complete disappearance of the propargylic
CH signal at
7.65 ppm.
δ 2.40 ppm and the appearance of the newly formed singlet of the triazole moiety at δ
2.4. Glycodendrimer Syntheses
Multivalent binding interactions of glycodendrimers constitute a well-established strategy to
enhanced binding efficacies toward their cognate lectins. We chose to prepare several dendrimers
harboring from 2 to 18 mannopyranoside moieties to be evaluated as inhibitors against E. coli
biofilm formation by using the highly efficient click chemistry. Azide-ending mannoside 22 was
anchored to bis-propargylated aromatic scaffold
, tetrapropargylated scaffold 10 (Scheme 6) and hexa-propargylated phosphorus core 15 (Scheme 7)
using CuAAC click reaction to afford 23 28 30 and 37 in good yields (75%, 92%, 82% and 96%,
7 (Scheme 5), tris-propargylated aromatic scaffold
2
,
,
respectively). Completion of the reaction was evidenced by proton and phosphorus NMR spectroscopy,
wherein complete disappearance of the propargylic CH signal and the appearance of the newly formed
characteristic singlet of the triazole moieties were observed. Additionally, mass spectral analysis
showed the presence of the required molecular ion peak (experimental section). All glycodendrimers
were fully deprotected using Zemplén conditions providing the desired glyco-ligands 24
, 29, 31 and
38 quantitatively.

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Scheme 5. Synthesis of dimeric (24) and trimeric (27) mannodendrons using click chemistry.
Scheme 6. Synthesis of tri- (29) and tetra-valent (31) pegylated mannosylated clusters using click
chemistry on trimer (2) and tetramer (10) scaffolds, respectively.

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Scheme 7. Synthesis of hexavalent phosphorus glycodendrimers 34
, 36 and 38 using click chemistry
on hexapropagylated core 15 and azido sugars 19, 32 and 22 respectively.
Hexameric mannosylated clusters possessing a short (19) and the long pegylated (22) azido
mannosides were anchored onto hexapropargylated phosphorus core 15 using CuAAC click reaction
to afford 34 and 38 in good yields of 91% and 96% respectively (Scheme 7). In order to compare
the role played by the sugar moiety in the subsequent bioassays, the known short 2-azidoehyl
β-D-glucopyranoside 32 [41] was also coupled to the above scaffold 15 under the same reaction
conditions to provide 34 also in an excellent 96% yield. Completion of the reactions was again readily
evidenced through their respective proton and phosphorus NMR spectroscopy, wherein complete
disappearance of the propargylic CH signal and the appearance of the newly formed characteristic
singlet of the triazole moieties are showed. Additionally, mass spectral analysis showed the presence
of the required molecular ion peak (experimental section). All clusters were fully deprotected
using Zemplén reaction (NaOMe, MeOH) to provide the desired glycosylated hexamers 34
38 quantitatively.
, 36 and
A G1 glycodendrimer 39 possessing 18 mannoside moieties was synthesized in good yield of
97%, by anchoring the azide-ending tri-valent mannosylated dendron 26 to the hexa-propargylated
phosphorus core 15 using CuAAC click reaction. Completion of the reaction was evidenced by
proton and phosphorus NMR spectroscopy, wherein complete disappearance of the propargylic
CH signal
δ 2.52 ppm and the appearance of the newly formed characteristic singlet of the triazole

Molecules 2018, 23, 1890
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moieties
δ
7.82 ppm, also the presence of one singlet phosphorus pick at
δ
9.54 ppm fully support
the homogeneity of the fully substituted cyclophosphazene core. Additionally, mass spectral analysis
showed the presence of the required molecular ion peak (experimental section). Compound 39
was fully deprotected by using Zemplén reaction providing the desired G1 glycodendrimer 40
quantitatively (Scheme 8).
Scheme 8. Synthesis of phosphorus glycodendrimer 40 having 18 α-D-mannopyranoside residues
using click chemistry and built around a cyclotriphosphazene core and a pentaerythritol scaffold at the
next generation level.
2.5. Dynamic Light Scattering (DLS) Studies
The relative ability of the above glycodendrimers to react with a homotetrameric leguminous
lectin such as Concanavalin A (ConA) from Canavalia ensiformis (jack bean) taken as a model
and rapidly forming stable cross-linked lattices was monitored using dynamic light scattering
(DLS). It was anticipated that the multivalent sugars at the peripheries of the dendrimers would
facilitate the multidirectional interactions with its associated tetrameric protein as seen before with
several other mannosylated structures [32
complexes were shown to rapidly form large aggregates as a function of time. As seen in Figure 1,
the complex resulting from the mannosylated dendrimers 24 27 29 31 38 40 and ConA resulted,
within 1~3 min to nanometer sizes clusters having sizes ranging from 1400 to 4600 nm. When using the
-D-glucopyranoside dendrimer 36, taken as negative control, no appreciable cross-linked lattice was
,42–47]. Indeed, these multivalent glycodendrimer–protein
,
,
,
,
,
β
observed. Clearly, the more highly dense mannodendrimer 40 (18 Man) reacted faster but also formed
larger size aggregates than their hexameric cluster counterparts 34 and 38. Interestingly, tetrameric
mannoside 31 formed larger aggregates faster than trimer 29 but both smaller clusters plateaued at the
same level of ~3000 nm size. Alternatively, tetramer 31 and hexamer 38 formed cross-linked lattices

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almost equally rapidly but the denser structure 38 formed larger aggregates at the end of the process.
As anticipated, hexamer 34, harboring short mannopyranoside aglycone, formed aggregates of smaller
size (2530 nm) when compared with hexamer 38 containing the tetraethyleneglycol linker (4090 nm)
(see Supplementary Materials). In addition, 38 reached its plateau faster (204 s) in comparison to 34
(230 s), indicating the higher adaptability of the longer spacer to form stable cross-linked lattices.
Figure 1. 3D DLS plots of the cross-linking kinetics of ConA in the presence of mannosylated
dendrimers 24, 27, 29, 31, 34, 38 and 40 and the glucosylated dendrimer 36 taken as negative control.
3. Materials and Methods
All reactions in organic medium were performed in standard oven dried glassware under an
inert atmosphere of nitrogen using freshly distilled solvents stored over molecular sieves. Solvents
were deoxygenated when necessary by bubbling nitrogen through the solution. All reagents were
used as supplied without prior purification and obtained from Sigma-Aldrich Chemical Co. (Toronto,
ON, Canada) Reactions were monitored by analytical thin-layer chromatography (TLC) using silica
gel 60 F254 pre-coated plates (Merck, Darmstadt, Germany) and compounds were visualized by
254 nm light and/or by dipping into a mixture of sulfuric acid and methanol in water or into a
mixture of KMin.O4 and K2CO3 in water followed by gentle warming with a heat-gun. Purifications
were performed by flash column chromatography using silica gel from Canadian Life Science (60 Å,
1
40–63
µ
m) (Peterborough, ON, Canada) with the indicated eluent. H-NMR, 13C-NMR and 31P-NMR
spectra were recorded at 300 and/or 600 MHz, 75 and/or 150 MHz and 122 and/or 243 MHz,
respectively, on a Bruker spectrometer (300 MHz and 600 MHz) (Milton, ON, Canada) and Varian

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◦
spectrometer (600 MHz) (Milton, ON, Canada). All NMR spectra were measured at 25 C in indicated
deuterated solvents. Proton and carbon chemical shifts ( ) are reported in ppm and coupling constants
δ
(J) are reported in Hertz (Hz). The resonance multiplicity in the 1H-NMR spectra are described as “s”
(singlet), “d” (doublet), “t” (triplet) and “m” (multiplet) and broad resonances are indicated by “broad.”
Residual protic solvent of CDCl3 (1H, 7.27 ppm; 13C, 77.0 ppm (central resonance of the triplet)), D2O
(1H, 4.80 ppm and 30.9 ppm for CH3 of acetone for 13C spectra), MeOD (1H, 3.30 ppm and 13C,
49.0 ppm), 85% H3PO4 was used as an external reference for 31P-NMR. Two-dimensional homonuclear
correlation 1H-1H COSY and 1H-13C HSQC experiments were used to confirm NMR peak assignments.
Letters are used to NMR assignment. Accurate mass measurements (HRMS) were performed on a
LC-MSD-TOF instrument from Agilent Technologies (Santa Clara, CA, USA) in positive electrospray
mode. Either protonated molecular ions [M + nH]n+ or adducts [M + nX]n+ (X = Na, K, NH4) were used
for empirical formula confirmation. Light Scattering-Multiangle Laser Light Scattering (LS-MALLS)
detection with performances verified with polystyrene 100 kDa and 2000 kDa were used to determine
the number-average molecular weight (Min.) and polydispersity index (MW/Min.). Calculations
were performed with Zimm Plot (model). MALDI-TOF MS data were acquired on a Bruker Microflex
LRF system (Bruker Daltonics, Billerica, MA, USA) equipped with a Compass 3.1 software platform.
Acquisitions were performed in positive ion mode. Reflector mode was utilized for samples below
5 kDa and linear mode for samples above 5 kDa. Samples were dissolved in water (1 mg/mL) and
mixed with 10 volumes of
ACN/H2O 0.1% TFA. A volume of 2
α
-cyano-4-hydroxycinnamic acid matrix prepared at 10 mg/mL with 50%
L was deposited on the target plate and dried. Representative
µ
acquisition parameters were: ion source 1, 19.5 kV; ion source 2, 18.05 kV; lens, 7.0 V; pulse ion
extraction, 240 ns; detector, 1.905 kV. Approximately 200 laser shot/spectra were obtained with a
60 Hz N2-cartridge-laser set at 337 nm and the laser intensity adjusted according to the signal intensity.
Sugar monomers were synthesized following the typical procedures found in literatures with a slight
modification as describe bellow.
Dynamic Light Scattering
Particle size distribution (DLS) was measured in water with the help of Zetasizer Nano S90 from
Malvern Instruments. Crosslinking studies were carried out in 1 mol/L phosphate buffered saline
(PBS) for the plant lectin Concavalin A (Sigma-Aldrich).
General procedure for the Cu(I) azide-alkyne cycloaddition reaction (I) (CuAAc) [28]. Solutions of alkyne
(1 equiv.) and azide (1.5 equiv. per alkyne site) were prepared in minimum amount of THF.
The resulting solutions were treated with an aqueous solution of CuSO4 (0.3 equiv. per alkyne
site) and sodium ascorbate solution in THF (0.35 equiv. per alkyne site). Biphasic mixture was then
stirred at 50 ◦C for 2 h then left at room temperature until completion of the reaction as judged by the
complete conversion of the limiting regents (alkyne). After completion of the reaction, EtOAc was
added, then Na2SO4 was added to quench the reaction by regenerating the CuSO4 5H2O. The mixture
·
was allowed to stir for 3 min then filtered before the solvent was removed under reduced pressure
and the resulting viscous oil was subjected to silica gel column chromatography using the appropriate
eluent system (0–10% MeOH in CH2Cl2) to afford the triazole products.
General procedure for the azide substitution (II) [28]. To a solution of the appropriate bromo/tosyl
derivatives (1 equiv.) in DMF was added NaN3 (1.5 equiv. per bromide). The reaction mixtures were
stirred at 80 ◦C until completion of reaction as judged by TLC. The excess solvent was next removed
under reduced pressure with heating at 60 ◦C until dryness. The residues were dissolved in EtOAc
and washed successively with water and brine. The organic layer was separated and dried on Na2SO4.
The residue was subjected to silica gel flash chromatography to afford the desired azido derivative.

Molecules 2018, 23, 1890
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General Procedure for De-O-acetylation (III) (Zemplén reaction) [28]. An acetylated glycocluster (0.1 mmol)
was dissolved in dry MeOH (3 mL) and a solution of sodium methoxide (1 M in MeOH, 0.5 equiv.) was
added. The reaction mixture was stirred at room temperature until the starting material disappeared.
The solution was neutralized by the addition of a cationic ion-exchange resin (H+), filtered and
washed with MeOH and then the solvent was removed in vacuo. The residue was lyophilized to yield
quantitatively the fully deprotected glycoclusters.
Synthesis of 2. Benzene-1,3,5-triol (1) [48] (2 g, 15.86 mmol) was dissolved in DMF (10 mL) followed by
the addition of K2CO3 (3 g, 14.87 mmol). The mixture was reflux for 30 min, propargyl bromide (8.5 mL,
79.3 mmol, 5 equiv.) was added dropwise, then stirred over night at room temperature. The residue
was dissolved in dichloromethane (DCM) and washed with water and brine. The organic layer was
dried over Na2SO4, filtered, then concentered under vacuum. The crude product was purified by silica
gel flash chromatography using 0–20% EtOAc in Hexane as eluents to afford compound
2
as a white
1
powder (2 g, 53%). Rf = 0.28 (EtOAc/Hexane, 1:4). H-NMR (300 MHz, CDCl3)
J = 2.4 Hz, 6H, b), 2.53 (t, J = 2.4 Hz, 3H, c).
δ 6.27 (s, 3H, k), 4.65 (d,
Synthesis of compound 9. To a solution of pentaerythritol 8 (2 g, 14.7 mmol) in dimethylsulfoxide (DMSO,
15 mL) were added an aqueous solution of NaOH (40 wt%, 10 mL). The solution was kept under
magnetic stirring at room temperature for 30 min Propargyl bromide (97%, 12 mL, 135.18 mmol) was
then added and the solution was kept at room temperature for an additional 24 h. Ethyl ether
was added to the reaction mixture then washed with water. The organic layer was dried over
Na2SO4. The crude product was purified by silica gel flash chromatography using 0–20% EtOAc in
Hexane as eluents to afford compound
1:4). Compound 10 was obtained as a white powder (1.2 g, 28%). Rf = 0.35 (EtOAc/Hexane, 1:4).
Tetrapropargylpentaerythritol 10 4.08 (d, J = 2.3 Hz, 8H, OCH2CCH),
49]. 1H-NMR (300 MHz, CDCl3)
9 as a colorless oil (2.2 g, 60%). Rf = 0.25 (EtOAc/Hexane,
[
δ
3.48 (s, 8H, CH2OCH2CCH), 2.42 (d, J = 2.2 Hz, 4H, OCH2CCH).
1
Tripropargylpentaerythritol
9
[
49]. H-NMR (300 MHz, CDCl3)
δ
4.13 (d, J = 2.4 Hz, 6H, OCH2CCH),
3.69 (s, 2H, CH2OH), 3.56 (s, 6H, CH2OCH2CCH), 2.42 (t, J = 2.4 Hz, 3H, CCH).
Synthesis of compound 11. A solution of pentaerythritol propargyl ether ( ) (2 g, 7.9 mmol, 1 equiv.),
9
Bu3NHSO4 (5 g, 14.7 mmol, 1.8 equiv.), Bis(2-chloroethyl) ether (22 mL, 187.61 mmol) in NaOH 50%
(30 mL), was stirred at room temperature for 24 h. DCM was added to the reaction mixture and the
organic layer was washed successfully with water and brine, then dried over anhydrous sodium
sulfate. The crude product was purified by silica gel flash chromatography using 0–20% EtOAc in
Hexane as eluents to afford compound 11 as a colorless oil (2.1 g, 73%). Rf = 0.25 (EtOAc/Hexane, 1:4).
1H-NMR (300 MHz, CDCl3)
δ 4.11 (d, J = 2.4 Hz, 6H, OCH2CCH), 3.76 (dd, J = 8.8, 3.2 Hz, 2H, CH2-Cl),
3.67–3.56 (m, 6H, OCH2-CH2), 3.52 (s, 6H, CH2OCH2CCH), 3.46 (s, 2H, CH2OCH2), 2.40 (t, J = 2.4 Hz,
3H, CCH).
Monopropagyloxyphenol (13) [50]. Hydroquinone (10.28 g, 93.37 mmol) was dissolved in acetone
(150 mL) followed by the addition of K2CO3 (15.42 g, 111.58 mmol). The mixture was reflux for 30 min,
propargyl bromide (10.28 mL, 93.50 mmol, 0.98 equiv.) was added dropwise, then stirred over night at
room temperature. Residue was dissolved in DCM (100 mL) and washed with water (3
×
50 mL) and
brine (3 50 mL). The organic layer was dried over Na2SO4, filtered and concentered under vacuum.
×
The crude product was purified by silica gel flash chromatography using 0–30% EtOAc in Hexane
as eluents to afford the mono substituted propargyl hydroquinone 13 as a brownish powder (3.11 g,
1
20.96 mmol, 43%). Rf = 0.35 (EtOAc/Hexane, 3:7). H-NMR (300 MHz, CDCl3)
δ
6.85 (d, J = 9.1 Hz, 2H,
CH).
aromatic), 6.78 (d, J = 9.1 Hz, 2H, aromatic), 4.66 (d, J = 1.4 Hz, 2H, CH2), 2.53 (t, J = 2.4 Hz, 1H,
≡

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Synthesis of 15
[
50–
55]. Cs2CO3 (3.4 g, 10.4 mmol) is added into a reaction mixture of
4-(prop-2-yn-1-yloxy)phenol (13) (6.7 mmol) and N3P3Cl6 (14) (0.6 mmol) in THF (20 mL). The mixture
◦
was heated at 40 C for 17 h, then filtered on Celite and concentrated. The pure 15 was isolated
as a white crystal yielding 98% after crystallization in EtOH. 1H-NMR (300 MHz, CDCl3)
δ
6.87 (d,
6.80 (d, J = 9.65 Hz, 6H, OC6H4O), 4.65 (d, J = 2.4 Hz, 6H, OCH2), 2.52 (t,
J = 2.4 Hz, 3H, CH). 31P-NMR (122 MHz, CDCl3) δ 9.82 (s).
J = 9.65 Hz, 6H, OC6H4O),
δ
1,2,3,4,6-Penta-O-acetyl-α/β-D-mannopyranose (17). 1,2,3,4,6-penta-O-acetyl-α/β-D-mannopyranose
was prepared according to the procedure reported [52] with a slight modification. D-Mannose 16
(3 g, 16.6 mmol), pyridine (40 mL) and acetic anhydride (32 mL, 333 mmol) were stirred at room
temperature. After stirring for 12 h, the reaction mixture was diluted with ice-water and extracted with
DCM. The combined organic layer was washed with 1 M aqueous HCl, saturated sodium bicarbonate
solution (NaHCO3), H2O and Brine. The organic layer was dried over Na2SO4 and the solvent was
removed under reduced pressure. The product 17 was obtained as colorless syrup (6 g, 15.4 mmol,
90%) which was a mixture of
synthetic step without any further purification. H-NMR (300 MHz, CDCl3)
α
and
β
anomers with a ratio of 3:1. This product was used in the next
1
δ
6.09 (d, J = 1.8 Hz, 3H,
H-1a), 5.86 (d, J = 1.1 Hz, 1H, H-1b), 5.49 (dd, J = 3.3, 1.1 Hz, 1H), 5.38–5.33 (m, 6H), 5.32 (d, J = 2.8 Hz,
1H), 5.26 (t, J = 2.0 Hz, 3H), 5.13 (dd, J = 10.0, 3.3 Hz, 1H), 4.30 (ddd, J = 12.4, 7.5, 5.1 Hz, 5H), 4.18–4.02
(m, 8H), 3.80 (ddd, J = 9.8, 5.3, 2.4 Hz, 1H), 2.22 (s, 3H), 2.18 (s, 9H), 2.17 (s, 9H), 2.10 (s, 3H), 2.09 (s,
12H), 2.05 (s, 12H), 2.01 (s, 12H).
2-Bromoethyl 2,3,4,6-tetra-O-acetyl-α-D-manno-pyranoside (18) [27,51]. Compound 17 (5.37 g, 13.8 mmol)
and 2-bromoethanol (0.98 mL, 13.8 mmol) were dissolved in DCM (50 mL). Then, boron trifluoride
etherate (5.8 mL, 47.2 mmol) was added to the solution and stirred under a nitrogen atmosphere
for 3 h and monitored by TLC (EtOAc/Hexane, 1:1). After addition of DCM (100 mL), the reaction
mixture was neutralized by adding saturated sodium bicarbonate solution (100 mL) and the resulting
solution was washed with water (2
×
200 mL). The combined organic layers were dried over Na2SO4,
filtered and concentrated to dryness under reduced pressure. The resulting oil was then purified using
silica gel chromatography (EtOAc/Hexane, 1:1). The relevant fractions were collected, combined
and concentrated to dryness under reduced pressure to yield 18 as a colorless powder (2.2 g, 40%).
1H-NMR (300 MHz, CDCl3)
δ 5.39–5.18 (m, 4H, H-2, 3 and 4), 4.85 (d, J = 1.6 Hz, 1H, H-1), 4.27 (dd,
J = 12.2, 5.3 Hz, 1H, H-6), 4.16–3.98 (m, 2H, H-6, H-5), 3.85 (ddd, J = 10.6, 6.6, 4.0 Hz, 1H, j), 3.72–3.60
(m, 1H, j0), 3.54–3.34 (m, 2H, CH2Br), 2.12 (s, 3H, COCH3), 2.08 (s, 3H, COCH3), 2.03 (s, 3H, COCH3),
1.97 (s, 3H, COCH3).
2-Azidoethyl 2,3,4,6-tetra-O-acetyl-α-D-manno-pyranoside (19) [27,51]. Compound 18 (1.40 g, 3.0 mmol)
and sodium azide (1.00 g, 15.4 mmol) were dissolved in anhydrous DMF (30 mL) and stirred at 80 ◦C
for 5 h. The reaction mixture was filtered and concentrated to dryness under reduced pressure and
further processed as given in general procedure (II) to afford compound 19 (0.75 g, 60%) as white
1
powder. H-NMR (300 MHz, CDCl3)
δ
5.48–5.11 (m, 3H, H-2, 3 and 4), 4.87 (d, J = 1.6 Hz, 1H, H-1),
4.29 (dd, J = 12.3, 5.3 Hz, 1H, H-6), 4.20–3.99 (m, 2H, H-60 and 5), 3.87 (m, 1H, j), 3.76–3.60 (m, 1H, j0),
3.56–3.36 (m, 2H, CH2N3), 2.16 (s, 3H, COCH3), 2.11 (s, 3H, COCH3), 2.06–2.03 (s, 3H, COCH3), 2.00 (s,
3H, COCH3).
Monotosylated tetraethylene glycol (20) [53]. To a solution of tetraethylene glycol (26.3 g, 135.4 mmol,
10 equiv.) in THF (60 mL) was added a solution of sodium hydroxide (1.79 g, 44.7 mmol, 3.3 equiv.)
◦
dissolved in water (5 mL). The mixture was cooled to 0 C and toluensulfonyl chloride (2.57 g,
16.54 mmol, 1 equiv.) in THF (5 mL) was added dropwise. The reaction was stirred at 0 ◦C for 2 h.
The solution was poured into water and the aqueous layer was extracted with dichloromethane.
The organic layers were washed with water, dried over Na2SO4, filtered and concentrated under
reduced pressure to yield 20 as a colorless oil (4.69 g, 97%) yield based on the p-toluenesulfomyl

Molecules 2018, 23, 1890
13 of 21
1
chloride. Rf = 0.25 (in 100% EtOAc). H-NMR (300 MHz, CDCl3)
δ
7.79 (d, J = 8.0 Hz, 2H, b), 7.33 (d,
J = 8.0 Hz, 2H, d), 4.16 (t, J = 4.7 Hz, 2H, a), 3.75–3.55 (m, 14H, e), 2.44 (s, 3H, c).
2-(2-{2-[2-(2-Tosyloxy-ethoxy)-ethoxy]-ethoxy}-ethyl) 2,3,4,6-tetra-O-acetyl- -D-mannopyranoside (21) [53].
α
Into a solution of pentaacetate mannose 17 (1.68 g, 4.31 mmol, 1 equiv.) in anhydrous CH2Cl2 (20 mL)
was added boron trifluoride etherate (1.23 mL, 9.93 mmol, 2.3 equiv.) at room temperature under
nitrogen atmosphere. The solution was stirred for 4 h before compound 20 (3.76 g, 10.79 mmol,
◦
2.5 equiv.) was added. Glycosylation was completed after stirring at 40 C overnight. The crude
product was washed with NaHCO3 sat, dried over Na2SO4 and concentrated under reduced pressure.
Further purification was processed on silica gel flash chromatography using 0–20% EtOAc in Hexane as
1
eluents to afford compound 21 as a colorless oil (1.8 g, 60%). Rf = 0.35 (EtOAc/Hexane, 1:4). H-NMR
(300 MHz, CDCl3) δ 7.80 (d, J = 8.3 Hz, 2H, aromatic), 7.34 (d, J = 8.0 Hz, 2H, aromatic), 5.38–5.25 (m,
3H, H-3, 4 and 2), 4.87 (d, J = 1.6 Hz, 1H, H-1), 4.36–4.19 (m, 2H, H-6), 4.18–4.01 (m, 3H, c and H-5),
3.85–3.75 (m, 1H, j’), 3.73–3.56 (m, 13H, j, i, d, g, f, e), 2.45 (s, 3H, CH3), 2.16 (d, 3H, COCH3), 2.09 (s,
3H, COCH3), 2.03 (s, 3H, COCH3), 1.99 (s, 3H, COCH3).
2-(2-{2-[2-(2-Azido-ethoxy)-ethoxy]-ethoxy}-ethyl) 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (22) [53]. To a
solution of 21 (520 mg, 0.766 mmol, 1 equiv.) in dry DMF (15 mL) under a nitrogen atmosphere were
added sodium azide (996 mg, 15.3 mmol, 20 equiv.). After stirring at 80 ◦C for 2 h, the solution was
diluted in EtOAc and further processed as given in general procedure (II) to afford compound 22 as a
1
colorless oil (380 mg, 90%), Rf = 0.4 (EtOAc/Hexane, 1:4). H-NMR (300 MHz, CDCl3)
δ
5.43–5.24 (m,
0
3H, H-3, 4 and 2), 4.87 (d, J = 1.6 Hz, 1H, H-1), 4.27 (td, J = 12.6, 5.0 Hz, 1H, H-6), 4.14–4.03 (m, 2H, H-6
and 5), 3.86–3.76 (m, 1H, j0), 3.71–3.64 (m, 13H, j, i, d, g, e, h, f), 3.39 (t, J = 5.1 Hz, 2H, CH2-N3), 2.15 (s,
3H, COCH3), 2.10 (s, 3H, COCH3), 2.04 (s, 3H, COCH3), 1.99 (s, 3H, COCH3).
Synthesis of 23. Into a solution of bispropargylated core (
mixture of THF/H2O (4 mL, 3:1, v/v), was added 2-(2-{2-[2-(2-Azido-ethoxy)-ethoxy]-ethoxy}-ethyl)
2,3,4,6-tetra-O-acetyl- -D-mannopyranoside (22) (220 mg, 0.39 mmol, 3 equiv.), Na-ascorbate (18 mg,
0.09 mmol, 0.7 equiv.) and CuSO4 5H2O (19 mg, 0.08 mmol, 0.6 equiv.). The mixture was stirred at
7) (30 mg, 0.13 mmol, 1.0 equiv.) in a
α
·
50 ◦C for 2 h, then at room temperature for 5 h. Upon completion of the reaction, EtOAc was added
to the reaction mixture and further processed as given in general procedure (I) to afford compound
23 (136 mg, 75%) as a white powder; Rf = 0.35 (with 5% MeOH in CH2Cl2 as eluents). 1H-NMR
(600 MHz, CDCl3)
δ 7.74 (s, 2H, H-triazole), 6.77 (s, 1H, HP-aromatic), 6.74 (s, 2H, HO-aromatic),
5.30 (dd, J = 10.0, 3.4 Hz, 2H, H-3), 5.25 (d, J = 9.9 Hz, 2H, H-4), 5.23–5.20 (m, 2H, H-2), 4.82 (s,
2H, H-1), 4.61 (s, 4H, b), 4.52–4.49 (m, 4H, c), 4.47 (s, 4H, m), 4.24 (dd, J = 12.2, 4.8 Hz, 2H, H-6),
4.05 (dd, J = 12.3, 4.1 Hz, 2H, H-60), 4.03–3.99 (m, 2H, H-5), 3.83 (t, J = 4.9 Hz, 4H, d), 3.77–3.73 (m,
2H, j), 3.61 (m, 2H, j0), 3.60 (s, 4H, i), 3.58 (s, 16H, g, f, e, h), 2.11 (s, 6H, COCH3), 2.05 (s, 6H, COCH3,
1.99 (s, 6H, COCH3), 1.94 (s, 6H, COCH3). 13C-NMR (150 MHz, CDCl3)
δ 170.6 (C=O), 169.9 (C=O),
169.8 (C=O), 169.6 (C=O), 156.8 (COH), 144.7 (C-triazole), 139.5 (Cm-aromatic), 123.8 (CH-triazole),
118.4 (Cp-aromatic), 113.9 (CO-aromatic), 97.5 (C-1), 71.9 (Cm), 70.5 (Cd), 70.4 (Cg), 70.3 (Cf), 69.8 (Ce),
69.4 (Ch), 69.4 (Cj), 69.3 (C-2), 68.9 (C-3), 68.2 (C-4), 67.2 (C-5), 65.9 (Ci), 63.4 (Cb), 62.2 (C-6), 50.1 (Cc),
20.8 (COCH3), 20.6 (COCH3), 20.6 (COCH3). ESI+-HRMS: [M + Na]+ calcd for C56H86N6NaO29,
1329.5331; found: 1329.5395.
Synthesis of 24. Compound 23 (50 mg, 0.036 mmol) and sodium methoxide (16 µL from 1 M solution in
MeOH) in 3 mL of methanol were stirred for 4 h and the mixture was treated following the general
procedure (III) described above. Deprotected compound 24 (38 mg, quant.) was obtained as a colorless
solid. 1H-NMR (600 MHz, D2O)
δ
8.04 (s, 2H), 6.89 (s, 1H), 6.82 (s, 2H), 4.84 (s, 2H), 4.70 (s, 4H),
4.66–4.59 (m, 4H), 4.57 (s, 4H), 3.98–3.94 (m, 4H), 3.93–3.80 (m, 4H), 3.80–3.72 (m, 4H), 3.67–3.60 (m,
12H), 3.60–3.55 (m, 8H), 3.55–3.52 (m, 4H). 13C-NMR (150 MHz, D2O)
215.4, 155.9, 143.9, 139.3, 125.5,
δ

Molecules 2018, 23, 1890
14 of 21
120.1, 114.7, 99.8, 72.7, 71.7, 70.5, 69.9, 69.7, 69.6, 69.5, 69.4, 69.4, 68.7, 66.7, 66.3, 62.4, 60.9, 49.9, 30.2.
ESI+-HRMS: [M + Na] + calcd for C40H70N6NaO21, 993.4486; found: 993.4545.
Synthesis of 25
1.0 equiv.)
.
Into a solution of tripropargyl pentaerythritol core (11) (125 mg, 0.35 mmol,
in a mixture of THF/H2O (4 mL, 3:1, v/v), was added 2-azidoethyl
2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (19) (745 mg, 1.42 mmol, 5.1 equiv.), Na-ascorbate (70 mg,
0.2◦9 mmol, 1.1 equiv.) and CuSO4
·
5H2O (78 mg, 0.31 mmol, 0.9 equiv.). The mixture was stirred at
50 C for 2 h, then at room temperature for 5 h. Upon completion of the reaction, EtOAc was added to
the reaction mixture and further processed as given in general procedure (I) to afford compound 25
1
(480 mg, 82%) as a white powder; Rf = 0.3 (with 5% MeOH in CH2Cl2 as eluents). H-NMR (300 MHz,
CDCl3)
δ 7.70 (s, 3H, HB-triazole), 5.31–5.19 (m, 9H, H-3, 4 and 2), 4.81 (d, J = 1.3 Hz, 3H, H-1), 4.61
(m, 6 H, i), 4.59 (s, 6H, h), 4.21 (dd, J = 12.3, 5.1 Hz, 3H, H-6), 4.13 (dt, J = 10.4, 4.9 Hz, 3H, j0), 4.04 (dd,
J = 12.3, 2.3 Hz, 3H, H-60), 3.91 (dt, J = 10.6, 5.2 Hz, 3H, j), 3.74 (m, 2H, d), 3.61 (m, 7 H, H-5, f and
CH2-Cl), 3.57–3.53 (m, 2H, e), 3.49 (s, 6H, g ), 3.44 (s, 2H, g), 2.13 (s, 9H, COCH3), 2.09 (s, 9H, COCH3),
0
2.04 (s, 9H, COCH3), 1.99 (s, 9H, COCH3).
Synthesis of 26. To a solution of compound 25 (480 mg, 0.29 mmol, 1.0 equiv.) in dry DMF (2 mL) under
◦
a nitrogen atmosphere were added sodium azide (186 mg, 2.9 mmol, 10 equiv.). After stirring at 80 C
for 2 h, the solution was diluted in EtOAc and further processed as given in general procedure (II)
1
to afford compound 26 as a white powder (430 mg, 90%). H-NMR (300 MHz, CDCl3)
δ
7.65 (s, 3H,
HB-triazole), 5.28–5.09 (m, 9H, H-3, 4 and 2), 4.76 (d, J = 1.1 Hz, 3H, H-1), 4.6–4.57 (m, 6H, i), 4.53 (s,
6H, h), 4.15 (dd, J = 12.4, 5.1 Hz, 3H, H-6), 4.07 (dt, J = 10.5, 5.7 Hz, 3H, j’), 3.98 (dd, J = 12.3, 2.3 Hz, 3H,
H-60), 3.86 (dt, J = 10.4, 5.1 Hz, 3H, j), 3.63–3.59 (m, 2H, d), 3.56–3.53 (m, 5H, H-5, f), 3.49 (m, 2H, e),
3.44 (s, 6H, g0), 3.39 (s, 2H, g), 3.33–3.27 (m, 2H, c, CH2-N3), 2.07 (s, 9H, COCH3), 2.03 (s, 9H, COCH3),
1.98 (s, 9H, COCH3), 1.94 (s, 9H, COCH3). 13C-NMR (75 MHz, CDCl3)
δ 170.6 (C=O), 170.0 (C=O),
169.6 (C=O), 169.1 (C=O), 145.6 (C-triazole), 123.7 (CHB-triazole), 97.6 (C-1), 77.2 (Cd), 71.0 (Cf), 70.3 (Ce),
70.0 (Cg), 69.2 (C-2), 69.1 (C-3), 68.9 (C-4), 66.3 (C-5), 65.7 (Cj), 64.8 (Ch), 62.2 (C-6), 51.8 (Ci), 50.8 (Cc),
49.5 (Cq), 20.8 (COCH3), 20.7 (COCH3).
Synthesis of 27. Compound 26 (200 mg, 0.02 mmol) and sodium methoxide (16 µL from 1 M solution in
MeOH) in 3 mL of methanol were stirred for 4 h and the mixture was treated following the general
procedure (III) described above. Deprotected compound 27 (150 mg, quant.) was obtained as a white
1
powder. H-NMR (300 MHz, MeOD)
δ
8.00 (s, 3H), 4.69–4.60 (m, 9H), 4.55 (s, 6H), 4.11 (dt, J = 10.3,
4.9 Hz, 3H), 3.88 (dt, J = 5.8, 4.6 Hz, 3H), 3.81–3.71 (m, 6H), 3.65 (ddd, J = 18.4, 7.4, 5.7 Hz, 18H),
3.58–3.49 (m, 4H), 3.46 (s, 6H), 3.40 (dd, J = 9.5, 4.3 Hz, 6H), 3.21 (dd, J = 7.6, 5.1 Hz, 4H). 13C-NMR
(75 MHz, MeOD) δ 146.08, 126.31, 101.33, 79.41, 74.58, 72.26, 72.06, 71.69, 71.35, 70.99, 70.1, 68.1, 66.9,
65.2, 62.4, 51.8, 51.4, 50.1, 49.9, 49.6, 49.3, 49.0, 49.0, 48.7, 48.4, 46.5, 31.3, 24.6. ESI+-HRMS: [M + 2H]2+
calcd for C42H72N12O23, 556.2418; found: 556.2470.
Synthesis of 28. Into a solution of trispropargylated core (
mixture of THF/H2O (4 mL, 3:1, v/v), was added 2-(2-{2-[2-(2-Azido-ethoxy)-ethoxy]-ethoxy}-ethyl)
2,3,4,6-tetra-O-acetyl- -D-mannopyranoside (22) (281 mg, 0.5 mmol, 5 equiv.), Na-ascorbate (22 mg,
0.11 mmol, 0.5 equiv.) and CuSO4 5H2O (23 mg, 0.09 mmol, 0.9 equiv.). The mixture was stirred at
2) (25 mg, 0.104 mmol, 1.0 equiv.) in a
α
·
50 ◦C for 2 h, then at room temperature for 5 h. Upon completion of the reaction, EtOAc was added
to the reaction mixture and further processed as given in general procedure (I) to afford compound
28 (188 mg, 92%) as a white powder; Rf = 0.32 (with 5% MeOH in CH2Cl2 as eluents). 1H-NMR
(600 MHz, CDCl3)
δ 7.83 (s, 3H, H-triazole), 6.28 (s, 3H, k) 5.32 (dd, J = 10.0, 2.8 Hz, 3H, H-3), 5.27
(t, J = 10.0 Hz, 3H, H-4), 5.25 (dd, J = 3.0, 1.7 Hz, 3H, H-2), 5.12 (s, 6H, b), 4.85 (d, J = 1.4 Hz, 3H,
H-1), 4.55 (t, J = 4.9 Hz, 6H, c), 4.27 (dd, J = 12.4, 5.1 Hz, 3H, H-6), 4.07 (d, J = 12.8 Hz, 3H, H-60), 4.03
(dd, J = 5.7, 3.5 Hz, 3H, H-5), 3.89 (t, J = 4.9 Hz, 6H, d), 3.82–3.73 (m, 3H, j), 3.65 (m, 3H, j0), 3.63 (s,
6H, i), 3.61 (s, 24H, g, e, f, h), 2.13 (s, 9H, COCH3), 2.07 (s, 9H, COCH3), 2.01 (s, 9H, COCH3), 1.96

Molecules 2018, 23, 1890
15 of 21
(s, 9H, COCH3). 13C-NMR (150 MHz, CDCl3)
δ
170.8 (C=O), 170.1 (C=O), 170.0 (C=O), 169.8 (C=O),
160.3 (COCb-aromatic), 143.7 (C-triazole), 124.2 (CH-triazole), 97.8 (C-1), 95.1 (Ck-aromatic), 70.8 (Cd),
70.7 (Cg), 70.6 (Cf), 70.1 (Ce), 69.7 (Ch), 69.6 (Cj), 69.5 (C-2), 69.2 (C-3), 68.5 (C-4), 67.5 (C-5), 66.2 (Ci),
62.5 (Cb), 62.1 (C-6), 50.4 (Cc), 21.0 (COCH3), 20.9 (COCH3), 20.8 (COCH3), 20.8 (COCH3). MALDI-TOF:
[M + H]+ calcd for C81H118N9O42: 1888.737; found, 1888.789.
Synthesis of 29. Compound 28 (100 mg, 0.05 mmol) and sodium methoxide (16 µL from 1 M solution
in MeOH) in 3 mL of methanol were stirred for 4 h and the mixture was treated following the
general procedure (III) described above. Deprotected compound 29 (74 mg, quant.) was obtained as
1
a white solid. H-NMR (600 MHz, MeOD)
δ
8.16 (s, 3H), 6.37 (s, 3H), 5.19 (s, 6H), 4.65–4.63 (m, 8H),
3.95–3.92 (m, 4H), 3.88–3.87 (m, 2H), 3.82 (m, 10.0, 3.1 Hz, 4H), 3.77–3.71 (m, 4H), 3.68–3.54 (m, 23H).
13C-NMR (150 MHz, MeOD)
δ 159.9, 143.3, 125.4, 100.1, 95.5, 72.9, 70.9, 70.3, 69.9, 69.8, 69.8, 68.9, 66.9,
66.4, 61.1, 50.2, 48.5, 47.9, 47.9, 47.8, 47.8, 47.8. MALDI-TOF: [M]+ calcd for C57H92N9O30: 1382.592;
found, 1382.801.
Synthesis of 30
0.23 mmol, 1.0 equiv.)
2-(2-{2-[2-(2-Azido-ethoxy)-ethoxy]-ethoxy}-ethyl) 2,3,4,6-tetra-O-acetyl-
.
Into a solution of tetrapropargyl pentaerythritol core (10) (67 mg,
in a mixture of THF/H2O (4 mL, 3:1, v/v), was added
-D-mannopyranoside (22
α
)
(790 mg, 1.4 mmol, 6 equiv.), Na-ascorbate (65 mg,◦0.32 mmol, 1.4 equiv.) and CuSO4
·
5H2O (70 mg,
0.28 mmol, 1.2 equiv.). The mixture was stirred at 50 C for 2 h, then at room temperature for 5 h. Upon
completion of the reaction, EtOAc was added to the reaction mixture and further processed as given
in general procedure (I) to afford compound 30 as a white powder (484 mg, 82%). Rf = 0.27 (with 5%
1
MeOH in CH2Cl2 as eluents). H-NMR (600 MHz, CDCl3)
δ 7.68 (s, 4H, H-triazole), 5.32 (dd, J = 10.0,
3.4 Hz, 4H, H-3), 5.26 (t, J = 10.0 Hz, 4H, H-2), 5.24 (dd, J = 3.2, 1.6 Hz, 4H, H-4), 4.85 (s, 4H, H-1), 4.52 (s,
0
8H, b), 4.51 (t, J = 5.5 Hz, 8H, c), 4.27 (dd, J = 12.2, 4.9 Hz, 4H, H-6), 4.07 (dd, J = 12.2, 2.2 Hz, 4H, H-6 ),
4.04 (ddd, J = 9.8, 4.8, 2.3 Hz, 4H, H-5), 3.87 (t, J = 5.3 Hz, 8H, d), 3.81–3.75 (m, 4H, j), 3.66 (dd, J = 5.8,
3.7 Hz, 4H, j0), 3.64–3.63 (m, 8H, i), 3.62–3.58 (m, 32H, g, f, e, h), 3.45 (s, 8H, k), 2.13 (s, 12H, COCH3),
2.08 (s, 12H, COCH3), 2.01 (s, 12H, COCH3), 1.96 (s, 12H, COCH3). 13C-NMR (150 MHz, CDCl3)
δ 170.7
(C=O), 170.0 (C=O), 169.9 (C=O), 169.7 (C=O), 145.1 (C-triazole), 123.6 (CH-triazole), 97.7 (C-1), 70.7
(Cd), 70.6 (Cg), 70.5 (Cf), 69.9 (Ce), 69.6 (Ch), 69.5 (Cj), 69.5 (C-2), 69.2 (Cq), 69.1 (C-3), 68.4 (C-4), 67.4
(C-5), 66.1 (Ci), 64.9 (Cb), 62.4 (C-6), 50.1 (Cc), 45.3 (Ck), 20.9 (COCH3), 20.8 (COCH3), 20.7 (COCH3),
20.7 (COCH3). ESI+-HRMS: [M + 2H]2+ calcd for C105H162N12O56, 1244.5021; found: 1244.0172.
Synthesis of 31. Compound 30 (100 mg, 0.038 mmol) and sodium methoxide (16 µL from 1 M solution
in MeOH) in 3 mL of methanol were stirred for 4 h and the mixture was treated following the general
procedure (III) described above. Deprotected compound 31 (70 mg, quant.) was obtained as a colorless
1
solid. H-NMR (600 MHz, D2O)
δ 8.01 (s, 4H), 4.87 (s, 4H), 4.60 (t, J = 4.9 Hz, 8H), 4.52 (s, 8H), 3.95 (d,
J = 4.8 Hz, 12H), 3.90–3.86 (m, 4H), 3.85 (dd, J = 7.4, 3.7 Hz, 4H), 3.81 (dd, J = 9.1, 3.2 Hz, 4H), 3.75 (dd,
J = 12.2, 5.4 Hz, 4H), 3.72–3.65 (m, 12H), 3.65–3.60 (m, 24H), 3.60–3.57 (m, 16H), 3.40 (s, 8H). 13C-NMR
(150 MHz, D2O)
δ 144.1, 125.3, 99.9, 72.7, 70.5, 69.9, 69.9, 69.7, 69.6, 69.5, 69.5, 68.7, 68.1, 66.7, 66.3, 63.5,
60.9, 49.9, 44.6. ESI+-HRMS: [M + 2H]2+ calcd for C73H130N12O40, 907.4248; found: 907.4258.
Synthesis of 33. Into a solution of hexapropargylated core (15) (30 mg, 0.03 mmol, 1.0 equiv.) in a mixture
of THF/H2O (4 mL, 3:1, v/v), was added 19 (152 mg, 0.29 mmol, 9.6 equiv.), Na-ascorbate (12 mg,
0.0◦6 mmol, 1.98 equiv.) and CuSO4
·
5H2O (14 mg, 0.05 mmol, 1.8 equiv.). The mixture was stirred at
50 C for 2 h, then at room temperature for 5 h. Upon completion of the reaction, EtOAc was added to
the reaction mixture and further processed as given in general procedure (I) to afford compound 33 as
1
a white powder (97 mg, 91%); Rf = 0.3 (with 5% MeOH in CH2Cl2 as eluents). H-NMR (600 MHz,
CDCl3)
δ 7.83 (s, 6H, H-triazole), 6.85 (d, J = 9.0 Hz, 12H, aromatic), 6.79 (d, J = 9.1 Hz, 12H, aromatic),
5.31–5.19 (m, 18H, H-2, 3 and 4), 5.16–5.08 (m, 12H, Ar-OCH2), 4.82 (s, 6H, H-1), 4.64–4.54 (m, 12H,
Man-OCH2), 4.21 (dd, J = 12.3, 5.1 Hz, 6H, CHH0N), 4.16–4.09 (m, 6H, H-6), 4.04 (dd, J = 12.3, 2.2 Hz,

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6H, H-60), 3.90 (dt, J = 10.4, 5.1 Hz, 6H, CHH0N), 3.65–3.58 (m, 6H, H-5), 2.13 (s, 18H, COCH3), 2.09 (s,
18H, COCH3), 1.98 (s, 18H, COCH3), 1.97 (s, 18H, COCH3). 13C-NMR (151 MHz, CDCl3)
170.8 (C=O),
δ
170.2 (C=O), 170.2 (C=O), 169.9 (C=O), 155.7 (CO-aromatic), 145.1 (C-triazole), 144.5 (Cp-aromatic),
124.5 (CH-triazole), 122.2 (Cm-aromatic), 115.7 (CO-aromatic), 97.9 (C-1), 77.2 (C-2), 69.6 (C-3), 69.5
(C-4), 69.3 (C-5), 66.6 (Cj), 66.2 (Cb), 62.7 (C-6), 50.0 (Ci), 21.1 (COCH3), 21.0 (COCH3), 20.9 (COCH3).
31P-NMR (243 MHz, CDCl3)
found: 1174.6753.
δ
9.84. ESI+-HRMS: [M + 3H]3+ calcd for C150H183N21O72P3, 1174.34752;
Synthesis of 34. Compound 33 (97 mg, 0.03 mmol) and sodium methoxide (16 µL from 1 M solution in
MeOH) in 3 mL of methanol were stirred for 4 h and the mixture was treated following the general
procedure (III) described above. Deprotected compound 34 (69 mg, quant.) was obtained as a white
1
solid. H-NMR (600 MHz, D2O)
δ
7.80 (s, 6H), 6.56 (s, 24H), 4.84 (s, 18H), 4.65 (s, 6H), 4.36 (s, 12H),
3.87 (s, 6H), 3.74 (s, 6H), 3.71–3.50 (m, 24H), 3.05 (d, J = 3.2 Hz, 6H). 13C-NMR (151 MHz, D2O)
δ 154.9,
143.8, 143.1, 125.1, 121.6, 115.5, 99.5, 72.8, 70.5, 69.9, 66.4, 65.2, 61.3, 60.6, 49.8. 31P-NMR (243 MHz,
CDCl3) δ 10.16. ESI+-HRMS: [M + 3H]3+ calcd for C102H135N21O48P3, 838.26603; found: 838.5901.
Synthesis of 35. Into a solution of hexapropargylated core (15) (50 mg, 0.049 mmol, 1.0 equiv.) in a
mixture of THF/H2O (4 mL, 3:1, v/v), was added 32 (185 mg, 0.44 mmol, 6 equiv.), Na-ascorbate
(20 mg, 0.1 mmol, 0.1 equiv.) and CuSO4
at 50 C for 2 h, then at room temperature for 5 h. Upon completion of the reaction, EtOAc was added
·
5H2O (22 mg, 0.28 mmol, 0.09 equiv.). The mixture was stirred
◦
to the reaction mixture and further processed as given in general procedure (I) to afford compound 35
1
as a white powder (171 mg, 96%); Rf = 0.2 (with 5% MeOH in CH2Cl2 as eluents). H-NMR (600 MHz,
CDCl3)
δ 7.74 (s, 6H, H-triazole), 6.92–6.74 (m, 24H, aromatic), 5.16 (t, J = 9.5 Hz, 6H, H-3), 5.11 (d,
J = 6.3 Hz, 12H, b), 5.05 (t, J = 9.7 Hz, 6H, H-4), 4.98 (dd, J = 9.6, 8.0 Hz, 6H, H-2), 4.60 (dt, J = 14.4,
4.0 Hz, 6H, j), 4.53–4.47 (m, 12H, j0, H-1), 4.24 (dt, J = 8.4, 4.5 Hz, 12H, i, H-6), 4.13 (dd, J = 12.3, 2.2 Hz,
6H, H-60), 3.93 (ddd, J = 10.6, 8.8, 3.5 Hz, 6H, i0), 3.70 (ddd, J = 10.0, 4.6, 2.4 Hz, 6H, H-5), 2.07 (s, 18H,
COCH3), 2.01 (s, 18H, COCH3), 1.97 (s, 18H, COCH3), 1.92 (s, 18H, COCH3). 13C-NMR (150 MHz,
CDCl3)
δ 169.6 (C=O), 169.1 (C=O), 168.4 (C=O), 168.4 (C=O), 154.2 (CO-aromatic), 143.6 (C-triazole),
142.7 (Cp-aromatic), 123.3 (CH-triazole), 120.9 (Cm-aromatic), 114.3 (CO-aromatic), 99.5 (C-1), 71.5 (C-2),
70.9 (C-3), 69.9 (C-4), 67.2 (C-5), 66.7 (Cj), 61.2 (Cb), 60.7 (C-6), 48.9 (Ci), 19.7 (COCH3), 19.7 (COCH3),
19.6 (COCH3), 19.5 (COCH3). 31P-NMR (243 MHz, CDCl3)
δ
9.72. MALDI-TOF: [M]+ calcd for
C150H179N21O72P3: 3519.020; found, 3519.489.
Synthesis of 36. Compound 35 (100 mg, 0.03 mmol) and sodium methoxide (16 µL from 1 M solution in
MeOH) in 3 mL of methanol were stirred for 4 h and the mixture was treated following the general
procedure (III) described above. Deprotected compound 36 (78 mg, quant.) was obtained as a white
1
powder. H-NMR (600 MHz, MeOD)
δ 8.05 (s, 6H), 6.70 (dd, J = 40.7, 9.1 Hz, 24H), 5.02 (s, 12H), 4.51 (t,
J = 5.0 Hz, 12H), 4.26 (d, J = 7.9 Hz, 6H), 4.17–4.07 (m, 6H), 4.01–3.84 (m, 6H), 3.75 (dd, J = 12.1, 1.9 Hz,
6H), 3.55 (dd, J = 12.1, 5.8 Hz, 6H), 3.31 (t, J = 9.0 Hz, 6H), 3.27–3.22 (m, 6H), 3.18 (d, J = 9.6 Hz, 6H),
3.11 (dd, J = 9.2, 8.0 Hz, 6H). 13C-NMR (150 MHz, MeOD)
δ 156.3, 145.2, 144.2, 126.5, 122.7, 116.6,
103.8, 77.3, 77.1, 74.3, 70.9, 68.9, 62.4, 62.1, 51.4, 49.4, 49.0, 24.2. 31P-NMR (243 MHz, MeOD)
MALDI-TOF: [M + H]+ calcd for C102H132N21O48P3: 2512.778; found, 2512.851.
δ 10.25.
Synthesis of 37. Into a solution of hexapropargylated core (15) (25 mg, 0.025 mmol, 1.0 equiv.) in
a mixture of THF/H2O (4 mL, 3:1, v/v), was added compound 22 (140 mg, 0.25 mmol, 10 equiv.),
Na-ascorbate (10 mg, 0.05 mmol, 2.1 equiv.) and CuSO4
The mixture was stirred at 50 C for 2 h, then at room temperature for 5 h. Upon completion of
the reaction, EtOAc was added to the reaction mixture and further processed as given in general
·
5H2O (10.8 mg, 0.04 mmol, 1.8 equiv.).
◦
procedure (I) to afford compound 37 as a white powder (101 mg, 96%); Rf = 0.22 (with 4% MeOH in
1
CH2Cl2 as eluents). H-NMR (600 MHz, CDCl3)
δ 7.85 (s, 6H, H-triazole), 6.81 (dd, J = 29.7, 9.0 Hz,
24H, aromatic), 5.33 (dd, J = 10.0, 3.4 Hz, 6H, H-3), 5.28 (t, J = 10.0 Hz, 6H, H-2), 5.25 (dd, J = 3.2,

Molecules 2018, 23, 1890
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1.6 Hz, 6H, H-4), 5.13 (d, J = 7.6 Hz, 12H, b), 4.86 (s, 6H, H-1), 4.54 (t, J = 5.1 Hz, 12H, c), 4.28 (dd,
J = 12.2, 4.9 Hz, 6H, H-6), 4.08 (dd, J = 12.3, 2.2 Hz, 6H, H-60), 4.05 (ddd, J = 9.7, 4.8, 2.3 Hz, 6H, H-5),
3.88 (t, J = 5.2 Hz, 12H, d), 3.81–3.76 (m, 6H, j), 3.67–3.65 (m, 6H, j0), 3.63 (d, J = 3.7 Hz, 12H, i), 3.61
(d, J = 7.6 Hz, 48H, g, f, e, h), 2.14 (s, 18H, COCH3), 2.09 (s, 18H, COCH3), 2.02 (s, 18H, COCH3), 1.98
(s, 18H, COCH3). 13C-NMR (150 MHz, CDCl3)
δ 170.7 (C=O), 170.0 (C=O), 169.9 (C=O), 169.7 (C=O),
155.3 (CO-aromatic), 144.6 (C-triazole), 143.6 (Cp-aromatic), 124.2 (CH-triazole), 121.8 (Cm-aromatic),
115.3 (CO-aromatic), 97.7 (C-1), 70.7 (Cd), 70.6 (Cg), 70.5 (Cf), 69.9 (Ce), 69.6 (Ch), 69.5 (Cj), 69.4 (C-2),
69.1 (C-3), 68.4 (C-4), 67.4 (C-5), 66.1 (Ci), 62.4 (Cb), 62.3 (C-6), 50.2 (Cc), 20.9 (COCH3), 20.8 (COCH3),
20.7 (COCH3), 20.7 (COCH3). 31P-NMR (243 MHz, CDCl3) 9.68. ESI+-HRMS: [M + 2H]2+ calcd for
δ
C186H254N21O90P3, 2158.2604; found: 2158.2588.
Synthesis of 38. Compound 37 (100 mg, 0.023 mmol) and sodium methoxide (16 µL from 1 M solution
in MeOH) in 3 mL of methanol were stirred for 4 h and the mixture was treated following the general
procedure (III) described above. Deprotected compound 38 (77 mg, quant.) was obtained as a white
solid. 1H-NMR (300 MHz, D2O)
δ
7.96 (s, 6H, H-triazole), 6.71 (s, 24H, aromatic), 4.99 (s, 12H, b),
0
4.82 (s, 6H, H-1), 4.49 (s, 12H, c), 3.93–3.75 (m, 36H, H-2,3,4,5,6 and 6 ), 3.74–3.38 (m, 84H, d, e, f, g, h, i,
j). 31P-NMR (122 MHz, D2O)
found: 1653.6342.
δ
10.11. ESI+-HRMS: [M + 2H]2+ calcd for C138H206N21O66P3, 1653.6321;
Synthesis of 39. Into a solution of hexapropargylated core (15) (26 mg, 0.026 mmol, 1.0 equiv.) in a
mixture of THF/H2O (4 mL, 3:1, v/v), was added (26) (400 mg, 0.24 mmol, 9.6 equiv.), Na-ascorbate
(11 mg, 0.06 mmol, 2.1 equiv.) and CuSO4 5H2O (12 mg, 0.05 mmol, 1.8 equiv.). The mixture was
·
stirred at 50 ◦C for 2 h, then at room temperature for 5 h. Upon completion of the reaction, EtOAc
was added to the reaction mixture and further processed as given in general procedure (I) to afford
compound 39 as a white powder (272 mg, 97%); Rf = 0.2 (with 5% MeOH in CH2Cl2 as eluents).
1H-NMR (600 MHz, CDCl3)
δ 7.82 (s, 6H, HA-triazole), 7.69 (s, 18H, HB-triazole), 6.82 (dd, J = 32.8,
8.8 Hz, 24H, aromatic), 5.22 (t, J = 10.0 Hz, 18H, H-3), 5.19 (d, J = 3.2 Hz, 12H, H-4), 5.17 (s, 12H, H-2),
5.09 (s, 12H, b), 4.79 (s, 18H, H-1), 4.59 (dd, J = 10.5, 5.7 Hz, 36H, i), 4.54 (s, 36H, h), 4.49 (t, J = 5.0 Hz,
0
0
12H, c), 4.19 (dd, J = 12.3, 5.0 Hz, 18H, H-6), 4.14–4.07 (m, 18H, j ), 4.02 (dd, J = 12.3, 1.8 Hz, 18H, H-6 ),
3.89 (m, 18H, j), 3.86 (t, J = 5.2 Hz, 12H, d), 3.62–3.59 (m, 18H, H-5), 3.55–3.48 (m, 24H, e, f), 3.46 (s,
36H, g0), 3.40 (s, 36H, g), 2.11 (s, 54H, COCH3), 2.06 (s, 54H, COCH3), 2.01 (s, 54H, COCH3), 1.96 (s,
54H, COCH3). 13C-NMR (150 MHz, CDCl3)
δ 170.6 (C=O), 169.9 (C=O), 169.6 (C=O), 169.1 (C=O),
155.4 (CO-aromatic), 145.5 (CB-triazole), 144.6 (CA-triazole), 143.5 (Cp-aromatic), 124.2 (CAH-triazole),
123.7 (CBH-triazole), 121.8 (Cm-aromatic), 115.3 (CO-aromatic), 97.5 (C-1), 71.0 (Cf), 70.3 (Ce), 69.7 (Cg),
69.4 (Cd), 69.2 (C-2), 69.1 (C-3), 68.9 (C-4), 68.8 (C-5), 66.3 (Cj), 65.7 (Cb), 64.8 (Ch), 62.2 (C-6), 50.3 (Ci),
49.5 (Cc), 45.3 (Cq), 20.8 (COCH3), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3). 31P-NMR (243 MHz,
CDCl3) δ 9.54. MALDI-TOF: [M + H]+ calcd for C450H607N75O222P3: 10705.772; found, 10706.833.
Synthesis of 40. Compound 39 (200 mg, 0.02 mmol) and sodium methoxide (16 µL from 1 M solution in
MeOH) in 3 mL of methanol were stirred for 4 h and the mixture was treated following the general
procedure (III) described above. Deprotected compound 40 (150 mg, quant.) was obtained as a white
1
solid. H-NMR (600 MHz, D2O)
δ
8.05 (s, 1H), 7.92 (s, 3H), 6.74 (dd, J = 42.4, 7.8 Hz, 4H), 5.09 (s, 2H),
4.75 (s, 3H), 4.61–4.51 (m, 6H), 4.45 (s, 6H), 4.02 (s, 3H), 3.83 (s, 6H), 3.71 (d, J = 10.8 Hz, 3H), 3.63 (ddd,
J = 25.4, 15.8, 7.5 Hz, 8H), 3.36 (dd, J = 75.3, 44.3 Hz, 12H), 3.11–3.02 (m, 3H). 13C-NMR (150 MHz,
D2O) δ 215.7, 154.9, 144.3, 143.8, 142.9, 125.3, 125.1, 121.7, 115.8, 99.5, 72.8, 70.5, 69.9, 69.7, 68.9, 68.7,
68.3, 66.3, 65.4, 63.6, 61.4, 60.6, 50.1, 49.9, 44.8, 30.2, 29.7, 29.6, 29.4, 29.3, 29.2. 31P-NMR (243 MHz, D2O)
δ 10.06. MALDI-TOF: [M + H]+ calcd for C306H463N75O150P3: 7681.011; found, 7681.383.

Molecules 2018, 23, 1890
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4. Conclusions
The synthesis of a new library of glycodendrimers possessing pegylated sugars in the aglycones
with different valences from 2 to 18, has been achieved in satisfying yields provided by the highly
efficient click chemistry. The above set of assay is relevant in demonstrating the relative binding
abilities of these glycodendrimers for their respective lectins. In DLS measurements, the use of
kinetics of mannodendrimers-protein aggregation in presence of the mannose-specific lectin ConA
gave useful preliminary results. We noticed that, highly substituted glycodendrimers demonstrated a
higher capacity to crosslink ConA by forming insoluble complexes within a short time frame. Hence,
the results obtained herein suggest that dendrimers harboring pegylated mannosides provided gradual
and functional binding interactions with a model mannose-binding protein such as ConA. Perhaps,
more importantly, is the fact that the synthetic strategy that we recently coined “onion peel strategy”
offers advantages over classical ones [32,47,54–57]. Indeed, by choosing the cyclotriphosphazene core
having six functional groups available (an A6 core) together with an AB3 pentaerythritol scaffold at
the next generation, provided a dendrimeric architecture from which, a total of 18 surface groups
can be achieved by a convergent synthesis in a short synthetic sequence leading to a G1 generation.
Ongoing activities, aimed at measuring the inhibition of biofilm formation from uropathogenic E. coli,
are underway.
Supplementary Materials: Supplementary materials are available online.
Author Contributions: C.S. performed the experiments, analyzed the NMR data and DLS measurements and
wrote the paper. T.C.S. and L.M.S. contributed equally to this work. A.A. co-directed the student C.S. and
proofread the paper. R.R. conceived the paper, designed the thematic, wrote and finalized the paper.
Funding: This research was funded by Natural Science and Engineering Research Council of Canada (NSERC)
and the Fonds du Québec—Nature et Technologies to R.R.
Acknowledgments: This work was supported by grants from the Natural Science and Engineering Research
Council of Canada (NSERC) to R.R. including a Canadian Research Chair and the Fonds du Québec—Nature et
Technologies to R.R.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript and in the
decision to publish the results.
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Sample Availability: Samples of the compounds are not available from the authors.
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