Inhibition of adherence of the yeast Candida albicans to buccal epithelial cells by synthetic aromatic glycoconjugates

Article abstract of DOI:10.1016/j.ejmech.2018.10.011

The yeast Candida albicans is an opportunistic fungal pathogen which induces superficial and systemic infections in immunocompromised patients. Adherence to host tissue is critical to its ability to colonise and infect the host. The work presented here de

Full text of DOI:10.1016/j.ejmech.2018.10.011

Accepted Manuscript
Inhibition of adherence of the yeast Candida albicans to buccal epithelial cells by
synthetic aromatic glycoconjugates
Harlei Martin, Mairead Mc Govern, Lorna Abbey, Aisling Gilroy, Stephanie Mullins,
Sarah Howell, Kevin Kavanagh, Trinidad Velasco-Torrijos
PII:
S0223-5234(18)30873-0
10.1016/j.ejmech.2018.10.011
EJMECH 10797
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 April 2018
Revised Date: 19 September 2018
Accepted Date: 5 October 2018
Please cite this article as: H. Martin, M.M. Govern, L. Abbey, A. Gilroy, S. Mullins, S. Howell, K.
Kavanagh, T. Velasco-Torrijos, Inhibition of adherence of the yeast Candida albicans to buccal epithelial
cells by synthetic aromatic glycoconjugates, European Journal of Medicinal Chemistry (2018), doi:
https://doi.org/10.1016/j.ejmech.2018.10.011.
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ACCEPTED MANUSCRIPT
Inhibition of Adherence of the yeast Candida albicans to Buccal Epithelial Cells by
Synthetic Aromatic Glycoconjugates
1
2
1
2
2
Harlei Martin , Mairead Mc Govern , Lorna Abbey , Aisling Gilroy , Stephanie Mullins ,
2
2
1
Sarah Howell , Kevin Kavanagh * and Trinidad Velasco-Torrijos *
Graphical Abstract

ACCEPTED MANUSCRIPT
Inhibition of Adherence of the yeast Candida albicans to Buccal Epithelial Cells by
Synthetic Aromatic Glycoconjugates
1
2
1
2
2
Harlei Martin , Mairead Mc Govern , Lorna Abbey , Aisling Gilroy , Stephanie Mullins ,
2
2
1
Sarah Howell , Kevin Kavanagh * and Trinidad Velasco-Torrijos *
1
Department of Chemistry, Maynooth University, Maynooth, Co. Kildare, Ireland
2
Department of Biology, Maynooth University, Maynooth, Co. Kildare, Ireland.
Key words: Candida albicans, anti-adherence, multivalent glycoconjugates, Structure-
Activity Relationship study, Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC)
ABSTRACT: The yeast Candida albicans is an opportunistic fungal pathogen which induces
superficial and systemic infections in immunocompromised patients. Adherence to host tissue
is critical to its ability to colonise and infect the host. The work presented here describes the
synthesis of a small library of aromatic glycoconjugates (AGCs) and their evaluation as
inhibitors of C. albicans adherence to exfoliated buccal epithelial cells (BECs). We identified
a divalent galactoside, ligand 2a, capable of displacing over 50% of yeast cells already
attached to the BECs. Fluorescence imaging indicates that 2a may bind to structural
components of the fungal cell wall.
INTRODUCTION
The attachment of pathogenic microorganisms to the surface of the host cells is a prequisite
1
,2
for infection.
The mechanisms that mediate pathogen adherence often involve microbial
proteins, known as adhesins, that recognize cell surface biomolecules such as protein
3
receptors and glycans. The development of compounds capable of blocking the adherence of
pathogens to host cells is an attractive alternative to traditional antibiotic treatments that rely
4
,5
solely on killing the infective microorganisms. There are several examples in which the

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anti-adherence approach has been exploited successfully to design inhibitors of microbial
6
adherence. Some of these include glycoclusters capable of preventing lung infection caused
7
by Pseudomonas aeruginosa and glycoconjugates used in the treatment of conditions
8
caused by pathogenic strains of Escherichia coli, such as Crohn’s disease and urinary tract
9
, 10
infections.
The lectins involved in the adherence processes in these reports (Lec A and
Lec B from P. auroginosa and Fim H from E. coli, respectively) have been extensively
studied and detailed knowledge of their structure and binding specificities is available. This
has greatly facilitated the design of high affinity glycoconjugates that can effectively compete
1
1
with host cell surface ligands.
Candida albicans is a dimorphoric yeast that can interconvert from single cells to hyphal
1
2
forms, and exists in a commensal state in the mucosae and gastrointestinal tract. In
immunocompromised patients C. albicans induces a range of superficial and systemic
1
3
infections, and is the third leading cause of infections related to medical devices such as
1
4
catheters. Adherence of C. albicans to host cells plays an important role in pathogenesis, as
it allows the establishment of a strong link to host cell surfaces and provides a focal point for
15
infection by enabling persistence in harsh environments such as the mouth. Interestingly,
this highly adherent organism is also the most pathogenic Candida species and the major
1
6
cause of fungal infections, indicating that its highly infectious rate may be related to its
strong adherence capacity. In addition, C. albicans expresses host regulator binding proteins
1
7
18
such as phosphoglycerate mutase (gpm1) and pH-regulated antigen 1 (pra1) that bind to
immune regulators such as Factor H and FHL-1 to avoid immune detection, demonstrating
1
9
that through adherence, C. albicans can block activation of immune system regulators.
The oral cavity, in particular, provides surfaces to which C. albicans can adhere, such as
buccal epithelial cells (BECs). C. albicans is capable of adhering to host cells through the
1
6
interaction of the yeast cell wall and epithelial cell surfaces. Adherence to abiotic and biotic

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surfaces is achieved by both non-specific and specific mechanisms: non-specific interactions
2
0
involve cell surface hydrophobicity (CSH). CSH does not play a dominant role in the
adherence process but has been known to maintain specific interactions between the yeast
2
1
and the host. Specific adherence mechanisms occur via the binding of adhesins to receptors
2
2
on the host cell surface through lectin-like and protein-protein interactions. Initial reports
indicate that some C. albicans adhesins recognise and bind to a broad range of cell surface
2
3
24
glycans, which include glycosphingolipids such as lactosylceramide and asialo-GM1, and
2
5
26
host oligosaccharides containing fucose (Fuc) and N-acetyl-glucosamine (GlcNAc).
These findings provide evidence that cell surface glycans are important receptors for C.
albicans and warrant the development of anti-adherence ligands that can mimic them, thus
disrupting C. albicans - epithelial cell interactions. These compounds could represent a
promising strategy to overcome fungal infections. However, the lack of structural knowledge
of the fungal adhesins that recognise these carbohydrates hampers a focused design approach
such as those described earlier. In this study, we opted instead to screen a small library of
synthetic glycoconjugates with a diverse presentation of binding epitopes in order to identify
structural features that can lead to effective inhibition of fungal adherence. Thus, we herein
report the synthesis of aromatic-core glycoconjugates (AGC) which display some of the
glycan residues reported to mediate C. albicans adherence to epithelial cells and their
subsequent evaluation as inhibitors of the adherence of C. albicans to BECs.
RESULTS AND DISCUSSION
Synthesis: There are numerous examples of glycoconjugates built upon aromatic scaffolds,
2
7
28
many of which are intended as ligands for adhesins, carbohydrate-binding proteins or
2
9, 30
sensors for the detection of pathogens.
The popularity of AGCs is partly due to the
versatility in functionality and the substitution patterns that can be achieved from readily

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available starting materials. Thus, we decided to explore 1,3 and 1,3,5 functionalized
aromatic derivatives as the starting point in the design of the anti-adherence AGCs library
(Scheme 1, Figures SI.1-3).
O
O
CO H
2
N
R1
1a R =
D
1
N
N
-Gal
-Fuc
-Gal-β-OC H -
H
N
1
1
b R =
L
1
c R =
D
1
2 4
HN
NH2
O
O
2a R =
D
-Gal
-Fuc
-Gal-β-OC H -
2
2
b R =
L
2
HO C
CO H
2
2
N
H
N
H
R2
R2
2c R =
N
N
N
N
N
D
2
2 4
N
2d
R =
D
-Man
-GlcNAc
-Lac
2
2
2
e R =
D
2
HN
O
O
NH2
f R =
D
2
O
ClOC
COCl
N
H
N
H
R3
N
N
N R
3
N
N
3a
R = D
-Gal
-Fuc
-Gal-β-OC H -
3
N
3
3
b R =
c R =
L
3
D
3
2
4
COCl
O
N
R3
N
H
N
N
OH
OH OH
O
OH OH
O
OH
O
HO
O
OH
HO
OH
HO
O
HO
HO
OH
-Gal
OH
D
L
-Fuc
D
-Gal-β-OC H -
D-Man
2
4
OH
O
OH
HO
OH
O
OH
O
HO
HO
O
OH HO
NHAc
OH
D
-GlcNAc
D
-Lac
OH
OH
O
OH
OH OH
O
O
O
O
N
O
O
O
OH
OH
OH
HO
HO
N
H
N
N
HO
N N
N
HO
N
H
O
NH
HO
H
N
O
OH
HO
HO
HN
HN
4
D-Man/
D
-Gal
5
D-Gal
Scheme 1: Chemical structures and starting materials used in the synthesis of the anti-
adherence AGCs library.

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It is well known that multivalency can be an important factor that modulates carbohydrate-
3
1
protein interactions. Hence, mono- (compounds 1a-c), di- (compounds 2a-f) and trivalent
analogues (compounds 3a-c) were investigated. Carbohydrate moieties present in the
epithelial cell surface and reported to bind C. albicans adhesins were selected to be grafted
onto the aromatic scaffold: these included galactose, fucose, mannose, glucose, N-acetyl
glucosamine and lactose derivatives. Triazolyl-containing spacer groups of different lengths,
generated by means of Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) reactions,
connected the glycosides to the central aromatic core. This methodology has been found to be
3
2
extremely useful and reliable for the efficient synthesis of numerous glycoconjugates. With
this modular approach, we were able to readily assemble a small collection of
glycoconjugates in which (i) the carbohydrate moiety, (ii) the valency and (iii) the distance
between the binding epitopes were varied. This provided sufficient structural diversity for an
initial screening of the requirements for fungal anti-adherence activity.
The synthesis of one of the divalent analogues, galactoside 2a, depicted in Scheme 2, is
representative for the synthesis of the other members of the AGCs library. 5-
Aminoisophthalic acid was reacted with propanoyl chloride. The resulting dicarboxilic acid
was reacted with propargyl amine using freshly prepared 4-(4,6-Dimethoxy-1,3,5-triazin-2-
3
3
yl)-4-methylmorpholinium chloride (DMTMM) to give diamide 6 in 78% yield. The
attachment of the carbohydrate moiety to the aromatic scaffold was effected by means of the
3
4
CuAAC reaction: in this example, 2,3,4,6-tetra-O-acetyl-1-β-azido-galactoside was reacted
with compound 6 using copper sulphate and sodium ascorbate as the catalytic system. If the
cycloaddition was carried out using conventional heating, the reaction times were long (up to
4
days) and the yields were moderate. However, we found that if the reaction was carried out
using microwave (MW) irradiation, it proceeded with yields up to 82% for the protected
35
glycoconjugate 7a and with a drastic reduction in reaction times. The deacetylation of

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compound 7a was accomplished under mild basic conditions to give the galactosyl AGC 2a
in 96% yield. Following comparable synthetic schemes (see Supporting Information,
Schemes SI.1-3), the remaining mono-, di- and trivalent AGCs were readily prepared in
moderate to good yields.
Scheme 2: Synthesis of galactosyl AGC 2a. Reagents and conditions: i) C H COCl, NEt ,
2
5
3
THF, N , rt, 22 h, 77 %; ii) DMTMM, propargyl amine, DMF, N 16 h, 78 %; iii) 2,3,4,6-
2
2,
tetra-O-acetyl-1-β-azido-galactoside, CuSO .5H O/Na Asc, CH COCH /H O, 100 °C in
4
2
3
3
2
MW, 10 min, 84%; iv) methanol, NEt , H O, 45°C, 6 h, 94%.
3
2
Biological Evaluation: All of the glycoconjugates (with exception of monovalent fucosyl
derivative, compound 1b) were found to be soluble in water. The toxicity of the compounds
against C. albicans was firstly evaluated. None of the compounds showed significant ability
to inhibit the growth of the yeast cells at the range of concentrations used in the subsequent
adherence assays (see Figure SI.4). This implies that any reduction of adherence observed is
not due to toxic effects. The ability of the glycoconjugates to inhibit the adherence of C.
albicans was then evaluated in different assays:

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Exclusion assay: The initial adherence assay was performed by treating C. albicans with the
glycoconjugates, allowing for an incubation period and then exposing the treated yeast cells
to the exfoliated BECs. The percentage increase or decrease of the number of C. albicans
cells adhering to BECs compared to the adherence of the untreated yeast is represented in
Table 1.
Table 1: Effect of AGCs on adherence of C. albicans to BECs (estimated according to
exclusion assays, at AGCs concentration = 10 mg/mL. SE in all cases was less than 10% of
mean change in adherence).
AGCs
% Increase/
Decrease
of
Adherence
1
1
1
2
2
2
2
2
2
3
3
3
4
5
a
b
c
a
b
c
d
e
f
D
- Gal
- Fuc
- Gal-β-OC H4
- 14.5
- 2
L
D
-7.5
-80
-8
2
D
-Gal
-Fuc
-Gal-β-OC H4
L
D
-35
+3
2
D
-Man
-GlcNAc
-Lac
D
-45
+6.5
-45
-30
-42
-24
-26
D
a
b
c
D-Gal
L-Fuc
D-Gal-β-OC H4
2
D
-Gal/
D-Man
D
-Gal
These results show the impact of the valency effect in the anti-adherence ability of the AGCs:
monovalent compounds, in which only one carbohydrate moiety is present, are considerably
less active than their di- and trivalent counterparts. In addition, it can be clearly seen that the
galactosyl derivatives (2a, 2c, 3a and 3c) and the N-acetyl glucosamine derivative 2e are
much more efficient at preventing the yeast adherence than any of the other glycoconjugates:

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in particular, divalent galacoside 2a was identified as the most active compound of the AGCs
library screened, showing a remarkable 80% decrease in adherence of the yeast to the BECs
after treatment. Interestingly, compound 4, a structural analogue of 2a in which one of the
galactosyl moieties has been replaced by mannose, is only capable of producing a 24%
reduction in yeast adherence. This highlights the importance of a divalent galactosyl pattern
as a recognition motif. The linker connecting the galactosyl moieties to the aromatic scaffold
appears to also influence the anti-adherence ability of the glycoconjugates: the more flexible
O-galactosides 1c, 2c, 3c inhibited yeast adherence less effectively than their respective
analogues 1a, 2a, 3a, in which the triazolyl spacer group is directly attached to the anomeric
galactosyl carbon. In addition, divalent galactoside 5, which features no linkers, was only
able to induce a 26% reduction in adherence of C. albicans to BECs. The divalent galactoside
2
a was then evaluated at lower concentrations (Figure 1a). Significantly, the anti-adherence
ability of this compound was maintained at a 100-fold dilution concentration (0.1 mg/mL,
38 µM)).
1
Competitive assay: The best performing compound (divalent galactosides 2a) was then
evaluated in a competition assay, in which its anti-adherence ability was tested in the
presence of both C. albicans and BECs. Co-incubation with compounds 2a resulted in a
reduction in adherence of yeast cells to BECs of 65%, even at the lowest concentration
(
Figure 1b).
Displacement assay: A further assay was performed where glycoconjugate 2a (0.1 mg/mL,
38 µM)) was added to a mixture of C. albicans and BECs, which had been previously
1
incubated together. The ability of the compound to reverse the adherence of the yeast to the
BECs was then examined. Two controls were used in this assay: control 1 involved the
assessment of the binding of C. albicans to BECs prior to compound exposure; control 2
involved BECs and adherent yeast cells being re-incubated in PBS for 90 minutes prior to a

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second filtration step. It was found that 2a imparted a reduction in adherence of 56%
(compared to the control 1) and 31% (compared to the control 2) (Figure 1). These results
suggest that divalent galactoside 2a bind effectively to C. albicans preventing its interaction
with BECs (Figure 1d).
Figure 1. Effect of divalent galactoside 2a on the adherence of C. albicans to BECs. The data
displays average yeast adherence per BEC: a) Exclusion assay: C. albicans was preincubated
with 2a (concentrations 10, 1 and 0.1 mg/mL); b) Competition assay: C. albicans, BECs and
2
a (concentrations 10, 1 and 0.1 mg/mL) were co-incubated; c) Displacement assay: C.
albicans and BECs were co-incubated and compound 2a (concentration 0.1 mg/mL) was
subsequently added; control 1 involved the assessment of the binding of C. albicans to BECs
prior to compound exposure; control 2 involved BECs and adherent yeast cells being re-
incubated in PBS for 90 minutes prior to a second filtration step; d) Optical microscopy
image of C. albicans attached to BEC before (right) after (left) treatment with 2a;

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Fluorescence imaging: A fluorescently labelled analogue of galactosylated AGC 2a,
compound 8 (Figure 2), was synthesized to investigate possible sites of interaction of anti-
adherence AGCs with C. albicans (Scheme SI-6). As controls, C. albicans cells with no
treatment were imaged under an Olympus Fluoview 1000 confocal microscope to discard
yeast autofluorescence (Figure 2a). In addition, C. albicans cells were incubated with
fluorescein isothiocyanate (FITC, Figure 2b). Fluorescein derivative galactoside 8 was then
co-incubated with C. albicans cells and the cells were imaged (Figure 2c). In this case,
strong localized fluorescence can be clearly observed. From these images, it can be deduced
that compound 8 is interacting with the surface of the yeast cells.
Figure 2: Confocal microscope images of C. albicans cells: a) without treatment; b) co-
incubated with FITC; c) co-incubated with fluorescently labelled galactosyl AGC 8; d)
chemical structure of galactoside 8. A wavelength of 488 nm laser was used for excitation
and emission was detected at 500 - 600 nm.

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These results indicate that the valency of the AGCs strongly influences their anti-adherence
ability: the monovalent derivatives 1a-c did not show any significant activity while the
trivalent derivatives 3a-c were moderate inhibitors, with the galactosyl derivatives 3a and 3c
achieving as high as 45% and 42% reduction of adherence of C. albicans to BECs,
respectively. Nevertheless, this study was focused in the divalent AGCs based on a 5-
aminoisophthalic acid scaffold, which may allow for further synthetic versatility.
The results from the anti-adherence assays highlight the potential of divalent galactosyl AGC
2
a as an inhibitor of the adherence of C. albicans to BECs. This compound consistently
showed the best anti-adherence activity in the three types of assays performed (with up to
0% reduction of adherence in the exclusion assays). Interestingly, divalent compounds
analogue in structure to 2a but featuring carbohydrate moieties other than galactose
compounds 2b, 2d-f) were not as efficient adherence inhibitors as 2a: the second best
8
(
performing AGC was the N-acetyl glucosamine derivative 2f (45% reduction of adherence in
the exclusion assays). These results suggest that the divalent presentation of the galactose
epitopes achieved in compound 2a is important in mediating adherence to C. albicans.
Polysaccharides and adhesins present in C. albicans cell wall mediate many of the adhesion
3
6
processes of the yeast. Initial research indicated that the addition of galactose or
3
7
galactosamine reduced C. albicans attachment to buccal mucosal cells in vitro. However,
this is in contrast to a study in which the pre-treatment of C. albicans with galactose failed to
3
8
inhibit adherence. It has also been reported that C. albicans binds specifically to cell surface
glycosphingolipids with terminal galactosyl residues such as lactosylceramide [Gal-β-(1-4)-
2
3
Glc−β-(1-1)Cer]
)Cer] by means of fimbrial proteins. This study also reports that the synthetic disaccharide
derivative GalNAc-β-(1-4)-Gal-β-O(CH ) CO CH was able to inhibit the binding of C.
and asialo-GM1 [Gal-β-(1-3)-GalNAc-β-(1-4)-Gal-β-(1-4)-Glc−β-(1-
2
4
1
2
8
2
3
albicans fimbriae to BECs in vitro. More recently, a synthetic disaccharide (Fimbrigal-P) also

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featuring the terminal GalNAc-β-(1-4)-Gal motif, was found to reduce fungal burden in an in
39
vivo model of oral candidiasis. However, to the best of our knowledge, there are no
Structure-Activity Relationship (SAR) studies of synthetic glycoconjugates regarding
inhibition of C. albicans adherence that have identified terminal galactosides as key epitopes
mediating yeast adhesion. A recent study has linked the binding specificities of some C.
albicans adhesins (from the Als family) to human cell surface glycans based on glycan array
screening results: a predicted glycan determinant for some of these proteins was a di-LacNAc
4
0
(
Gal-β-1,4-GlcNAc) disaccharide. Further studies are currently going on in our laboratory
to identify the fungal adhesin that lead compound 2a may be binding to.
CONCLUSION
In conclusion, this work reports the synthesis of a small library of AGCs designed to conduct
a preliminary SAR study on their ability to inhibit the adherence of the pathogenic yeast C.
albicans to BECs. The anti- adherence assays allowed for the identification of divalent
galactosyl derivative 2a as an efficient inhibitor of C. albicans adherence, with 2a being able
to displace over 50% of yeast cells already attached to BECs. The precise three-dimensional
presentation of the galactosyl moieties in 2a appears to be a requirement for efficient
adherence inhibition, which suggest that AGC 2a is interfering with a specific recognition
process part of the complex C. albicans adherence mechanisms. Fluorescence studies suggest
that a potential target for 2a could be indeed a fungal cell wall adhesin. The synthetic
accessibility and high efficacy shown by 2a in the biological assays make this compound a
promising lead for development of new fungal anti-adherence agents, less prone to the
appearance of resistance mechanisms than conventional fungicidal treatments.
EXPERIMENTAL SECTION

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Chemistry. General Methods: All reagents for synthesis were bought commercially and
used without further purification. Tetrahydrofuran (THF) was freshly distilled over sodium
wire and benzophenone. Dichloromethane (DCM) was freshly distilled over CaH prior to
2
use. Reactions were monitored with thin layer chromatography (TLC) on Merck Silica Gel
F254 plates. Detection was effected by UV (λ=254nm) or charring in a mixture of 5% sulfuric
acid-ethanol. NMR spectra were recorded using Bruker Ascend 500 spectrometer at 293K.
All chemical shifts were referenced relative to the relevant deuterated solvent residual peaks.
1
13
Assignments of the NMR spectra were deduced using H NMR and C NMR, along with 2D
experiments (COSY, HSQC and HMBC). Chemical shifts are reported in ppm. Flash
chromatography was performed with Merck Silica Gel 60. Microwave reactions were carried
out using a CEM Discover Microwave Synthesizer. Optical rotations were obtained from an
−
1
2
−1
AA-100 polarimeter and [α] ꢁvalues are given in 10 cm ·g . High performance liquid
ꢀ
chromatography analysis (HPLC, Waters Alliance 2695) was performed in final compounds
and indicated purity of 95% based on integrations without the use of an internal standard.
High resolution mass spectrometry (HRMS) was performed on an Agilent-LC 1200 Series
coupled to a 6210 Agilent Time-Of-Flight (TOF) mass spectrometer equipped with an
electrospray source in both positive and negative (ESI+/−) modes. Infrared spectra were
−
1
obtained as a film on NaCl plates or as KBr disks in the region 4000–400 cm on a Perkin
Elmer Spectrum 100 FT-IR spectrophotometer. Synthetic schemes and spectroscopic data for
all members of the ACG library are provided in the SI.
General Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction procedures:
Method A: Copper sulphate pentahydrate (20 mg) and sodium ascorbate (40 mg) were added
to a solution of the acetylated sugar azide (1,25 equiv per propargyl group) and the
corresponding propargyl amide scaffold in acetone/water (2:1 ratio). The reaction was
allowed to stir at rt until deemed complete by TLC analysis (typically 16-24 h). The solvent

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was removed in vacuo. The residue was dissolved in DCM, washed with water (x3) and dried
(
MgSO ). The mixture was filtered and the solvent was removed in vacuo to yield the crude
4
product, which was purified by silica gel column chromatography (DCM:MeOH 98:2-93:7)
to give the corresponding product.
Method B: Copper sulphate pentahydrate (20 mg) and sodium ascorbate (40 mg) were added
to a solution of the acetylated sugar azide (1,25 equiv per propargyl group) and the
corresponding propargyl amide scaffold in acetonitrile/water (2:1 ratio). The reaction was
allowed to stir in the MW at 100 ⁰C until deemed complete by TLC analysis (typically 5-15
min). The solvent was removed in vacuo. The residue was dissolved in DCM, washed with
water (x3) and dried (MgSO ). The mixture was filtered and the solvent was removed in
4
vacuo to yield the crude product, which was purified by silica gel column chromatography
(DCM:MeOH 98:2-93:7) to give the corresponding product.
General acetyl ester hydrolysis procedure: The acetylated glycoconjugate was dissolved in
methanol/water (2:1 ratio). NEt (0.1 mL) was added and the reaction mixture was allowed to
3
+
stir at 45 ⁰C until completion (typically 6-18 h). The solution was cooled to rt, Amberlite H
was added and the mixture was allowed to stir for 30 min. The solution was filtered and the
solvent was removed in the rotatory evaporator and the residue was dried under high vacuum
or lyophilized to give the deprotected glycoconjugate.
N,N’-di(prop-2-yn-1-yl)-5-propionamidoisophthalamide (6). 5-aminoisophathalic acid (5
g, 27.6 mmol) was dissolved in anhydrous THF (60 mL) under N and propionyl chloride
2
(2.7 mL, 30.4 mmol) was added dropwise. The mixture was allowed to stir for 5 min and
NEt (5 mL, 35.8 mmol) was added slowly. The reaction was left to stir for 22 h. The solvent
3
was removed under reduced pressure, and the residue was dissolved in hot methanol. The
insoluble material was filtered off and the filtrate was evaporated in a rotatory evaporator to

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3
3
give 5-propionoamidoisophthalic acid, which was used without further purification. (5.03
g, 77 %). 5-propionoamidoisophthalic acid (0.78 g, 3.27 mmol) and DMTMM (1.99 g, 7.20
mmol) were suspended in anhydrous DMF (25 mL) under N . After 10 min, propargylamine
2
(0.46 mL, 7.2 mmol) was added and the reaction mixture went clear. It was left to stir at rt for
1
6 h. The reaction mixture was poured into ice/water (30 mL) and the precipitated formed
1
was then filtered and dried on the air to give 6: white amorphous solid (0.79 g, 78%). H
NMR (500 MHz, d -DMSO): δ 10.15 (s, 1H, NHCOC H ), 8.94 (t, J = 5.3 Hz, 2H,
6
2
5
NHCH CCH), 8.18 (s, 2H, Ar-H), 7.93 (s, 1H, Ar-H), 4.11-4.00 (m, 4H, NHCH CCH), 3.13
2
2
(
s, 2H, NHCH CCH), 2.35 (q, J = 7.5 Hz, 2H, CH CH ), 1.10 (t, J = 7.5 Hz, 3H, CH CH ).
2 2 3 2 3
1
3
C NMR (125 MHz, d -DMSO): δ 172.8 (COC H ), 166.2 (CONHCH CCH), 140.0 (Ar-C),
6
2
5
2
1
35.3 (Ar-C), 121.4 (Ar-CH), 120.7 (Ar-CH), 81.6 (CH CCH), 73.4 (CH CCH), 29.1
2 2
(
CH CH ), 10.0 (CH CH ). IR (KBr): 3289.16, 3241.00, 3093.06, 2977.14, 2116.87,
2 3 2 3
-1
+
+
1
682.50, 1570.58 cm . HRMS (ESI+): m/z calcd for C H N O +H [M+H] 312.1343,
17 17 3 3
found 312.1361.
N,N’-di-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-1,2,3-triazol-4-ylmethylamide)-
N’’-propyl-5-aminobenzene-1,3-dicarboxamide (7a). Prepared from 6 and 2,3,4,6-tetra-O-
3
4
acetyl-1-β-azido-
D
-galactopyranoside, according to Method B: pale yellow amorphous solid
608 mg, 84%). R = 0.29 (DCM: methanol 9:1).ꢁ[α] ꢂꢀ ꢃ -4.3 (c 0.7, DCM). H NMR (500
1
(
f
MHz, CDCl ): δ 9.09 (s, 1H, NHCOC H ), 8.21 (s, 2H, NHCH -triaz), 7.97-7.95
3
2
5
2
(overlapping of 2 s, 4H, Ar-H and triaz-H), 7.78 (s, 1H, Ar-H), 5.89 (d, J = 9.2 Hz, 2H, H-1),
5
3
4
2
1
.54 (t, J = 9.7 Hz, 2H, H-2), 5.49 (d, J = 3.2 Hz, 2H, H-4), 5.27 (dd, J = 10.3, 3.2 Hz, 2H, H-
), 4.59 (dd, J = 15.5, 5.2 Hz, 4H, CH -triaz), 4.29 (t, J = 6.5 Hz, 2H, H-5), 4.16 – 4.05 (m,
2
H, H-6 and H-6’), 2.30 (q, J = 7.5 Hz, 2H, CH CH ), 2.14 (s, 3H, OAc), 1.93 (s, 6H, OAc x
2
3
13
), 1.76 (s, 3H, OAc), 1.06 (t, J = 7.5 Hz, 3H, CH CH ). C NMR (125 MHz, CDCl ): δ
2
3
3
73.4 (COC H ), 170.4 (CO of OAc), 170.2 (CO of OAc), 169.9 (CO of OAc), 169.3 (CO of
2
5

ACCEPTED MANUSCRIPT
OAc), 166.9 (CONHCH -triaz), 145.4 (C-triaz), 139.1, 134.7 (each Ar-C), 121.7 (CH-triaz),
2
1
21.4, 120.9 (each Ar-CH), 86.0 (C-1), 73.8 (C-5), 70.8 (C-3), 68.1 (C-2), 67.0 (C-4), 61.2
C-6), 35.3 (CH -triaz), 30.2 (CH CH ), 20.6( CH of OAc), 20.6 (CH of OAc), 20.5 (CH
3
(
2
2
3
3
3
of OAc), 20.2 (CH of OAc), 9.4 (CH CH ). IR (film on NaCl): 3290, 2979, 2940, 2120,
3
2
3
-1
+
+
1
753, 1655, 1599, 1536 cm . HRMS (ESI+): m/z calcd. for C H N O +H [M+H]
45 56 9 21
1
058.3591, found 1058.3602.
N,N’-di-(2,3,4-tri-O-acetyl-β-L-fucopyranosyl-1,2,3-triazol-4-ylmethylamide)-N’’-
propyl-5-aminobenzene-1,3-dicarboxamide (7b). Prepared from 6 and 2,3,4-tri-O-acetyl-1-
4
1
β-azido-
L-fucopyranoside according to Method B: yellow amorphous solid (93 mg, 62 %).
ꢃꢄ
1
R =0.44 (DCM:MeOH 9:1). [α] +1.6 (c 0.9, DCM). H NMR (500 MHz, CDCl ): δ 8.37 (s,
f
ꢀ
3
1
H, NHCOC H ), 7.97 (s, 2H, triaz-H), 7.94 (s, 2H, Ar-H), 7.83 (t, J = 5.1 Hz, 2H,
2 5
CONHCH -triaz), 7.70 (s, 1H, Ar-H), 5.84 (d, J = 9.2 Hz, 2H, H-1), 5.58 – 5.51 (m, 2H, H-
2
2
), 5.38 (d, J = 3.3 Hz, 2H, H-4), 5.27 – 5.23 (m, 2H, H-3), 4.66 (dd, J = 15.3, 5.7 Hz, 4H,
CH -triaz), 4.15 (q, J = 6.4 Hz, 2H, H-5), 2.38 (qd, J = 7.7, 3.7 Hz, 2H, CH CH ), 2.22 (s,
2
2
3
6
7
H, OAc), 1.99 (s, 6H, OAc), 1.83 (s, 6H, OAc), 1.24 (d, J = 6.4 Hz, 6H, H-6), 1.17 (t, J =
13
.5 Hz, 3H, CH CH ). C NMR (125 MHz, CDCl ): δ 172.8 (NHCOC H ), 170.5 (CO of
2
3
3
2
5
OAc), 169.9 (CO of OAc), 169.4 (CO of OAc), 166.7 (CONHCH -triaz), 145.5 (C-triaz),
2
1
39.1 (Ar-C), 135.0 (Ar-C), 121.4 (CH-triaz), 121.2 (Ar-CH), 120.5 (Ar-CH), 86.3 (C-1),
2.8 (C-5), 71.3 (C-3), 69.9 (C-4), 68.2 (C-2), 35.5 (CH -triaz), 30.4 (CH CH ), 20.7 (CH of
7
2
2
3
3
OAc), 20.5 (CH of OAc), 20.3 (CH of OAc), 16.1 (C-6), 9.5 (CH CH ). IR (film on NaCl):
3
3
2
3
-1
+
+
3
318, 2924, 1749, 1656, 1535 cm . HRMS (ESI+): m/z calcd for C H N O + H [M+H]
41 51 9 17
9
42.9130, found 942.9142.
N,N’-di-[2-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-ethyl-1,2,3-triazol-4-
ylmethylamide)-N’’-propyl-5-aminobenzene-1,3-dicarboxamide (7c). Prepared from 6
4
2
and 2-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)ethyl azide according to Method B:

ACCEPTED MANUSCRIPT
ꢂꢅ
yellow amorphous solid (545 mg, 82 %). R = 0.38 (DCM:MeOH 9:1). [α]ꢀ -9.1 (c 1.1,
f
1
DCM). H NMR (500 MHz, CDCl ): δ 9.11 (s, 1H, NHCOC H ), 8.14 (s, 2H, CONHCH -
3
2
5
2
triaz), 7.96 (s, 2H, Ar-H), 7.64 (d, J = 16.6 Hz, 3H, Ar-H and triaz-H), 5.30 (d, J = 3.2 Hz,
2
4
4
7
H, H-4), 5.07 (dd, J = 10.3, 8.1 Hz, 2H, H-2), 4.94 (dd, J = 10.5, 3.2 Hz, 2H, H-3), 4.66 –
.36 (m, 10H, CH -triaz and CH CH O and H-1), 4.15 (dd, J = 13.6, 6.4 Hz, 2H, CHO-Gal),
2
2
2
.09-4.01 (m, 4H, H-6 and H-6’), 3.88 (ap t, J = 6.4 Hz, 4H, CHO-Gal and H-5), 2.27 (d, J =
.0 Hz, 2H, CH CH ), 2.06 (s, 6H, OAc), 1.95 (s, 6H, OAc), 1.89 (d, J = 2.1 Hz, 12H, OAc
2
3
13
x2), 1.03 (t, J = 7.3 Hz, 3H, CH CH ). C NMR (125 MHz, CDCl ): δ 173.2 (NHCOC H ),
2
3
3
2
5
1
70.4 (CO of OAc), 170.2 (CO of OAc), 170.0 (CO of OAc), 169.7 (CO of OAc), 166.8
(
CONHCH -triaz), 144.7 (C-triaz), 139.3 (Ar-C), 134.7 (Ar-C), 123.6 (CH-triaz), 121.1 (Ar-
2
CH), 120.2 (Ar-CH), 100.8 (C-1), 70.7 (C-5), 70.6 (C-3), 68.5 (C-2), 67.5 (CH CH O), 66.9
2
2
(
C-4), 61.1 (C-6), 50.00 (CH CH O), 35.5 (CH -triaz), 30.2 (CH CH ), 20.7 (CH of OAc),
2 2 2 2 3 3
2
0.6 (CH of OAc), 20.6 (CH of OAc), 20.5 (CH of OAc), 9.5 (CH CH ). IR (film on
3 3 3 2 3
-1
NaCl): 3311, 3148, 3071, 2980, 1750, 1656, 1599, 1543 cm . HRMS (ESI+): m/z calcd for
+
+
C H N O + H [M+H] 1146.4115, found 1146.4208.
49
64
9
23
N,N’-di-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl-1,2,3-triazol-4-ylmethylamide)-
N’’-propyl-5-aminobenzene-1,3-dicarboxamide (7d). Prepared from 6 and 2,3,4,6-tetra-O-
4
3
acetyl-1-α-azido-D-mannopyranoside according to Method B: sticky, yellow amorphous
ꢂꢂ
1
solid (110 mg, 82 %).R = 0.42 (DCM:MeOH 9:1). [α]ꢀ +3 (c 1, DCM). H NMR (500
f
MHz, CDCl ): δ 8.93 (s, 1H, NHCOC H ), 8.23 (s, 2H, NHCH -triaz), 7.94 (s, 2H, triaz-H),
3
2
5
2
7
.78 (s, 2H, Ar-H), 7.55 (s, 1H, Ar-H), 6.11 (s, 2H, H-1), 5.99 (s, 2H, H-2), 5.90 (d, J = 9.8
Hz, 2H, H-3), 5.41 (t, J = 9.6 Hz, 2H, H-4), 4.65 –4.54 (m, 4H, CH -triaz), 4.27 (dd, J = 12.5,
2
2
2
2
.9 Hz, 2H, H-6), 4.05 (dd, J = 12.4, 3 Hz, 2H, H-6’), 3.97 (dd, J = 6.0, 3.5 Hz, 2H, H-5),
.48 – 2.33 (m, 4H, CH CH ), 2.17 (d, J = 1.2 Hz, 6H, OAc), 2.06 (d, J = 1.6 Hz, 6H, OAc),
2
3
1
3
.02 – 1.96 (m, 12H, OAc x2), 1.14 (dd, J = 9.4, 5.5 Hz, 6H, CH CH ). C NMR (125 MHz,
2
3

ACCEPTED MANUSCRIPT
CDCl ): δ 173.5 (COC H ), 170.7 (CO of OAc), 170.0 (CO of OAc), 169.9 (CO of OAc),
3
2
5
1
1
2
69.7 (CO of OAc), 166.6 (CONHCH -triaz), 145.6 (C-triaz), 138.9 (Ar-C), 134.4 (Ar-C),
2
23.5 (CH-triaz), 121.4 (Ar-CH), 120.2 (Ar-CH), 84.2 (C-1), 71.8 (C-5), 69.3 (C-3), 68.3 (C-
), 65.6 (C-4), 61.7 (C-6), 35.1 (CH -triaz), 30.3 (CH CH ), 20.8 (CH of OAc), 20.7 (CH of
2
2
3
3
3
OAc), 20.7 (CH of OAc), 20.6 (CH of OAc), 20.4 (CH of OAc), 9.5 (CH CH ). IR (film
3
3
3
2
3
-1
+
on NaCl): 3429, 2115, 1748, 1646 cm . HRMS (ESI+): m/z calcd for C H N O + H
45
56
9
21
+
[
M+H] 1058.3591, found 1058.3593.
N,N’-di-(2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-β-
ylmethylamide)-1,2,3-triazol-4-ylmethylamide)-N’’-propyl-5-aminobenzene-1,3-
dicarboxamide (7e). Prepared from 6 and 2-acetamido-2-deoxy-,3,4,6-tri-O-acetyl-1-β-
D-glucopyranosyl-1,2,3-triazol-4-
4
4
azido-
D-glucopyranoside according to Method B: yellow amorphous solid (60 mg, 42 %).
ꢂꢆ
1
R = 0.36 (DCM:MeOH 9:1). [α] -30 (c 0.4, DCM). H NMR (500 MHz, d -Pyr): δ 10.87
f
ꢀ
5
(s, 1H, NH), 9.88 (t, J = 5.6 Hz, 2H, NH), 9.74 (d, J = 9.0 Hz, 2H, NH), 8.90 (s, 2H, triaz-H),
8
.62 (s, 2H, Ar-H), 8.57 (s, 1H, Ar-H), 6.77 (d, J = 9.9 Hz, 2H, H-1), 6.06 (t, J = 9.6 Hz, 2H,
H-3 or 4), 5.61 (t, J = 9.7 Hz, 2H, H-2), 5.23 – 5.14 (m, 2H, H-3 or 4), 4.48 (dd, J = 12.3, 5.0
Hz, 2H, H-6), 4.38 (d, J = 8.2 Hz, 2H, H-5), 4.32 (d, J = 12.3 Hz, 2H, H-6’), 2.43 (q, J = 7.5
Hz, 2H, CH CH ), 2.10 (s, 3H, OAc), 2.01 (s, 3H, OAc), 1.89 (s, 3H, OAc), 1.72 (s, 3H,
2
3
OAc), 1.19 (t, J = 7.6 Hz, 3H, CH CH ). IR (ATR): 3305, 3078, 2924, 2850, 1743, 1667,
2
3
-1
+
+
1
651, 1529 cm . HRMS (ESI+): m/z calcd for C H N O + H [M+H] 1056.3910, found
45 57 11 19
1
056.3942.
N,N’-di-[{4-O-(2,3,4,6-tetra-O-acetyl-β-
glucopyranosyl}-1,2,3-triazol-4-ylmethylamide)-N’’-propyl-5-aminobenzene-1,3-
dicarboxamide (7f). Prepared from 6 and 4-O-(2,3,4,6-tetra-O-acetyl-β- -galactopyranosyl)-
-glucopyranoside according to Method B: sticky, yellow
D-galactopyranosyl)-2,3,6-tri-O-acetyl-β-D-
D
4
5
2
,3,6-tri-O-acetyl-1-β-azido-
D
ꢂꢂ
1
amorphous solid (102mg, 72 %). R = 0.62 (DCM:MeOH 9:1). [α]ꢀ 11 (c 1, DCM). H
f

ACCEPTED MANUSCRIPT
NMR (500 MHz, CDCl ): δ 8.55 (s, 1H, NHCOC H ), 7.93 – 7.84 (m, 6H, NHCH -triaz,
3
2
5
2
triaz-H and Ar-H), 7.68 (s, 1H, Ar-H), 5.84 (d, J = 9.2 Hz, 2H, H-1 Gal), 5.53 – 5.45 (m, 2H,
H-2 Gal), 5.40 (dd, J = 11.2, 7.2 Hz, 2H, H-3 Gal), 5.35 (dd, J = 7.0, 3.5 Hz, 2H, H-4 Glc),
5
.11 (dd, J = 10.3, 7.9 Hz, 2H, H-2 Glc), 5.02 – 4.96 (m, 2H, H-3 Glc), 4.63 (dd, J = 6.8, 4.3
Hz, 4H, CH -triaz), 4.57 (d, J = 7.9 Hz, 2H, H-1 Glc), 4.47 (dd, J = 11.1, 7.8 Hz, 2H, H-6
2
Glc), 4.18 – 4.05 (m, 6H, H-6’ Glc and H-6 and H-6’ (Gal)), 4.04 – 3.99 (m, 2H, H-4 Gal),
3
.93 (dd, J = 14.4, 8.4 Hz, 4H, H-5 Gal and H-5 Glc), 2.40 (q, J = 7.2 Hz, 2H, CH CH ), 2.19
2 3
13
–
1.92 (m, 42H, OAc x14), 1.81 (s, 3H), 1.19 (t, J = 7.5 Hz, 2H, CH CH ). C NMR (125
2
3
MHz, CDCl ): δ 173.1 (COC H ), 170.4 (CO of OAc), 170.1 (CO of OAc), 170.1 (CO of
3
2
5
OAc), 169.6 (CO of OAc), 169.5 (CO of OAc), 169.1 (CO of OAc), 166.6 (CONHCH2-
triaz), 145.6 (C-triaz), 138.9 (Ar-C), 134.7 (Ar-C), 121.5 (CH-triaz), 121.2 (Ar-CH), 120.7
(
Ar-CH), 101.1 (C-1 Glc), 85.5 (C-1 Gal), 76.0 (C-5 Gal), 75.6 (C-4 Gal), 72.6 (C-3 Gal),
0.9 (C-3 Glc), 70.8 (C-2 Gal), 70.7 (C-5 Glc) , 69.1 (C-2 Glc), 66.7 (C-4 Glc), 61.9 (C-6
Glc), 60.8 (C-6 Gal), 35.5 (CH -triaz), 30.4 (CH CH ), 20.7 (CH of OAc), 20.6 (CH of
7
2
2
3
3
3
OAc), 20.6 (CH of OAc), 20.5 (CH of OAc), 20.4 (CH of OAc), 20.3, 9.5 (CH CH ). IR
3
3
3
2
3
-1
(
film on NaCl): 3293, 2942, 1749, 1656, 1599, 1537 cm . HRMS (ESI+): m/z calcd for
+
+
C H N O + H [M+H] 1634.5281, found 1634.5287.
69
88
9
37
N,N’-di-(β-D-galactopyranosyl-1,2,3-triazol-4-ylmethylamide)-N’’-propyl-5-
ꢂꢅ
aminobenzene-1,3-dicarboxamide (2a). White amorphous solid (60 mg, 94 %). [α] +12.7
ꢀ
1
(
c 0.5, H O). H NMR (500 MHz, D O): δ 8.24 (s, 2H, triaz-H), 7.85 (s, 2H, Ar-H), 7.79 (s,
2
2
1
2
4
H, Ar-H), 5.66 (d, J = 8.8 Hz, 2H, H-1), 4.64 (s, 4H, CH -triaz), 4.20 (t, J = 9.2 Hz, 2H, H-
2
), 4.08 (d, J = 8.6 Hz, 2H, H-4), 3.97 (s, 2H, H-5), 3.86 (d, J = 9.7 Hz, 2H, H-3), 3.75 (d, J =
.7 Hz, 2H, H-6 and H-6’), 2.37 (d, J = 7.4 Hz, 2H, CH CH ), 1.12 (t, J = 7.3 Hz, 3H,
2
3
13
CH CH ). C NMR (125 MHz, D O): δ 176.8 (COC H ), 169.0 (CONHCH -triaz), 145.2
2
3
2
2
5
2
(C-triaz), 138.2, 134.6 (Ar-C), 123.0 (CH-triaz), 122.9, 122.1 (Ar-CH), 88.1 (C-1), 78.3 (C-

ACCEPTED MANUSCRIPT
5
), 72.9 (C-3), 69.8 (C-2), 68.6 (C-4), 60.9 (C-6), 35.1 (CH -triaz), 29.9 (CH CH ), 9.2
2
2
3
-1
(
CH CH ). IR (KBr): 3368, 2940, 2121, 1649, 1598, 1546 cm . HRMS (ESI+): m/z calcd. for
2 3
+
+
C H N O +H [M+H] 722.2746, found 722.2730.
29
40
9
13
N,N’-di-(β-L-fucopyranosyl-1,2,3-triazol-4-ylmethylamide)-N’’-propyl-5-aminobenzene-
1
,3-dicarboxamide (2b). Pale yellow amorphous solid (76 mg, 92 %). [α] ꢂꢀ ꢇ +4.3 (c 0.4,
1
H O). H NMR (500 MHz, D O): δ 8.22 (d, J = 4.1 Hz, 2H, triaz-H), 7.88 (d, J = 1.5 Hz, 2H,
2
2
Ar-H), 7.81 (s, 1H, Ar-H), 5.65 – 5.61 (d, J = 9.2 Hz, 2H, H-1), 4.65 (s, 4H, CH -triaz), 4.15
2
(t, J = 9.5 Hz, 2H, H-2), 4.08 – 4.02 (m, 2H, H-5), 3.90 – 3.82 (m, 4H, H-3 and H-4), 2.37 (q,
13
J = 7.6 Hz, 2H, CH CH ), 1.25 – 1.22 (m, 6H, C6-H ), 1.12 (t, J = 7.6 Hz, 3H, CH CH ). C
2
3
3
2
3
NMR (125 MHz, D O): δ 176.6 (COC H ), 168.7 (CONHCH -triaz), 144.8 (C-triaz), 138.2
2
2
5
2
(Ar-C), 134.4 (Ar-C), 123.0 (CH-triaz), 122.7 (Ar-CH), 121.9 (Ar-CH), 88.1 (C-1), 74.4 (C-
5
), 73.1 (C-3), 71.2 (C-4), 69.5 (C-2), 35.0 (CH -triaz), 29.9 (CH CH ), 15.5 (C-6), 9.1
2
2
3
-1
(
CH CH ). IR (ATR): 3261, 2917, 2851, 1646, 1601, 1536 cm . HRMS (ESI+): m/z calcd for
2 3
+
+
C H N O + H [M+H] 690.6910, found 690.6923.
29
39
9
11
N,N’-di-[2-O-(β-D-galactopyranosyl)-ethyl-1,2,3-triazol-4-ylmethylamide)-N’’-propyl-5-
aminobenzene-1,3-dicarboxamide (2c). Pale brown amorphous solid (55 mg, 91 %). [α] ꢀꢂ ꢈ
1
+
2.9 (c 0.3, H O). H NMR (500 MHz, D O): δ 8.10 (s, 2H, triaz-H), 8.00 (d, J = 1.5 Hz, 2H,
2
2
Ar-H), 7.93 (s, 1H, Ar-H), 4.73 – 4.67 (m, 8H, CH -triaz and CH CH ), 4.39 – 4.27 (m, 4H,
2
2
2
H-1 and CHCH ), 4.18 – 4.10 (m, 2H, CHCH ), 3.90 (dd, J = 3.4, 0.8 Hz, 2H, H-4), 3.77 –
2
2
3
.68 (m, 4H, H-6 and H-6’), 3.68 – 3.61 (m, 2H, H-5), 3.59 (dd, J = 9.9, 3.4 Hz, 2H, H-3),
.48 (dd, J = 10.0, 7.8 Hz, 2H, H-2), 2.47 (q, J = 7.6 Hz, 2H, CH CH ), 1.20 (td, J = 7.6, 1.6
3
2
3
13
Hz, 3H, CH CH ). C NMR (125 MHz, D O): δ 176.8 (COC H ), 168.8 (CONHCH -triaz),
2
3
2
2
5
2
1
44.3 (C-triaz), 138.1 (Ar-C), 134.6 (Ar-C), 124.8 (CH-triaz), 123.0 (Ar-CH), 122.1 (Ar-
CH), 103.0 (C-1), 75.1 (C-5), 72.6 (C-3), 70.6 (C-2), 68.5 (C-4), 60.9 (C-6), 35.0 (CH -triaz),
2
2
9.9 (CH CH ), 9.2 (CH CH ). IR (KBr): 3365, 3323, 3117, 3053, 2977, 2942, 2882, 1691,
2 3 2 3

ACCEPTED MANUSCRIPT
-1
+
+
1
651, 1614, 1564 cm . HRMS (ESI+): m/z calcd for C H N O + H [M+H] 810.3270,
33 48 9 15
found 810.3322.
N,N’-di-(α-D-mannopyranosyl-1,2,3-triazol-4-ylmethylamide)-N’’-propyl-5-
aminobenzene-1,3-dicarboxamide (2d). Pale yellow amorphous solid (42 mg, 88 %). [α] ꢀꢂ ꢂ
1
+
19.1 (c 0.4, H O). H NMR (500 MHz, D O): δ 8.14 (s, 2H, triaz-H), 7.85 (s, 2H, Ar-H),
2
2
7
9
2
.78 (s, 1H, Ar-H), 6.08 (s, 2H, H-1), 4.75 (s, 2H, H-2), 4.64 (s, 4H, CH -triaz), 4.14 (dd, J =
2
.0, 3.2 Hz, 2H, H-3), 3.86 – 3.74 (m, 6H, H-4 and H-6 and H-6’), 3.38 – 3.29 (m, 2H, H-5),
13
.37 (q, J = 7.6 Hz, 2H, CH CH ), 1.12 (t, J = 7.6 Hz, 3H, CH CH ). C NMR (125 MHz,
2
3
2
3
D O): δ 176.4 (COC H ), 168.5 (CONHCH -triaz), 145.0 (C-triaz), 138.2 (Ar-C), 134.4 (Ar-
2
2
5
2
C), 123.7 (CH-triaz), 122.4 (Ar-CH), 121.7 (Ar-CH), 86.7 (C-1), 76.2 (C-5), 70.6 (C-3), 68.3
(
C-2), 66.6 (C-4), 60.5 (C-6), 35.0 (CH -triaz), 29.9 (CH CH ), 9.1 (CH CH ). IR (KBr):
2 2 3 2 3
-1
+
+
3
375, 2941, 1649, 1555 cm . HRMS (ESI+): m/z calcd for C H N O + H [M+H]
29 40 9 13
7
22.2746, found 722.2740.
N,N’-di-(2-Acetamido-2-deoxy-β-D-glucopyranosyl-1,2,3-triazol-4-ylmethylamide)-1,2,3-
triazol-4-ylmethylamide)-N’’-propyl-5-aminobenzene-1,3-dicarboxamide (2e). Pale
ꢂꢂ
1
yellow amorphous solid (34 mg, 75 %). [α] -5.2 (c 0.3, H O). H NMR (500 MHz, D O): δ
ꢀ
2
2
8
.20 (s, 2H, triaz-H), 8.01 (t, J = 3.9 Hz, 2H, Ar-H), 7.96 – 7.89 (m, 1H, Ar-H), 5.86 (d, J =
9
.7 Hz, 2H, H-1), 4.72 – 4.66 (m, 4H, CH -triaz), 4.27 (t, J = 10.0 Hz, 2H, H-2), 4.01 – 3.64
2
(
m, 10H, H-3, H-4, H-5, H-6 and H-6’), 2.48 (q, J = 7.6 Hz, 2H, CH CH ), 1.79 (s, 3H,
2 3
1
3
NHAc), 1.21 (t, J = 7.6 Hz, 3H, CH CH ). C NMR (125 MHz, D O): δ 176.8 (COC H ),
2
3
2
2
5
1
74.1 (CO of NHAc), 168.9 (CONHCH -triaz), 123.2 (Ar-CH), 122.8 (CH-triaz), 122.2 (Ar-
2
CH), 86.4 (C-1), 78.9 (C-5), 73.5 (C-3), 69.3 (C-4), 60.4 (C-6), 55.4 (C-2), 35.0 (CH -triaz),
2
-
3
0.0 (CH CH ), 21.6 (CH of NHAc), 9.2 (CH CH ). IR (ATR): 3370, 2943, 1648, 1557 cm
2 3 3 2 3
1
+
+
.
HRMS (ESI+): m/z calcd for C H N O + Na [M+Na] 826.3096, found 826.3102.
33 45 11 13

ACCEPTED MANUSCRIPT
N,N’-di-[{4-O-β-D-galactopyranosyl-β-D-glucopyranosyl}-1,2,3-triazol-4-
ylmethylamide)-N’’-propyl-5-aminobenzene-1,3-dicarboxamide (2f). White amorphous
ꢃꢉ
1
(
65 mg, 90 %). [α] +1.5 (c 0.6, H O). H NMR (500 MHz, D O): δ 8.16 (s, 2H, triaz-H),
ꢀ
2
2
7
.91 (s, 2H, Ar-H), 7.84 (s, 1H, Ar-H), 5.72 (d, J = 9.2 Hz, 2H, H-1 Glc), 4.64 (s, 4H, CH2-
triaz), 4.45 (d, J = 7.8 Hz, 2H, H-1 Gal), 4.00 (t, J = 9.0 Hz, 2H, H-2 Glc), 3.93 – 3.87 (m,
3
3
3
H, H-6 Glc and H-4 Gal), 3.85 – 3.79 (m, 4H, H-3 Glc, H-4 Glc, H-5 Glc and H-6’ Glc),
.78 – 3.65 (m, 4H, H-6 Gal, H-6’ Gal and H-5 Gal), 3.65 – 3.57 (m, 2H, H-3 Gal), 3.56 –
.49 (m, 2H, H-2 Gal), 2.38 (q, J = 7.6 Hz, 1H, CH CH ), 1.12 (dd, J = 9.4, 5.8 Hz, 3H,
2
3
13
CH CH ). C NMR (125 MHz, D O): δ 168.8 (CONHCH -triaz), 145.0 (C-triaz), 138.2 (Ar-
2
3
2
2
C), 134.4 (Ar-C), 123.1 (CH-triaz), 122.8 (Ar-CH), 102.8 (C-1 Gal), 87.2 (C-1 Glc), 77.6 (C-
4
7
/5 Glc), 77.2 (C-4/5 Glc), 75.3 (C-5 Gal), 74.4 (C-3 Glc), 72.4 (C-3 Gal), 71.9 (C-2 Glc),
0.9 (C-2 Gal), 68.5 (C-4 Gal), 61.0 (C-6 Gal), 59.7 (C-6 Glc), 35.0 (CH -triaz), 29.8
2
-1
(
CH CH ), 9.1 (CH CH ). IR (KBr): 3412, 2923, 2125, 1644, 1548 cm . HRMS (ESI+): m/z
2 3 2 3
+
+
calcd for C H N O + H [M+H] 1046.3802, found 1046.2788.
4
1
60
9
23
N-(prop-2-yn-1-yl)-3-propionamidobenzamide (9). 3-aminobenzoic acid (2 g, 14.6 mmol)
was dissolved in anhydrous THF (15 mL) under N and propionyl chloride (3.19 mL, 36.5
2
mmol) was added dropwise. The mixture was allowed to stir for 5 min and NEt (6.1 mL,
3
4
3.8 mmol) was added slowly. The reaction was left to stir for 16 h. The solvent was
removed in vacuo. The crude mixture was dissolved in ethyl acetate (30 mL), washed with
.5 M HCl (30 mL), and dried (MgSO ). The mixture was filtered and the solvent was
0
4
removed under reduced pressure to yield the product 3-(propionylamino)benzoic acid as an
off-white amorphous (0.637 g, 23 %) which was used without further purification. This acid
(0.307 g, 1.59 mmol) and TBTU (0.56 g, 1.75 mmol) were dissolved in anhydrous DMF (15
mL) under N . NEt (0.3 mL, 2.38 mmol) was added, and the reaction mixture was stirred for
2
3
1
0 min on ice. Propargylamine (0.15 mL, 2.38 mmol) was added, and the reaction was stirred

ACCEPTED MANUSCRIPT
for 16 h at rt. The solvent was removed in vacuo. The crude mixture was dissolved in ethyl
acetate (30 mL), washed with 0.5 M HCl (30 mL), sat. NaHCO (30 mL) and brine (30 mL),
3
and dried (MgSO ). The mixture was filtered and the solvent was removed in the rotatory
4
1
evaporator to yield product 9: pale yellow amorphous (0.359 g, 98 %). H NMR (500 MHz,
d -DMSO): δ 9.99 (s, 1H, NHCH CCH), 8.86 (s, 1H, NHCOC H ), 8.04 (s, 1H, Ar-H), 7.77
6
2
2
5
(d, J = 8.2 Hz, 1H, Ar-H), 7.48 (d, J = 7.7 Hz, 1H, Ar-H), 7.37 (t, J = 7.9 Hz, 1H, Ar-H), 4.03
(
dd, J = 5.5, 2.4 Hz, 2H, CH CCH), 3.10 (t, J = 2.3 Hz, 1H, CH CCH), 2.32 (q, J = 7.5 Hz,
2
2
1
3
2
H, CH CH ), 1.08 (t, J = 7.5 Hz, 3H, CH CH ). C NMR (125 MHz, d -DMSO): δ 172.6
2 3 2 3 6
(
COC H ), 166.4 (CONHCH -triaz), 139.9 (Ar-C), 135.0 (Ar-C), 129.1 (Ar-CH), 122.3 (Ar-
2 5 2
CH), 121.8 (Ar-CH), 118.9 (Ar-CH), 81.8 (CH CCH), 73.2 (CH CCH), 30.0 (CH CH ), 29.0
2
2
2
3
(
CH CCH), 10.1 (CH CH ). IR (KBr): 3365, 3321, 3298, 3117, 2977, 2942, 1690, 1652,
2 2 3
-1
+
+
1
562 cm . HRMS (ESI+): m/z calcd for C H N O +H [M+H] 231.1134, found 231.1135.
13 15 2 2
N-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-1,2,3-triazol-4-ylmethylamide)-N’-
propyl-3-aminobenzene-1-carboxamide (10a). Prepared from 9 and 2,3,4,6-tetra-O-acetyl-
3
4
1
-β-azido-D-galactopyranoside, according to Method A: Off-white amorphous solid (235
ꢃꢄ
1
mg, 83%). R = 0.45 (DCM:MeOH 9:1). [α]ꢀ -6.9 (c 0.9, DCM). H NMR (500 MHz,
f
CDCl ): δ 8.62 (s, 1H, NHCOC H ), 7.95 (s, 1H, triaz-H), 7.89 – 7.78 (m, 2H, Ar-H x2), 7.72
3
2
5
(
s, 1H, NHCH -triaz), 7.48 (d, J = 7.4 Hz, 1H, Ar-H), 7.28 (t, J = 7.8 Hz, 1H, Ar-H), 5.91 (d,
2
J = 9.2 Hz, 1H, H-1), 5.56 (m, 2H, H-2 and H-4), 5.34 – 5.24 (m, 2H, H-3), 4.68 (dd, J =
1
6
4.5, 3.5 Hz, 2H, CH -triaz), 4.31 (t, J = 6.1 Hz, 1H, H-5), 4.15 (dd, J = 11.5, 6.8 Hz, 2H, H-
2
and H-6’), 2.39 (q, J = 7.4 Hz, 2H, CH CH ), 2.18 (s, 3H, OAc), 2.00 (s, 6H, OAc x2), 1.82
2
3
13
(
s, 3H, OAc), 1.18 (t, J = 7.5 Hz, 3H, CH CH ). C NMR (125 MHz, CDCl ): δ 173.0
2 3 3
(
NHCOC H ), 170.4 (CO of OAc), 170.1 (CO of OAc), 169.9 (CO of OAc), 169.1 (CO of
2
5
OAc), 167.5 (CONHCH -triaz), 145.4 (C-triaz), 138.8 (Ar-C), 134.5 (Ar-C), 129.1 (Ar-CH),
2
1
23.1 (Ar-CH), 122.3 (Ar-CH), 121.6 (CH-triaz), 118.5 (Ar-CH), 86.1 (C-1), 73.9 (C-5), 70.8

ACCEPTED MANUSCRIPT
(
C-3), 68.0 (C-2), 66.9 (C-4), 61.2 (C-6), 35.3 (CH -triaz), 30.4 (CH CH ), 20.6 (CH of
2
2
3
3
OAc), 20.6 (CH of OAc), 20.5 (CH of OAc), 20.2 (CH of OAc), 9.6 (CH CH ). IR (film
3
3
3
2
3
-1
on NaCl): 3311, 2980, 1753, 1652, 1591, 1553 cm . HRMS (ESI+): m/z calcd for C H N-
2
7
34
+
+
O +H [M+H] 604.2255, found 604.2262.
5
11
N-(2,3,4-tri-O-acetyl-β-
L-fucopyranosyl-1,2,3-triazol-4-ylmethylamide)-N’-propyl-3-
aminobenzene-1-carboxamide (10b). Prepared from 9 and 2,3,4-tri-O-acetyl-1-β-azido-L-
4
1
fucopyranoside according to Method A: Off-white amorphous solid (90 mg, 76 %). R =0.56
f
DCM:MeOH 9:1). [α] ꢂꢀ ꢈ +16.1 (c 1, DCM). H NMR (500 MHz, CDCl ): δ 8.32 (s, 1H,
1
(
:
3
NHCOC H ), 7.90 (s, 1H, triaz-H), 7.87 (d, J = 8.0 Hz, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.46
2
5
(
m, 2H, Ar-H and NHCH -triaz), 7.28 (t, J = 8.0 Hz, 1H, Ar-H), 5.78 (d, J = 9.2 Hz, 1H, H-
2
1
), 5.52 – 5.46 (m, 1H, H-2), 5.35 (d, J = 2.9 Hz, 1H, H-4), 5.24 (dd, J = 10.3, 3.4 Hz, 1H, H-
3
), 4.66 (dd, J = 15.2, 5.6 Hz, 2H, CH -triaz), 4.11 (q, J = 6.4 Hz, 1H, H-5), 2.37 (q, J = 7.5
2
Hz, 2H, CH CH ), 2.20 (s, 3H, OAc), 1.97 (s, 3H, OAc), 1.81 (s, 3H, OAc), 1.26 – 1.13 (m,
2
3
1
3
6
H, C6-H and CH CH ). C NMR (125 MHz, CDCl ): δ 171.8 (COC H ), 169.5 (CO of
3 2 3 3 2 5
OAc), 168.9 (CO of OAc), 168.2 (CO of OAc), 166.4 (CONHCH -triaz), 144.3 (CH-triaz),
2
1
37.8 (Ar-C), 133.6 (Ar-C), 128.2 (Ar-CH), 122.1 (Ar-CH), 121.4 (Ar-CH), 120.4 (CH-
triaz), 117.4 (Ar-CH), 85.3 (C-1), 71.7 (C-5), 70.2 (C-3), 68.9 (C-4), 67.2 (C-2), 34.4 (CH2-
triaz), 29.5 (CH CH ), 19.7 (CH of OAc), 19.5 (CH of OAc), 19.3 (CH of OAc), 15.0 (C-
2
3
3
3
3
6
1
5
), 8.6 (CH CH ). IR (film on NaCl): 3308, 3146, 3085, 2985, 2941, 2248, 1750, 1647, 1591,
2 3
-1
+
+
553 cm . HRMS (ESI+): m/z calcd for C H N O + H [M+H] 546.2200, found
2
5
32
5
9
46.2197.
N-[2-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-ethyl-1,2,3-triazol-4-
ylmethylamide)-N’-propyl-3-aminobenzene-1-carboxamide (10c). Prepared from 9 and 2-
4
2
O-(2,3,4,6-tetra-O-acetyl-β-
D-galactopyranosyl)ethyl azide according to Method A: Off-
ꢂꢇ
1
white amorphous solid (97 mg, 88 %). R =0.36 (DCM:MeOH 9:1). [α] -3.1 (c 1, DCM). H
f
ꢀ

ACCEPTED MANUSCRIPT
NMR (500 MHz, CDCl ): δ 8.41 (s, 1H, NHCOC H ), 7.90 (d, J = 8.0 Hz, 1H, Ar-H), 7.80
3
2
5
(
s, 1H, Ar-H), 7.63 (s, 1H, triaz-H), 7.56 (t, J = 5.1 Hz, 1H, NHCH -triaz), 7.46 (d, J = 7.7
2
Hz, 1H, Ar-H), 7.27 (t, J = 8 Hz, 1H, Ar-H), 5.34 (dd, J = 3.4, 1.0 Hz, 1H, H-4), 5.11 (dd, J =
1
2.5, 6.2 Hz, 1H, H-2), 4.96 (dd, J = 10.5, 3.4 Hz, 1H, H-3), 4.69 –4.59 (m, 2H, CH -triaz),
2
4
.56 – 4.44 (m, 2H, CH CH O), 4.42 (d, J=7.9, 1H, H-1), 4.18 (dt, J = 10.5, 4.1 Hz, 1H,
2
2
CHO-Gal), 4.08 (dd, J = 11.3, 6.6 Hz, 2H, H-6 and H-6’), 3.95 – 3.85 (m, 2H, CHO-Gal and
H-5), 2.36 (q, J = 7.5 Hz, 2H, CH CH ), 2.09 (s, 3H, OAc), 1.99 (s, 3H, OAc), 1.93 (s, 3H,
2
3
13
OAc), 1.90 (s, 3H, OAc), 1.16 (t, J = 7.6 Hz, 3H, CH CH ). C NMR (125 MHz, CDCl ): δ
2
3
3
1
72.8 (COC H ), 170.4 (CO of OAc), 170.2 (CO of OAc), 170.0 (CO of OAc), 169.7 (CO of
2 5
OAc), 167.3 (CONHCH -triaz), 144.5 (C-triaz), 138.9 (Ar-C), 134.6 (Ar-C), 129.1 (Ar-CH),
2
1
7
3
23.8 (CH-triaz), 123.0 (Ar-CH), 122.2 (Ar-CH), 118.5 (Ar-CH), 100.9 (C-1), 70.9 (C-5),
0.6 (C-3), 68.6 (C-2), 67.5 (CH CH O-Gal), 66.9 (C-4), 61.2 (C-6), 50.1 (CH CH O-Gal),
2
2
2
2
5.5 (CH -triaz), 30.5 (CH CH ), 20.6 (CH of OAc), 20.6 (CH of OAc), 20.6 (CH of
2
2
3
3
3
3
OAc), 20.5 (CH of OAc), 9.6 (CH CH ). IR (film on NaCl): 3312, 3146, 2980, 2941, 2250,
3
2
3
-1
+
+
2
111, 1750, 1649, 1591, 1552 cm . HRMS (ESI+): m/z calcd for C H N O + H [M+H]
29 37 5 12
6
48.2517, found 648.2581.
-galactopyranosyl-1,2,3-triazol-4-ylmethylamide)-N’-propyl-3-aminobenzene-1-
N-(β-
D
carboxamide (1a). White amorphous solid (73 mg, 92 %). [α] ꢃꢀ ꢄ +11.6 (c 0.7, H O). H
1
2
NMR (500 MHz, D O): δ 8.09 (s, 1H, triaz-H), 7.55 (t, J = 1.8 Hz, 1H, Ar-H), 7.34 (ddd, J =
2
8
.0, 2.1, 1.0 Hz, 1H, Ar-H), 7.32 – 7.29 (m, 1H, Ar-H), 7.21 (t, J = 7.9 Hz, 1H, Ar-H), 5.53
(
d, J = 9.2 Hz, 1H, H-1), 4.48 (s, 2H, CH -triaz), 4.07 (t, J = 9.5 Hz, 1H, H-2), 3.93 (dd, J =
2
3
3
7
.3, 0.6 Hz, 1H, H-4), 3.83 (td, J = 6.0, 0.8 Hz, 1H, H-5), 3.72 (dd, J = 9.8, 3.3 Hz, 1H, H-3),
.62 (d, J = 6.1 Hz, 2H, H-6 and H-6’), 2.24 – 2.18 (q, J = 7.7 Hz, 2H, CH CH ), 0.99 (t, J =
2
3
1
3
.6 Hz, 3H, CH CH ). C NMR (125 MHz, D O): δ 176.5 (NHCOC H ), 169.7 (CONHCH -
2
3
2
2
5
2
triaz), 145.0 (C-triaz), 137.5 (Ar-C), 133.7 (Ar-C), 129.4 (Ar-CH), 124.8 (Ar-CH), 123.5 (Ar-

ACCEPTED MANUSCRIPT
CH), 123.1 (CH-triaz), 119.8 (Ar-CH), 88.2 (C-1), 78.3 (C-5), 72.9 (C-3), 69.8 (C-2), 68.6
(
C-4), 60.9 (C-6), 34.9 (CH -triaz), 29.8 (CH CH ), 9.2 (CH CH ). IR (ATR): 3268, 1643,
2 2 3 2 3
-
1
+
+
1
4
588, 1542 cm . HRMS (ESI+): m/z calcd for C H N O +H [M+H] 436.1882, found
19 26 5 7
36.1826.
-fucopyranosyl-1,2,3-triazol-4-ylmethylamide)-N’-propyl-3-aminobenzene-1-
N-(β-
L
ꢂꢇ
1
carboxamide (1b). Yellow amorphous solid (63 mg, 94 %). [α] -6.3 (c 0.6, H O). H NMR
ꢀ
2
(
500 MHz, d -DMSO): δ 10.02 (s, 1H, NHCOC H ), 9.01 (t, J = 5.6 Hz, 1H, NHCH -triaz),
6 2 5 2
8
.07 (m, 2H, triaz-H and Ar-H), 7.82 (d, J = 8.2 Hz, 1H, Ar-H), 7.56 (d, J = 7.7 Hz, 1H, Ar-
H), 7.40 (t, J = 7.9 Hz, 1H, Ar-H), 5.47 (d, J = 9.2 Hz, 1H, H-1), 5.20 (d, J = 5.9 Hz, 1H,
OH), 4.96 (d, J = 5.4 Hz, 1H, OH), 4.67 (d, J = 5.7 Hz, 1H, OH), 4.61 – 4.49 (m, 2H, CH2-
triaz), 3.99 (dd, J = 15.0, 9.1 Hz, 1H, H-2), 3.89 (q, J = 6.4 Hz, 1H, H-5), 3.56 (m, 2H, H-3
and H-4), 2.36 (q, J = 7.6 Hz, 2H, CH CH ), 1.16 (d, J = 6.4 Hz, 3H, C6-H ), 1.12 (t, J = 7.5
2
3
3
13
Hz, 3H, CH CH ). C NMR (125 MHz, d -DMSO): δ 172.6 (COC H ), 166.7 (CONHCH -
2
3
6
2
5
2
triaz), 145.6 (C-triaz), 139.9 (Ar-C), 135.3 (Ar-C), 129.1 (Ar-CH), 122.3 (Ar-CH), 122.0 (Ar-
CH), 122.0 (CH-triaz), 119.0 (Ar-CH), 88.5 (C-1), 74.4 (C-3), 73.7 (C-5), 71.6 (C-4), 69.5
(
C-2), 35.4 (CH -triaz), 30.0 (CH CH ), 16.9 (C-6), 10.1 (CH CH ). IR (KBr): 3401, 2925,
2 2 3 2 3
-1
+
+
1
645, 1589, 1542 cm . HRMS (ESI+): m/z calcd for C H N O + H [M+H] 436.1832,
19 25 7 7
found 436.1849.
N-[2-O-(β-D-galactopyranosyl)-ethyl-1,2,3-triazol-4-ylmethylamide)-N’-propyl-3-
ꢂꢊ
aminobenzene-1-carboxamide (1c). Off-white amorphous solid (104 mg, 87 %). [α] +3.8
ꢀ
1
(
c 1, MeOH). H NMR (500 MHz, D O): δ 8.00 (s, 1H, triaz-H), 7.75 (s, 1H, Ar-H), 7.56-
2
7
.53 (m, 2H, Ar-H), 7.40 (t, J = 7.8 Hz, 1H, Ar-H), 4.50-4.46 (m, 4H, CH -triaz and
2
CH CH ), 4.29-4.20 (m, 2H, H-1 and CHCH ), 4.15-4.10 (m, 1H, CHCH ), 3.89 (s, 1H, H-4),
2
2
2
2
3
.80-3.40 (m, 5H, H-6, H-6’, H-2, H-3 and H-5), 2.33 (s, 2H, CH CH ), 1.08 (s, 3H,
2 3
13
CH CH ). C NMR (125 MHz, D O) δ 176.9 (COC H ), 168.8 (CONHCH -triaz), 144.4 (C-
2
3
2
2
5
2

ACCEPTED MANUSCRIPT
triaz), 137.3 (Ar-C), 134.0 (Ar-C), 130.7 (Ar-CH), 129.6, 127.8 (Ar-CH), 126.6 (Ar-CH),
1
25.2 (CH-triaz), 123.8, 122.1 (Ar-CH), 103.0 (C-1), 75.1 (C-5), 72.6 (C-3), 70.6 (C-2), 68.5
(
(
C-4), 67.8 (CH CH ), 60.9 (C-6), 50.8 (CH CH ), 34.8 (CH -triaz), 29.8 (CH CH ), 9.3
2
2
2
2
2
2
3
-1
CH CH ). IR (KBr): 3400, 2934, 2615, 1648, 1590, 1549 cm . HRMS (ESI+): m/z calcd for
2
3
+
+
C H N O + H [M+H] 480.2094, found 480.2107.
21
29
5
8
N,N’,N’’-tri-(2,3,4,-tri-O-acetyl-β-L-fucopyranosyl-1,2,3-triazol-4-ylmethylamide)-
4
6
benzene-1,3,5-tricarboxamide (12b). Prepared from 11 and 2,3,4-tri-O-acetyl-1-β-azido-
4
1
ꢂꢈ
ꢀ
L
-fucopyranoside according to Method A: yellow amorphous solid (93 mg, 72 %). [α]
1
+
24.7 (c 0.9, DCM). H NMR (500 MHz, CDCl ): δ 8.22 (s, 3H, Ar-H), 8.06 (s, 3H, NHCH -
3
2
triaz), 8.00 (s, 3H, triaz-H), 5.89 (d, J = 9.2 Hz, 3H, H-1), 5.55 (t, J = 9.7 Hz, 3H, H-2), 5.36
d, J = 3.0 Hz, 3H, H-4), 5.25 (dd, J = 10.2, 3.3 Hz, 3H, H-3), 4.68 (dd, J = 58.5, 10.9 Hz,
(
6
H, CH -triaz), 4.17 (q, J = 6.2 Hz, 3H, H-5), 2.21 (s, 9H, OAc), 1.98 (s, 9H, OAc), 1.77 (s,
2
13
9
H, OAc), 1.21 (d, J = 6.3 Hz, 9H, C6-H ). C NMR (125 MHz, CDCl ): δ 169.5 (CO of
3
3
OAc), 168.9 (CO of OAc), 168.2 (CO of OAc), 165.1 (CONHCH -triaz), 144.4 (C-triaz),
2
1
27.7 (Ar-CH), 120.6 (CH-triaz), 85.3 (C-1), 71.7 (C-5), 70.2 (C-3), 68.9 (C-4), 67.2 (C-2),
4.4 (CH -triaz), 19.7 (CH of OAc), 19.6 (CH of OAc), 19.3 (CH of OAc), 15.0 (C-6). IR
3
2
3
3
3
-1
(
KBr): 3411, 2989, 2942, 2115, 1751, 1659, 1537 cm . HRMS (ESI+): m/z calcd for
+
+
C H N O + H [M+H] 1179.4595, found 1179.4610.
52
67 12 20
N,N’,N’’-tri-[2-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-ethyl-1,2,3-triazol-4-
ylmethylamide)-benzene-1,3,5-tricarboxamide (12c). Prepared from 11 and 2-O-(2,3,4,6-
4
2
tetra-O-acetyl-β-
D
-galactopyranosyl)ethyl azide according to Method A: Off-white
amorphous solid (69 mg, 50 %). [α] ꢂꢀ ꢇ -5.8 (c 0.7, DCM). H NMR (500 MHz, CDCl ): δ
1
3
8
.22 (s, 3H, NHCH -triaz), 8.16 (s, 3H, triaz-H), 7.67 (s, 3H, Ar-H), 5.34 (d, J = 3.2 Hz, 3H,
2
H-4), 5.12 (dd, J = 10.4, 8.0 Hz, 3H, H-2), 4.98 (dd, J = 10.5, 3.4 Hz, 3H, H-3), 4.79 – 4.50
m, 12H, CH -triaz and CH CH O), 4.47 (d, J = 7.9 Hz, 3H, H-1), 4.28 – 4.18 (m, 3H, CHO-
(
2
2
2

ACCEPTED MANUSCRIPT
Gal), 4.09 (dd, J = 11.3, 6.6 Hz, 6H, H-6 and H-6’), 4.00-3.86 (m, 6H, CHO-Gal and H-5),
1
3
2
.11 (s, 9H, OAc), 2.00 (s, 9H, OAc), 1.93 (s, 18H, OAc x 2). C NMR (125 MHz, CDCl ):
3
δ 170.4 (CO of OAc), 170.1 (CO of OAc), 170.0 (CO of OAc), 169.7 (CO of OAc), 165.9
CONHCH -triaz), 144.7 (C-triaz), 134.6 (Ar-C), 128.5 (CH-triaz), 123.6 (Ar-CH), 100.9 (C-
(
2
1
), 70.6 (C-5), 68.6 (C-3), 67.5 (C-2), 67.0 (CH CH O-Gal), 61.2 (C-4), 50.1 (C-6), 35.5
2 2
(
CH -triaz), 20.7 (CH of OAc), 20.6 (CH of OAc), 20.6 (CH of OAc), 20.5 (CH of OAc).
2 3 3 3 3
-1
IR (film on NaCl): 3391, 2939, 1748, 1661, 1537 cm . HRMS (ESI+): m/z calcd for
+
+
C H N O + H [M+H] 1573.5342, found 1574.5422.
66
84 12 33
N,
N’,
N’’-tri-(β-
D-galactopyranosyl-1,2,3-triazol-4-ylmethylamide)-benzene-1,3,5-
tricarboxamide
(3a).
Prepared
from
N,N’,N’’-tri-(2,3,4,6-tetra-O-acetyl-β-
D
-
4
7
galactopyranosyl-1,2,3-triazol-4-ylmethylamide)-benzene-1,3,5-tricarboxamide
(12a).
ꢂꢇ
1
Yellow amorphous solid (63 mg, 89 %). [α] +11.1 (c 0.6, H O). H NMR (500 MHz, D O):
ꢀ
2
2
δ 8.24 (s, 3H, triaz-H), 8.19 (s, 3H, Ar-H), 5.65 (d, J = 9.2 Hz, 3H, H-1), 4.64 (s, 6H, CH2-
triaz), 4.19 (t, J = 9.5 Hz, 3H, H-2), 4.06 (d, J = 3.2 Hz, 3H, H-4), 3.96 (t, J = 6.1 Hz, 3H, H-
1
3
5
), 3.85 (dd, J = 9.8, 3.3 Hz, 3H, H-3), 3.74 (d, J = 6.0 Hz, 6H, H-6 and H-6’). C NMR (125
MHz, D O): δ 168.1 (CONHCH -triaz), 144.8 (C-triaz), 134.3 (Ar-C), 129.2 (Ar-CH), 123.2
2
2
(CH-triaz), 88.2 (C-1), 78.3 (C-5), 73.0 (C-3), 69.8 (C-2), 68.6 (C-4), 60.9 (C-6), 35.1 (CH2-
-1
+
triaz). IR (KBr): 3402, 1658, 1539, cm . HRMS (ESI+): m/z calcd for C H N O + H
36
48 12 18
+
[
M+H] 937.3288, found 937.3201.
N,N’,N’’-tri-(β- -fucopyranosyl-1,2,3-triazol-4-ylmethylamide)-benzene-1,3,5-
L
tricarboxamide (3b). Yellow amorphous solid (53 mg, 88 %). [α] ꢂꢀ ꢃ.ꢅ -5.6 (c 0.5, H O). H
1
2
NMR (500 MHz, D O): δ 8.26 (s, 3H, Ar-H), 8.25 (s, 3H, triaz-H), 5.66 (d, J = 9.2 Hz, 3H,
2
H-1), 4.70 (s, 6H, CH -triaz), 4.19 (t, J = 9.4 Hz, 3H, H-2), 4.10 – 4.06 (m, 3H, H-5), 3.91
2
(dd, J = 3.4, 0.8 Hz, 3H, H-4), 3.88 (dd, J = 9.7, 3.4 Hz, 3H, H-3), 1.28 – 1.26 (m, 9H, C6-
1
3
H ). C NMR (125 MHz, D O): δ 168.3 (CONHCH -triaz), 144.9 (C-triaz), 134.3 (Ar-C),
3
2
2