Unique tetrameric and hexameric mannoside clusters prepared by click chemistry

Article abstract of DOI:10.1016/j.carres.2015.07.014

The synthesis of novel tetrameric and hexameric mannoside clusters bearing 1,2,3-trizole linkages via Cu(I)-catalyzed azide-alkyne cycloaddition reaction ("click chemistry") is described. An attractive feature of these multiarmed mannoside clusters as potential inhibitors of uropathogenic Escherichia coli is the use of an aglycone whose length is designed to fit in the tyrosine gate. The acetylated mannosides were deprotected and the corresponding de-O-acetylated mannosides were found to exhibit good water solubility.

Full text of DOI:10.1016/j.carres.2015.07.014

Accepted Manuscript
Title: Unique tetrameric and hexameric mannoside clusters prepared by click
chemistry
Author: Hussein Al-Mughaid, Raed M. Al-Zoubi, Nawal K. Paul, T. Bruce
Grindley
PII:
S0008-6215(15)00218-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carres.2015.07.014
CAR 7040
To appear in:
Carbohydrate Research
Received date: 18-6-2015
Revised date: 16-7-2015
Accepted date: 19-7-2015
Please cite this article as: Hussein Al-Mughaid, Raed M. Al-Zoubi, Nawal K. Paul, T. Bruce
Grindley, Unique tetrameric and hexameric mannoside clusters prepared by click chemistry,
Carbohydrate Research (2015), http://dx.doi.org/doi:10.1016/j.carres.2015.07.014.
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Unique Tetrameric and Hexameric Mannoside Clusters Prepared by Click Chemistry
Hussein Al-Mughaida,b*, Raed M. Al-Zoubib, Nawal K. Paula, and T. Bruce Grindleya
aDepartment of Chemistry, Dalhousie University, Halifax, NS, B3H 4J3, Canada
bDepartment of Chemistry, Jordan University of Science and Technology, PO Box 3030, Irbid 22110, Jordan
Highlights



We have presented a facile synthesis of tetrameric and hexameric mannoside clusters
built on hydroquinone and 1,1,1-tris(4-hydroxyphenyl)ethane scaffolds.
The synthesis was achieved by using the copper mediated Huisgen 1,3-dipolar
cycloaddition, also referred to as click chemistry.
Attractive feature of the presented multiarmed mannoside clusters as a potential inhibitor
of uropathogenic, Escherichia coli is the use of an aglycone whose length is designed to
fit in the tyrosine gate.

The unprotected mannosides were found to exhibit good water solubility, a prerequisite
for biological activity.
Graphical Abstract
AcO
AcO
AcO
AcO
HO
O
HO
HO
HO
O
O(CH2)6N3 1.CuI/click coupling
O(CH2)6
N
N
+
phenolic
2.De-O-acetylation
N
phenolic
H
n
n
n =4, 6
n =4, 6
ABSTRACT
The synthesis of novel tetrameric and hexameric mannoside clusters bearing 1,2,3-trizole
linkages via Cu(I)-catalyzed azide-alkyne cycloaddition reaction ("click chemistry") is described. An
1
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attractive feature of these multiarmed mannoside clusters as potential inhibitors of uropathogenic
Escherichia coli is the use of an aglycone whose length is designed to fit in the tyrosine gate. The
acetylated mannosides were deprotected and the corresponding de-O-acetylated mannosides were
found to exhibit good water solubility.
Key words: -D-mannosides, click chemistry, UTIs, trizoles
*Corresponding Author: Hussein Al-Mughaid; Email: al-mughaid@just.edu.jo
Telephone: 962-2-7201000, ext-23587
Fax: 962-2-7201071
2
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1. Introduction
It has been widely accepted that the interactions between carbohydrates and their protein
receptors play a crucial biological role in cell recognition and adhesion.1 Unfortunately, the interaction
of single monosaccharide or oligosaccharides with their protein receptors is weak with dissociation
constants in the millimolar to micromolar range.2 However, the presence of multiple copies of the
carbohydrate (multivalency) strengthen the binding3,4 such that synthetic ligands can compete with the
natural multivalent ligands.
Urinary tract infections (UTIs), are one of the most common bacterial infections, affecting
millions of people around the world each year.5-7 They are mainly caused by strains of gram negative
uropathogenic bacteria, Escherichia coli (UPEC).8 The vast majority of E. coli express Type 1 fimbriae or
pili, which are hair-like protein structures present on the cell surfaces. UPEC bind to the urinary tract
endothelial surface through the 200 to 500 fimbriae which extend from the surface of each bacterial cell.
At the tip of each pili there is an adhesin protein known as FimH, a lectin that recognizes terminal -D-
mannopyranoside residues of the N-linked glycoprotein Uroplakin Ia (UPIa) on the endothelial surface,
enabling adherence and invasion of the host cell and at the same time preventing the rapid clearance of
E. coli from the UTI by the bulk flow of urine. UPIa is a membrane glycoprotein that is expressed
abundantly on superficial umbrella cells of the urinary tract on the endothelial surface.9 It was found
that; the mannose-binding pocket of FimH is adjacent to a hydrophobic area known as the tyrosine gate
that is formed by two tyrosines (Tyr48 and Tyr137) and one isoleucine (Ile52).10 In binding the UPI
oligosaccharides that are terminated by trimannosides, the tyrosine gate accommodates the non-polar
faces of the non-terminal mannopyranoses.11,12 The combination of a mannose-binding pocket with an
adjacent extended hydrophobic region on FimH has resulted in compounds that are designed to bind to
3
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this site being poorly recognized by the many other mannose-binding receptors in humans.13 Binding
occurs by a catch-bond mechanism that is critical for attachment in the high-sheer environment of the
urinary tract.14,15 Infection commences with FimH binding to UPIa that triggers a conformational change
in the cell surfaces,16 allowing the bacteria to enter and establish sub-surface colonies that are difficult
to eradicate.
Blocking the FimH–mediated bacterial adhesion is sufficient to prevent bacterial entry and
infection and a variety of α-D-mannopyranosides have been synthesized to evaluate the utility of anti-
adhesion strategies against E. coli. Firon et al. found that phenyl α-D-mannopyranosides, particularly the
2-chloro-4-nitro derivative, bound effectively,17 and recently Hultgren and coworkers have shown that
α-linked biphenyl derivatives are even more effective.18 Other phenyl glycosides with planar
substituents on the phenyl ring have also proved to be quite effective,19-21 and these compounds remain
attached to FimH surface long enough to be clinically relevant.22 In 2005, Bouckaert et al. discovered10
that heptyl α-D-mannopyranoside bound to FimH with strengths comparable to the better phenyl
glycosides.
Multivalent glycoconjugates have attracted considerable attention as anti-pathogenic agents
because of their improved biding characteristics.23 In particular, those derived from mannose have been
synthesized and investigated as inhibitors of bacterial adhesion by different research groups.24-37 We
aim to expand the repertoire of available polyvalent mannosides in order to determine which scaffolds
are most compatible with the FimH environment. We have chosen to use linker arms matching the
heptyl glycoside that binds well to FimH10,12 as others have done with different scoffolds.35 It was
thought that the open approximately spherical shape of the mannose-tipped dendrimers constructed
here would provide opportunities to binding to FimH from more bacteria than would similarly-linked
4
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mannosides that were attached to one rim of a cyclodextrin.35
The Cu(I)-catalyzed click reaction between terminal alkynes and azides to form a stable 1,4-
disubstituted trizole product with high regioselectivity in excellent yield under mild reaction conditions
has proven to be a powerful tool in the preparation of glycodendrimers.38 A number of mannose-tipped
dendrimers have been prepared using this reaction at some stage of their synthesis.24,25,28,30,31,35,37
Herein, we report the syntheses of unique tetrameric and hexameric mannoside clusters built on
hydroquinone and 1,1,1-tris(4-hydroxyphenyl)ethane scaffolds by a convergent strategy using click
chemistry.
2. Results and discussion
2.1. Synthesis of alkynyl-terminated dendritic cores 9 and 10
The synthesis of useful bromide 4 is depicted in Scheme 1. 5-Hydroxyisophthalic acid 1 was
converted to the corresponding dimethyl 5-hydroxyisophthalate 2 using a catalytic amount of H2SO4 in
refluxing methanol in 85% yield. Reduction of the diester 2 by LiAlH4 in refluxing THF provided the
known benzyl alcohol 3 in 92% yield.39 Compound 3 was then selectively O-alkylated at the phenolic
position using 1,3-dibromopropane, K2CO3, and a catalytic amount of KI in refluxing acetone to give the
corresponding bromo derivative 4 in good yield. Its 1H-NMR spectrum contained two triplets at 4.09 and
3.64 ppm for the CH2OAr and CH2Br groups, respectively. The structure was also confirmed by the
presence of peaks at 66.0 ppm for the CH2OAr group and 33.2 ppm for the CH2Br group in the 13C-NMR
spectrum. It should be noted that 1,3-dibromopropane was used in excess to avoid the formation of
undesired dimeric or oligomeric products.
5
Page 5 of 26

The tetrol 6 and hexol 8 were obtained by the reaction of phenols 5 and 7 with bromide 4 in the
presence of K2CO3 and a catalytic amount of KI in refluxing acetone in yields of 69% and 70%,
respectively (Scheme 2). The 1H-NMR spectra confirmed the structure of alkylated phenols 5 and 7 from
4 by the disappearance of the triplet at 3.64 ppm of the CH2Br group. The 13C-NMR spectra were a good
indicator for the formation of polyols 6 and 8, as they showed the appearance of a new peak around
64.0 ppm for the CH2OAr group and the disappearance of the peak at 33.2 ppm that belongs to the
CH2Br group.
6
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Finally, the propargyl ethers 9 and 10 were obtained by the reaction of compounds 6 and 8 with
propargyl bromide and NaH in dry DMF at low temperature in yields of 62% and 60%, respectively as
outlined in Scheme 3. A new signal, a narrow triplet, appeared in the 1H-NMR spectra of each of the
product propargyl ethers 9 and 10 at about 2.5 ppm corresponding to the acetylenic proton and a new
narrow doublet appeared at about 4.2 ppm corresponding to the propargylic CH2. The 13C-NMR spectra
also provided evidence for the formation of ethers 9 and 10, as they contained peaks for acetylenic
carbons at 79.7 ppm and about 74 ppm.
7
Page 7 of 26

2.2 Synthesis of 6-azidohexyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside 16
As shown in Scheme 4, the known azido mannoside 1640 was obtained in five steps from mannose.
Thus, D-mannose was converted to the corresponding peracetate derivative 12 using acetic anhydride
and a catalytic amount of I2, followed by anomeric de-O-acetylation using ammonium acetate in DMF.
Base catalyzed addition to trichloroacetonitrile afforded the acetylated imidate 14. Glycosylation using
6-chloro-1-hexanol afforded 15, which was transformed into the desired azido mannoside 16 after
sodium azide displacement of the chloride. This is the procedure followed by Öberg et al.40 but 16 was
not characterized previously. It is worth mentioning that direct glycosylation using 6-azido-1-hexanol
furnished the desired azido mannoside 16 in poor yield in agreement with Kaya et al.41
8
Page 8 of 26

2.3. Synthesis of multiarmed mannoside clusters 18 and 20 via click chemistry
Having successfully prepared mannoside 16 and the propargyl ethers 9 and 10, the next step
was to combine them via a CuI catalyzed click reaction.38,42 Thus, the cycloaddition of tetra-O-
propargyl ether 9 and azide 16 (4.2 equiv) under the promotion of sodium ascorbate, copper
sulfate pentahydrate in a mixture of THF and H2O at room temperature furnished the tetramer 17
in 86% yield, which after de-O-acetylation under Zemplén conditions gave unprotected tetramer
18 in 94% yield as shown in Scheme 5.
9
Page 9 of 26

The target hexamer 20 was also obtained by reacting azide 16 (8.8 equiv) and hexa-O-
propargyl ether 10 under the same conditions described for the synthesis of tetramer 17 in the
yield of 84%. De-O-acetylation as above furnished unprotected tetramer 20 in 95% yield as
illustrated in Scheme 6.
1
The structures of tetramer 17 and hexamer 19 was confirmed by their H NMR spectra,
which contained the characteristic trizole proton singlets at ppm for and ppm for.
In the 13C-NMR spectra, the triazole peaks appeared at 122.4and ppm for17and
9and ppm for 19, further confirming their structures. Removal of the acetates from
17 and 19 to give 18 and 20, respectively, was confirmed by the disappearance of methyl protons
in 1H-NMR spectra and the methyl carbons in 13C-NMR spectra.
The synthesis of new kinds of glycoside clusters should offer new opportunities for better
understanding of protein-carbohydrate interactions.
10
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11
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3. Conclusions
In summary, the click reaction of coupling of propargylated polyols 6 and 8 with acetyl-protected
azidohexyl mannoside 16 allowed an efficient synthesis of unique tetrameric and hexameric mannoside
clusters 17 and 19. Synthesized clusters were fully characterized by NMR spectroscopy and MALDI-TOF
MS. Deprotected clusters 18 and 20 show good water solubility and will be evaluated against urinary
tract infection in work to be presented later. We expect that these clusters, being based on a
tetrahedral carbon atom and the core, with three equivalent arms with adopt roughly spherical shapes
that are quite flexible allowing binding to FimH on pili from several bacteria.
4. Experimental Section
4.1. General methods
1H and 13C NMR spectra were recorded on a Bruker Avance 500 NMR spectrometer operating at
500.13 and 125.7 MHz respectively using the solvent resonances as secondary standards. The carbon
and hydrogen atoms were assigned following analysis of their one dimensional (1H, 13C) and two
dimensional (COSY, HSQC, and HMBC) NMR spectral data. Coupling constant (J) values are reported in
Hz. ESI high-resolution mass spectra were recorded on a Bruker Micro-TOF mass spectrometer using
electrospray ionization. MALDI mass spectra were obtained using an α-cyano-4-hydroxycinnamic acid
matrix with acquisition in the reflectron mode. Calibration was done with a peptide mixture. Melting
points were determined on a Fisher-John's melting point apparatus and are uncorrected. Acetone was
refluxed over K2CO3 and distilled over molecular sieves. Dichloromethane was first dried with calcium
chloride, and then refluxed over calcium hydride for one hour followed by distillation. Tetrahydrofuran
was refluxed over LiAlH4 and distilled over molecular sieves. N,N-Dimethylformamide (DMF) was stored
12
Page 12 of 26

over activated 4 Å molecular sieves for 72 h. It was then distilled under reduced pressure over freshly
activated 4 Å molecular sieves. Unless otherwise noted, non-aqueous reactions were carried out under a
nitrogen atmosphere. Compounds were visualized by quenching of fluorescence where applicable and/
or were located by spraying the TLC plate with a solution of 2 % ceric ammonium sulfate in 0.5 M H2SO4
followed by heating on a hot plate until color developed. Solid compounds were purified on silica gel
using flash column chromatography and specified eluents, or by crystallization. Liquids and oils were
purified using flash column chromatography.
4.2. 3,5-Bis(hydroxymethyl)-1-(3-bromopropoxy)benzene (4)
a
HO
OH
c
O
Br
d
b
To a stirred solution of 3,5-dihydroxymethylphenol (1,39 20 g, 0.13 mol) in dry acetone (800 mL)
was added anhydrous K2CO3 (72 g, 0.52 mol) and the resulting mixture was mechanically stirred for 1 h.
1,3-Dibromopropane (66 mL, 0.65 mol) was then added and the resulting reaction mixture was refluxed
for 24 h. After cooling, the solid was filtered off and the filtrate was concentrated to dryness.
Purification using column chromatography (hexanes/ EtOAc; 1: 2, RF 0.34) afforded a colorless solid (28 g,
78% yield): mp 77 – 79 °C ; 1H NMR (500.13 MHz, CD3CN) δ 2.27 (quint, J = 6 Hz, 2H, Hc), 3.23 (t, J = 6 Hz,
2H, 2OH), 3.64 (t, J = 6.5 Hz, 2H, Hd), 4.09 (t, J = 6 Hz, 2H, Hb), 4.53 (d, J = 6 Hz, 4H, Ha), 6.80 (s, 2H, PhH),
6.89 (s, 1H, PhH); 13C NMR (125.7 MHz, CD3CN) δ 160.0, 144.8, 118.3, 112.3 (PhC), 66.4 (Cb), 64.6 (Ca),
33.2 (Cd), 31.5 (Cc). HR ESI MS m/z calcd for C11H15BrNaO3 297.0097, found: 297.0094.
4.3. Bis-1,4-[(3,5-dihydroxymethylphenoxy)-3-propoxy]benzene (6)
13
Page 13 of 26

O
O
OH
OH
HO
c
d
O
O
b
HO
a
To a stirred solution of hydroquinone (1.0 g, 9.1 mmol) in dry acetone (150 mL) was added
anhydrous K2CO3 (5.0 g, 36 mmol) and the resulting mixture was stirred for 30 min. 3,5-
bis(hydroxymethyl)-1-(3-bromopropoxy)benzene 4 (5.5 g, 20 mmol) was then added and the resulting
reaction mixture was heated at reflux temperature for 24 h. The reaction mixture was allowed to cool to
rt and then filtered. The filtrate was concentrated to give a crude product that was then crystallized
from EtOAc to afford off-white solid (3.1 g, 69% yield): mp 116 - 118 °C; 1H NMR (500.13 MHz, DMSO-d6)
δ 2.13 (quint, J = 6.5 Hz, 4H, Hc), 4.05 (t, J = 6.5 Hz, 4H, Hd), 4.10 (t, J = 6.5 Hz, 4H, Hb), 4.44 (d, J = 5.5 Hz,
8H, Ha), 5.15 (t, J = 5.5 Hz, 4H, OH), 6.76 (s, 4H, PhH), 6.83 (s, 2H, PhH), 6.87 (s, 4H, PhH); 13C NMR (125.7
MHz, DMSO-d6) δ 158.4, 152.6, 143.9, 116.6, 115.4, 110.6 (PhC), 64.6, 64.1 (Cb, Cd), 62.8 (Ca), 28.8 (Cc).
HR ESI MS m/z calcd for C28H34NaO8 521.2146, found: 521.2121.
4.4. 1,1,1-Tris[4-(3-[3,5-dihydroxylmethylphenoxy]propoxy)phenyl]ethane (8)
OH
OH
OH
HO
O
O
OH
HO
a
14
O
c
O
O
b
d
O
e
Page 14 of 26

To a stirred solution of 1,1,1-tris(4-hydroxyphenyl)ethane (1.5 g, 4.9 mmol) in dry acetone (250
mL) was added anhydrous K2CO3 (4.7 g, 34 mmol) and the resulting mixture was stirred for 1 h. 3,5-
bis(hydroxymethyl)-1-(3-bromopropoxy)benzene 4 (4.9 g, 18 mmol) was then added and the resulting
reaction mixture was refluxed for 24 h. After cooling, the solid was filtered off and the filtrate was
concentrated to an oily residue. Purification using column chromatography (pure EtOAc→ 2% CH3OH in
EtOAc; RF 0.12 EtOAc) afforded 8 a pale yellow viscous oil (3.1 g, 70% yield): ): 1H NMR (500.13 MHz,
acetone-d6) δ 2.06 (s, 3H, He), 2.21 (quint, J = 6.5 Hz, 6H, Cc), 4.13 – 4.19 (m, 12H, Hb, Hd), 4.23 (t, J = 5 Hz,
6H, OH), 4.57 (d, J = 5 Hz, 12H, Ha), 6.80 – 6.98 (m, 21H, PhH); 13C NMR (125.7 MHz, acetone-d6) δ 160.0,
157.9, 144.8, 142.7, 130.4, 117.8, 114.5, 111.9 (PhC), 65.12, 65.1 (Cb, Cd), 64.6, (Ca), 51.3 (Cquat), 31.1 (Cc),
30.1 (Ce). HR ESI MS m/z calcd for C53H60NaO12 911.3977, found: 911.3954.
4.5. Bis-1,4-[(3,5-bis(propargyloxymethylphenoxy)-3-propoxy]benzene (9)
O
f
O
O
O
e
O
O
d
O
O
c
a
b
15
Page 15 of 26

To a stirred solution of the tetrol 6 (0.5 g, 1 mmol) in dry DMF (30 mL) was added NaH (0.28 g,
7.2 mmol, 60% in mineral oil) in portions at -10 °C. After 15 min propargyl bromide (1.2 g, 8 mmol, 80%
in toluene) was added, and the reaction mixture was stirred for 3 h at rt. Water (15 mL) was carefully
added and the product was extracted using Et2O (3 x 30 mL). The combined organic extracts were dried
(MgSO4) and concentrated. The resulting oily residue was purified using column chromatography
(EtOAc/ hexanes; 1:4 → 1:3, RF 0.5 in EtOAc/hexanes 1:2) to yield compound 9 as brown oil (0.4 g, 62%
yield): 1H NMR (500.13 MHz, CDCl3) δ 2.23 (quint, J = 6 Hz, 4H, He), 2.47 (t, J = 2.5 Hz, 4H, Ha), 4.10 (t, J =
6 Hz, 4H, Hd), 4.16 (t, J = 6 Hz, 4H, Hf), 4.17 (d, J = 2.5 Hz, 8H, Hb), 4.57 (s, 8H, Hc), 6.83 (s, 4H, PhH), 6.86
(s, 4H, PhH), 6.92 (s, 2H, PhH); 13C NMR (125.7 MHz, CDCl3) δ 159.4, 153.2, 139.2, 120.0, 115.6, 113.7
(PhC), 79.7 (HCC), 74.9 (HCC), 71.4 (Cc), 65.1, 64.7 (Cd, Cf), 57.3 (Cb), 29.5 (Ce). HR ESI MS m/z calcd for
C28H34NaO8 673.2772, found: 673.2764.
4.6. 1,1,1-Tris[4-(3-[3,5-dipropargyloxymethylphenoxy]propoxy)phenyl]ethane (10)
O
O
a
O
O
O
O
b
c
O
O
O
e
O
O
d
f
O
g
16
Page 16 of 26

To a stirred solution of the hexol 8 (0.55 g, 0.62 mmol) in dry DMF (60 mL) was added NaH (0.22
g, 5.6 mmol, 60% in mineral oil) in portions at -10 °C. After 15 min propargyl bromide (0.88 g, 7.4 mmol,
80% in toluene) was added, and the reaction mixture was stirred for 3 h at rt. Water (20 mL) was
carefully added and the product was extracted using Et2O (3 x 30 mL). The combined organic extracts
were dried (MgSO4) and concentrated to give an oily residue that was purified using column
chromatography (EtOAc/ hexanes; 1:2, RF 0.3) to yield 10 as a brown oil (1.2 g, 60% yield): 1H NMR
(500.13 MHz, CDCl3) δ 2.09 (s, 3H, Hg), 2.54 (quint, J = 6 Hz, 6H, He), 2.46 (t, J = 2.5 Hz, 6H, Ha), 4.11 –
4.19 (m, 24H, Hd, Hf, Hb), 4.57 (s, 12H, Hc), 6.78 – 6.80 (m, 6H, PhH), 6.86 (s, 6H, PhH), 6.92 (s, 3H, PhH),
6.96 – 6.99 (m, 6H, PhH); 13C NMR (125.7 MHz, CDCl3) δ 159.4, 156.9, 142.0, 139.2, 129.8, 120.0, 113.73,
113.7 (PhC), 79.7 (CCH), 74.9 (CCH), 71.4 (Cc), 64.6, 64.3 (Cd, Cf), 57.3 (Cb), 50.9 (Cquat), 31.2 (Cg), 29.5 (Ce).
HR ESI MS m/z calcd for C71H72NaO12 1139.5022, found: 1139.5038.
4.7. 6-Azidohexyl 2,3,4,6-tetra-O-acetyl--D-mannoside (16)
OAc
AcO
O
AcO
AcO
O
N3
To a stirred solution of 6-chlorohexyl 2,3,4,6-tetra-O-acetyl--D-mannopyranoside (6.0 g, 13
mmol) in dry DMF (100 mL) was added NaN3 (1.7 g, 26 mmol) followed by a catalytic amount of
tetrabutylammonium bromide (50 mg) and the resulting mixture was stirred at 100 ºC for 16 h. After
17
Page 17 of 26

cooling, water (30 mL) was added and the product was extracted with Et2O (3 x 40 mL). The combined
organic extracts were dried over MgSO4 and concentrated. The resulting oily residue was purified by
flash column chromatography (hexanes/ EtOAc; 2: 1, RF 0.33) to yield the title compound as a pale
yellow oil (4.5 g, 73% yield). 1H NMR: δ 1.25-1.27 (m, 4H, N3CH2(CH2)4CH2O), 1.45-1.49 (m, 4H,
N3CH2(CH2)4CH2O), 1.84, 1.90, 1.94, 2.00 (4s, 12H, 4 x COCH3), 3.14 (t, J = 7 Hz, 2H, CH2N3), 3.29-3.34 (m,
1H, OCH2), 3.53-3.57 (m, 1H, OCH2), 3.83 (m, 1H, H-5), 3.95 (dd, J6a,5 = 2.5 Hz, J6a,b = 12 Hz, 1H, H-6a), 4.12
(dd, J6b,5 = 5.5 Hz, J6a,b = 12 Hz, 1H, H-6b), 4.65 (d, J = 1.5 Hz, 1H, H-1), 5.06-5.13 (m, 3H, H-2, H-3, H-4);
13C NMR: δ 170.2, 169.7, 169.5, 169.4 (4 x C=O), 97.2 (C-1), 69.3 (C-2), 68.8(C-3), 68.1 (C-5), 67.9 (C-4),
65.9 (OCH2), 62.2 (C-6), 51.0 (CH2N3), 28.4, 28.5, 26.1, 25.4 (N3CH2(CH2)4CH2O), 20.5, 20.4, 20.3 (4 x
COCH3). HR ESI MS: m/z calculated for C20H31N3NaO10 496.1898, found 496.1895.
4.8. Tetrameric mannoside cluster (17)
18
Page 18 of 26

To a stirred solution of tetra-O-propargyl ether 9 (0.12 g, 0.18 mmol) and 6-azidohexyl 2,3,4,6-O-
acetyl-α-D-mannopyranoside 16 (0.36 g, 0.76 mmol, 4.2 equiv) in DMF (30 mL) was added sodium
ascorbate (75 mg) followed by copper(II) sulfate pentahydrate (19 mg, in 10 mL of H2O) in one portion.
The heterogeneous mixture was vigorously stirred for 24 h. Most of the DMF was then removed under
reduced pressure and CH2Cl2 (30 mL) was added. The organic layer was separated, dried (MgSO4),
filtered, and concentrated. The resulting residue was purified using column chromatography (EtOAc →
5 % CH3OH in EtOAc; RF 0.12 in EtOAc) to yield compound 17as pale yellow oil (0.41 g, 86% yield). 1H
NMR (500.13 MHz, CDCl3) δ 1.19 - 1.33 (m, 16H, Hc, Hd), 1.43 - 1.51 (m, 8H, Hb), 1.75 - 1.83 (m, 8H, He),
1.86 (s, 12H, CH3), 1.91 (s, 12H, CH3), 1.96 (s, 12H, CH3), 2.02 (s, 12H, CH3), 2.06 - 2.13 (m, 4H, Hj), 3.28 -
3.35 and 3.51 - 3.60 (m, 8H, Ha), 3.82 - 3.88 (m, 4H, H-5), 3.92 - 3.99 (m, 8H, H-6, H-6’), 4.01 - 4.07 and
4.11 – 4.18 (m, 8H, Hi ,Hk) 4.19 - 4.29 (m, 8H, Hf), 4.42 (s, 8H, Hg), 4.53 (s, 8H, Hh), 4.67 (br, 4H, H-4), 5.09
- 5.20 (m, 12H, H-1, H-2, H-3), 6.70 (s, 4H, PhH), 6.73 (s, 4H, PhH), 6.81 (s, 2H, PhH), 7.51 (s, 4H, C=CH);
13C NMR (125.7 MHz, CDCl3) δ 170.3, 169.8, 169.6, 169.5 (C=O), 159.0, 152.8, 139.4, 119.2, 115.2, 112.9
(PhC), 144.6 (C=CH), 122.4 (C=CH), 97.2 (C-1), 71.9 (C-5), 69.3 (Ch), 68.9 (C-3), 68.1 (C-2), 67.9 (C-4), 65.9
(Ca), 64.7, 64.2 (Ck, Ci), 63.4 (Cg), 62.2 (C-6), 49.9 (Cf), 29.9 (Cb), 29.1 (Cj), 28.7 (Ce), 26.0 (Cd), 25.4 (Cc),
20.6, 20.5, 20.4 (CH3). ESI MS m/z calcd for C120H166N12Na3O48/3 870.69, found: 871.02.
OH
HO
HO
HO
OH
O
HO
HO
HO
O
4.9. De-O-acetylated
tetrameric mannoside cluster (18)
f
b
d
O
O
N
N
N
N
a
j
c
e
N
g
N
h
O
O
k
O
O
O
O
i
19
HO
O
O
HO
HO
HO
O
N
O
O
N
N
N
N
OH
OH
OH
N
O
OH
Page 19 of 26

To a stirred solution of the tetravalent 17 (1.0 g, 0.39 mmol) in methanol (15 mL) was added 3
drops of a freshly prepared sodium methoxide solution (0.5 M in MeOH). The reaction mixture was
stirred at rt for 1 h. The mixture was then neutralized (Amberlite IR-120 (H+), filtered, and concentrated.
The resulting residue was dissolved in methanol (3 mL) and EtOAc (10 mL) was added slowly. The cloudy
solution was allowed to stand and the product precipitated as thick syrup. The clear solvent was
decanted and the thick syrup was dried under reduced pressure to give the completely the de-O-
1
acetylated 18 as a colorless solid (0.69 g, 94% yield). H NMR (500.13 MHz, CDCl3/ CD3OD) δ 0.85 – 0.95
(m, 8H, Hd), 0.95 – 1.05 (m, 8H, Hc), 1.09 – 1.22 (m, 8H, Hb), 1.42 – 1.53 (quint, J = 7 Hz, 8H, He), 1.74 –
1.84 (quint, J = 6 Hz, 4H, Hj), 2.93 – 3.01 (m, 4H, Ha), 3.08 – 3.14 (br, 4H, H-5), 3.22 – 3.45 (m, 24H, H-2,
H-3, H-4, H-6, H-6’, Ha), 3.67 (t, J = 6 Hz, 4H, Hk), 3.74 (t, J = 6 Hz, 4H, Hi), 3.95 (t, J = 7 Hz, 8H, Hf), 4.14 (s,
8H, Hg), 4.23 (s, 8H, Hh), 4.41 (br, 4H, H-1), 6.42 (s, 4H, PhH), 6.46 (s, 4H, PhH), 6.53 (s, 2H, PhH), 7.44 (s,
4H, C=CH); 13C NMR (125.7 MHz, CDCl3/ CD3OD) δ 158.7 , 152.5 (PhC), 143.9 and 123.0 (C=CH), 138.9,
20
Page 20 of 26

118.9, 114.8, 112.6 (PhC), 99.6 (C-1), 72.3 (C-5), 71.5 (C-3), 70.8 (C-2), 70.3 (Ch), 66.6 (C-4), 66.4 (Ca),
64.4, 63.9 (Ck, Ci), 62.5 (Cg), 60.7 (C-6), 49.6 (Cf), 29.4 (Cb), 28.6 (Cj), 28.5 (Ce), 25.5 (Cd), 24.9 (Cc). MALDI
12
MS m/z calcd for C89H134N12O32 1872.00, found 1872.25; calcd for
C
87
13CH134N12O32Na 1894.92, found:
1895.05.
4.10. Hexameric mannoside cluster (19)
R
O
AcO
AcO
AcO
O
c
e
a
g
AcO
N
j
h
O
f
d
O
O
L
b
O
N
N
i
k
AcO
R
AcO
AcO
O
O
O
R
AcO
N
O
N
N
To a stirred solution of hexa-O-propargy ether 10 (0.80 g, 0.72 mmol) and 6-azidohexyl 2,3,4,6-
O-acetyl-α-D-mannopyranoside 16 (3.0 g, 6.3 mmol, 8.8 equiv) in DMF (30 mL) was added sodium
ascorbate (80 mg) followed by copper(II) sulfate pentahydrate (30 mg, in 10 mL of H2O) in one portion.
The heterogeneous mixture was vigorously stirred for 24 h. Most of the DMF was then removed under
reduced pressure. The resulting residue was purified using column chromatography (EtOAc → 5 %
MeOH in EtOAc; RF 0.38 (5% MeOH in EtOAc/ DCM; 1:1) to yield compound 19 as a yellow oil (2.38 g, 84%
yield). NMR (500.13 MHz, CDCl3) δ 1.31 – 1.43 (m, 24H, Hc, Hd), 1.58 (quint, J = 7 Hz, 12H, Hb), 1.81 (s,
3H, CH3), 1.92 (quint, J = 7 Hz, 12H, He), 1.98 (s, 18H, CH3), 2.03 (s, 18H, CH3), 2.08 (s, 18H, CH3), 2.14 (s,
18H, CH3), 2.22 (quint, J = 6 Hz, 6H, Hj), 3.39 – 3.44 (td, J = 6, 10 Hz, 6H, Ha), 3.64 – 3.68 (td, J = 6, 10 Hz,
6H, Ha), 3.93 – 3.97 (m, 6H, H-5), 4.07 – 4.15 (m, 18H, H-6, Hi, Hk), 4.25 – 4.28 (m, 6H, H-6), 4.35 (t, J = 7
21
Page 21 of 26

Hz, 12H, Hf), 4.54 (s, 12H, Hg), 4.65 (s, 12H, Hh), 4.77 (d, J = 1 Hz, 6H, H-4), 5.21 – 5.33 (m, 18H, H-1, H-2,
H-3), 6.77 (d, J = 9 Hz, 6H, PhH), 6.83 (s, 6H, PhH), 6.93 (s, 3H, PhH), 6.96 (d, J = 9 Hz, 6H, PhH), 7.58 (s,
6H, C=CH); 13C NMR (125.7 MHz, CDCl3) δ 170.8, 170.2, 170.1, 169.9 (C=O), 159.3, 156.9 (PhC), 145.1
(C=CH), 141.9, 139.7 (PhC), 129.7 (C=CH), 122.6, 119.6, 113.7, 113.3 (PhC), 97.6 (C-1), 72.3 (C-5), 69.7
(Ch), 69.2 (C-3), 68.5 (C-2), 68.3 (C-4), 66.3 (Cg), 64.6, 64.4 (Ci, Ck), 63.8 (Ca), 62.6 (C-6), 50.7 (Cquat), 50.3
(Cf), 30.9 (CH3), 30.3 (Cb), 29.4 (Cj), 29.1 (Ce), 26.4 (Cd), 25.8 (Cc), 21.0, 20.9, 20.8 (4 CH3). MALDI MS m/z
calcd for C191H258N18O72 3978.69, found: 3978.71.
4.11. De-O-acetylated hexameric cluster (20)
R
O
HO
HO
HO
HO
O
c
e
a
g
j
N
h
O
f
O
d
O
L
b
O
N
N
i
k
HO
R
HO
HO
HO
O
O
O
R
N
O
N
N
To a stirred solution of the hexavalent 19 (1.5 g, 0.38 mmol) in methanol (15 mL) and THF (15
mL) was added 3 drops of a freshly prepared sodium methoxide solution (0.5 M in MeOH). The reaction
22
Page 22 of 26

mixture was stirred at rt for 1 h. The mixture was then neutralized (Amberlite IR-120 (H+), filtered, and
concentrated. The resulting residue was dissolved in methanol (5 mL) and EtOAc (15 mL) was added
slowly. The cloudy solution was allowed to stand and the product precipitated as thick syrup. The clear
solvent was decanted and the thick syrup was dried under reduced pressure to give the completely de-
O-acetylated 20 as a colorless solid (1.1 g, 95% yield): NMR (500.13 MHz, CD3OD/ CDCl3) δ 1.21 – 1.32 (m,
12H, Hd), 1.33 – 1.43 (m, 12H, Hc), 1.45 – 1.58 (m, 12H, Hb), 1.85 (quint, J = 7.5 Hz, 12H, He), 2.00 (s, 3H,
CH3), 2.17 (quint, J = 6 Hz, 6H, Hj), 3.32 – 3.39 (m, 6H, Ha), 3.46 – 3.52 (m, 6H, Ha), 3.59 – 3.83 (m, 36H, C-
2, C-3, C-4, C-5, C-6, C-6’), 4.07 (t, J = 6 Hz, 6H, Hi), 4.12 (t, J = 6 Hz, 6H, Hk), 4.33 (t, J = 7 Hz, 12H, Hf), 4.50
(s, 12H, Hg), 4.59 (s, 12H, Hh), 4.71 (s, 6H, C-1), 6.72 (d, J = 8.5 Hz, 6H, PhH), 6.84 (s, 6H, PhH), 6.86 (d, J =
8.5 Hz, 9H, PhH), 7.87 (s, 6H, C=CH); 13C NMR (125.7 MHz, CD3OD/ CDCl3) δ 160.6, 158.2 (PhC), 145.8 and
130.7 (C=CH), 143.1, 140.9, 125.0, 120.6, 114.8, 114.4 (PhC), 101.4 (C-1), 74.4 (C-5), 73.2 (Ch), 72.6 (C-3),
72.2 (C-2), 68.6 (C-4), 68.3 (Cg), 65.6, 65.4 (Ci, Ck), 64.2 (Ca), 62.9 (C-6), 51.7 (Cquat), 51.3 (Cf), 31.4 (CH3),
31.2 (Cb), 30.4 (Cj), 30.3 (Ce), 27.3 (Cd), 26.7 (Cc). MALDI MS m/z calcd for C143H210N18O48 2948.46, found
2948.38; calcd for C143H210N18O48Na 2970.44, found: 2970.42.
Acknowledgements
HA and RA thank Jordan University of Science and Technology (Jordan) for Financial Support and HA
thanks the Department of Chemistry at Dalhousie University (Canada) for laboratory space. TBG thanks
NSERC for financial support.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at XXXXXX
23
Page 23 of 26

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Scheme 1. Synthesis of bromide 4. Reagents and conditions: (a) CH3OH, H2SO4/reflux, 85%; (b)
LiAlH4,THF, reflux, 92%; (c) K2CO3, acetone, Br(CH2)3Br, reflux, 24h, 78%.
Scheme 2. Synthesis of benzyl alcohols 6 and 8. Reagents and conditions: (a) 4, acetone,
K2CO3, KI(cat), reflux, 24h, 69 % for 6 and 70% for 8.
Scheme 3. Synthesis of propargyl ethers 9 and 10. Reagents and conditions: (a) i. NaH,
DMF; ii. propargyl bromide, -10-0 oC, 3h, 62% for 9 and 60% for 10.
Scheme 4. Synthesis of azido mannoside 16. Reagents and conditions: (a) Ac2O, I2 (cat), 85%; (b)
ammonium acetate, DMF, rt, 90%; (c) CCl3CN, DBU, CH2Cl2, 68%; (d) 6-chlorohexanol, TMSOTf, CH2Cl2,
72%; (e) NaN3, DMF, 100 oC, 4h, 73%.
Comment [E1]: AUTHOR: Two different
version of Scheme 4 captions were
provided and the one in the ms has
been retained. Please check that it
is correct.
Scheme 5. Synthesis of the tetrameric mannoside cluster 18. Reagents and conditions: (a) CuSO4 .5H2O,
sodium ascorbate, H2O-THF (1:1), rt, overnight, 86%; (b) MeONa, MeOH, rt, 1h, 94%.
Scheme 6. Synthesis of the hexameric mannoside cluster 20. Reagents and conditions: (a) CuSO4 .5H2O,
sodium ascorbate, H2O-THF (1:1), rt, overnight, 84%; (b) MeONa, MeOH, rt, 1h, 95%.
26
Page 26 of 26