Multivalent Presentations of Glycomimetic Inhibitor of the Adhesion of Fungal Pathogen Candida albicans to Human Buccal Epithelial Cells

Article abstract of DOI:10.1021/acs.bioconjchem.1c00115

Candida albicans causes some of the most prevalent hospital-acquired fungal infections, particularly threatening for immunocompromised patients. C. albicans strongly adheres to the surface of epithelial cells so that subsequent colonization and biofilm fo

Full text of DOI:10.1021/acs.bioconjchem.1c00115

pubs.acs.org/bc
Article
Multivalent Presentations of Glycomimetic Inhibitor of the Adhesion
of Fungal Pathogen Candida albicans to Human Buccal Epithelial
Cells
Harlei Martin, David Goyard, Anatte Margalit, Kyle Doherty, Olivier Renaudet,* Kevin Kavanagh,*
and Trinidad Velasco-Torrijos*
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ABSTRACT: Candida albicans causes some of the most prevalent
hospital-acquired fungal infections, particularly threatening for
immunocompromised patients. C. albicans strongly adheres to the
surface of epithelial cells so that subsequent colonization and
biofilm formation can take place. Divalent galactoside glycomi-
metic 1 was found to be a potent inhibitor of the adhesion of C.
albicans to buccal epithelial cells. In this work, we explore the effect
of multivalent presentations of glycomimetic 1 on its ability to
inhibit yeast adhesion and biofilm formation. Tetra-, hexa-, and
hexadecavalent displays of compound 1 were built on RAFT
cyclopeptide- and polylysine-based scaffolds with a highly efficient
and modular synthesis. Biological evaluation revealed that the
scaffold choice significantly influences the activity of the lower
valency conjugates, with compound 16, constructed on a tetravalent polylysine scaffold, found to inhibit the adhesion of C. albicans
to human buccal epithelial cells more effectively than the glycomimetic 1; however, the latter performed better in the biofilm
reduction assays. Interestingly, the higher valency glycoconjugates did not outperform the anti-adhesion activity of the original
compound 1, and no significant effect of the core scaffold could be appreciated. SEM images of C. albicans cells treated with
compounds 1, 14, and 16 revealed significant differences in the aggregation patterns of the yeast cells.
arrangements of recognition epitopes have been developed to
increase the affinity of carbohydrates for their target proteins.17
These multivalent constructs can have well-defined molecular
structures and display a specific number of carbohydrate
ligands when they are built around scaffolds such as
calixarenes,18 dendrimers,19 cyclodextrins,20 and fullerenes;21
higher valencies with more variable degrees of substitution are
achieved when using polymers,22 nanoparticles,23 and quantum
dots.24 Even with the extensive research carried out in this
field, it still remains extremely challenging to predict optimal
multivalent presentations that result in enhanced binding
affinities for a specific target.
INTRODUCTION
■
The initiation of a multitude of human diseases is mediated by
protein−carbohydrate recognition.1,2 For a microbe to infect
its host, it first adheres to the host cell using adhesion proteins,
many of which recognize carbohydrate epitopes displayed on
the host cell surface.3 The development of small-molecule
inhibitors of this adhesion process has been extensively
studied. Glycoconjugates that mimic the glycans displayed on
host cell surfaces are attractive candidates for the development
of anti-adhesion compounds.4 Numerous ligands for microbial
lectins found in pathogens such as Pseudomonas aeruginosa,
Aspergillus fumigatus, and Escherichia coli have been
reported.5−10 Significantly, the crystal structures of these
adhesins have been solved, which has facilitated structure-
based ligand design.11−13 However, carbohydrates interact with
their protein receptors with low affinity, with dissociation
constants typically in the millimolar to micromolar range.14
Consequently, a major focus of glycoscience research involves
the development of strategies for increasing the lectin-ligand
binding affinities to levels required for therapeutic use.15 This
can be achieved through the glycomimetic approach or
through utilizing the multivalent or cluster glycoside effect.5,16
Multivalent glycoconjugates with diverse valencies and spatial
Lysine-containing cyclodecapeptides called “regioselectively
addressable functionalized templates” (RAFT) were first
described as stable scaffolds for the de novo design of proteins
Received: March 6, 2021
Revised: April 8, 2021
© XXXX The Authors. Published by
American Chemical Society
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Figure 1. Structure of lead compound 1 and multivalent displays on tetra-, hexa-, and hexadecavalent RAFT cyclopeptides (R4 11, R6 14, RR16 18)
and dendrimeric polylysine (D4 16, DD16 19) scaffolds.
or as peptidomimics.25 These scaffolds have a defined and
constrained structure, and the presence of the lysine residue in
the amino acid sequence has allowed for further functionaliza-
tion and display of carbohydrates in a multivalent manner. In
order to improve the recognition properties of the cyclo-
peptide-based glycoclusters toward lectins, newer generations
with higher valency and varying levels of rigidity have been
developed by Renaudet and co-workers. A modular chemo-
selective strategy was used to introduce either a flexible
polylysine framework or a constrained cyclopeptide onto the
RAFT core, providing two different hyperbranched skeletons
in a controlled manner. Biologically relevant carbohydrates
were then conjugated to obtain a new series of glycoden-
drimer-like structures.26 These structures have been used to
generate highly potent ligands for interactions with different
lectins.27 For example, multivalent cyclopeptide scaffolds
presenting N-acetyl glucosamine (GlcNAc), N-acetyl galactos-
amine (GalNAc), and, more recently, mannose (Man)
moieties have been shown to bind the lectins wheat germ
agglutinin,28 soybean agglutinin,29 and DC-SIGN,30 respec-
tively, at micro- and nanomolar concentrations. Glycoclusters
based on RAFT and polylysine scaffolds displaying several
copies of α-fucose (Fuc) and β-galactose (Gal) have also been
found to have high affinity for LecA and LecB, important
adhesins from Pseudomonas aeruginosa.31,32 A glycodendron
displaying 24 α-mannosides was found to bind BC2L-A, a
lectin from respiratory pathogen Burkholderia cenocepacia, at
nanomolar concentration.33 Interestingly, glycomimetics with
affinity toward fucose-binding receptors from emerging
pathogens Aspergillus fumigatus and Burkholderia ambifaria
were grafted onto hexavalent RAFT scaffolds and showed
dissociation constants in the low nanomolar range.34
Candida albicans is an opportunistic pathogenic yeast and is
the most prevalent cause of fungal infections worldwide,
particularly in hospital-acquired infections.35−37 The yeast is
capable of causing a wide range of superficial and systemic
infections in the immunocompromised patient, and there is
now also growing concern for the coinfection of fungal
pathogens in Covid-19 patients.38−40 It is therefore extremely
important that new treatments are developed for these fungal
infections, some of which are now showing resistance to
conventional antifungal therapies.41 Compound 1 (Figure 1)
was previously reported by us as a successful inhibitor of C.
albicans adhesion to human buccal epithelial cells (BECs).42,43
The presence of the two triazolyl galactosides in compound 1
was found to be critical for its activity, as glycoconjugates with
carbohydrates other than galactose displayed on the same
aromatic scaffold had reduced the anti-adhesion effect or
provided no effect at all. Given the versatility of the RAFT
cyclopeptide and dendrimeric polylysine scaffolds, herein we
describe their use to prepare multivalent displays of the
glycomimetic 1 (shown in Figure 1) and investigate their
efficacy at inhibiting the adhesion of C. albicans to BECs. Lead
compound 1 was modified with a suitable triethylene glycol
linker to facilitate connection with these scaffolds using the
highly efficient copper-catalyzed azide−alkyne cycloaddition
(CuAAC) methodology. Since the structure of the target lectin
in C. albicans is not known, ligand optimization requires the
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a
Scheme 1. Inset Shows the Structure of Lead Compound 1. Synthesis of Compound 9
a
Reagents and conditions: (i) Di-tert-butyl dicarbonate, NaOH, 1,4-dioxane, 0 °C to rt, 3 h, 86%; (ii) DMTMM, propargylamine, THF, 48 h, 95%;
(iii) 2,3,4,6-tetra-O-acetyl-1-β-azido-galactoside, CuSO4·5H2O/sodium ascorbate (Na Asc), CH3COCH3/H2O, rt, 16 h, 71%; (iv) TFA, DCM, 2 h,
rt, 99%; (v) bromoacetyl bromide, NEt3, anhydrous DCM, 16 h, 83%; (vi) NaN3, anhydrous DMF, N2, 80 °C, 16 h, quant %; (vii) 7, CuSO4·
5H2O/Na Asc, CH3CN/H2O, MW, 100 °C, 10 min, 45%; (ix) MeOH, NEt3, H2O, 45 °C, 6 h, 90%.
screening of a diverse range of multivalent presentations to
identify the optimum structural parameters (i.e., valency,
flexibility, and three-dimensional orientation) to achieve
increased anti-adhesion activities.
We next prepared three different glycoclusters with valencies
of four and six. Azide-containing cyclopeptide scaffolds R4 10
and R6 13 and lysine-based dendron D4 15 were prepared
using previously reported procedures.32,33,44 Coupling of these
three scaffolds with compound 9 using the CuAAC protocol
gave glycoclusters 11, 14, and 16, respectively. In order to
prepare glycodendrimers with higher valencies, compounds 11
and 16 were further functionalized by reacting them with N-
succinimidyl pentynoate under basic conditions to yield
compounds 12 and 17, respectively (Scheme 2).
Compound 12 was then coupled to another azide-function-
alized RAFT cyclopeptide 10 using the same CuAAC
conditions to give glycocluster 18. This compound features
16 copies of the lead compound attached to rigid cyclopeptide
cores, resulting in the display of 32 galactose residues.
Similarly, compound 17 was then coupled to another azide-
functionalized polylysine scaffold 15 to give the glycoden-
drimer 19, which, like compound 18, has 16 copies of the lead
compound. However, compound 19 provides a more flexible
presentation of the 32 galactose moieties (Scheme 3). All the
final compounds were purified by preparative HPLC and
characterized by mass spectrometry.
Biological Evaluation. Adhesion assays to assess the
ability of the multivalent compounds to inhibit C. albicans
adhesion to human BECs were carried out with all
glycoclusters and dendrimers, using the original lead
compound 1 as a positive control (Figure 2). An exclusion
assay was first performed where the yeast cells were pretreated
with the glycoconjugates, prior to exposure to BECs. As seen in
Figure 2, the tetravalent polylysine glycodendrimer 16
achieved better results than lead compound 1, inhibiting the
yeast adhesion by 64%. Interestingly, tetravalent glycocuster
11, based on a RAFT cyclopeptide scaffold, showed only 30%
inhibition, approximately half of the activity of analogue 16,
highlighting the critical role of the core scaffolds on the overall
presentation of the recognition epitopes. Compound 14, based
RESULTS AND DISCUSSION
■
Chemical Synthesis. The scaffolds chosen for this study
included RAFT cyclopeptides containing four (R4, 10) or six
(R6, 13) lysines oriented out of the upper domain of the
scaffold, and a flexible lysine-based dendron (D4, 15), all
previously reported by Renaudet and co-workers.32,33,44 All
these scaffolds featured azide groups, which required the
functionalization of lead compound 1 with an alkyne group in
order to use CuAAC protocols. Lead compound 1 was
therefore modified with a triethylene glycol linker with a
terminal propargyl group as shown in Scheme 1. Based on our
previously reported synthesis,42 5-amino-isophthalic acid was
N-Boc protected, followed by reaction with propargylamine
and TBTU to give aromatic-centered diamide scaffold 2.
CuAAC chemistry was used to conjugate the acetylated
galactose azides to the scaffold to give compound 3. N-Boc-
deprotection with TFA yielded compound 4, which was
reacted with bromoacetyl bromide to give 5 and subsequently
converted to compound 6 by reaction with sodium azide.
Separately, triethylene glycol was reacted with propargyl
bromide to give dialkyne linker 7.45 This was then reacted
with the azide derivative 6 using CuAAC methodology. To
promote reaction with only one alkynyl group, this reaction
was carried out in dilute conditions and with a large excess of
linker 7, giving the desired monoalkynyl compound 8 and
unreacted linker 7 as the major outcomes of the reaction after
column chromatography. Deacetylation of 8 under mild basic
conditions gave key synthetic intermediate 9, as it presents an
alkyne that will allow for conjugation to the peptidic scaffold
functionalized with azido groups.
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a
Scheme 2. Synthesis of Glycoclusters
a
Reagents and conditions: (i) 9, CuSO4·5H2O/Na Asc, THPTA, DMF/PBS buffer (pH 7.5), rt, 1 h, 34−78%; (ii) N-succinimidyl pentynoate,
DIPEA, DMF (pH 9), rt, 1 h, 82−97%. All amino acids have L-configuration.
on a RAFT hexavalent scaffold, had very comparable results to
the original compound 1 (47% and 45%, respectively). On the
other hand, the higher valency glycoconjugates 18 and 19,
displaying 16 copies of the lead compound 1, performed very
similarly, independently of the structure of the core scaffolds
used: neither of them surpassed the inhibition activity of
compound 1, with 33% inhibition for RAFT-based 18 and 34%
inhibition for glycodendrimer 19.
The competition assays, where C. albicans yeast cells, BECs,
and glycoclusters/dendrimers were coincubated, showed a
similar trend to the previous assays (Figure 2): Again,
compound 16 based on the tetravalent polylysine scaffold
was the most effective inhibitor, with a 62% reduction of
adhesion, outperforming lead compound 1 (48%). Both tetra-
and hexavalent glycoconjugates built on cyclic RAFT scaffolds
(compounds 11 (33%) and 14 (42%), respectively) did not
inhibit the adhesion as well as compound 1. The higher
valencies displayed by both the hexadecavalent glycoconjugates
18 (38%) and 19 (40%) resulted once again in comparable
inhibition of adhesion of C. albicans, irrespectively of the core
scaffold architectures.
SEM Imaging. SEM was used to observe the morphology
of C. albicans cells after 24 h exposure to the lead compound 1
and glycoclusters 14 and 16, the best performing of the
multivalent glycoconjugates (Figure 3). In the control (not
treated) samples, the transition of some of the yeast cells to
hyphae and pseudohyphae can be observed, while these
morphologies were not evident in the samples treated with the
D
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a
Scheme 3. Synthesis of Glycodendrimers 18 and 19
a
Reagents and conditions: (i) CuSO4·5H2O/Na Asc, THPTA, DMF/PBS buffer (pH 7.5), rt, 1 h, 87−89%.
glycoconjugates. Interestingly, while larger clumps of yeast cells
were observed for the control sample, the cells treated with
compound 1 appeared more dispersed. The yeast exposed to
glycodendrimers 14 and 16 showed just a few cells aggregating
tightly. These images suggest that, although both compound 1
and 16 display the same divalent galactoside glycomimetic
epitope, the mechanisms by which they interact with structural
components of the cell wall in C. albicans may be different.
Yeast adhesion processes are critical in the development of
biofilms.46 The ability of these compounds to interfere with
adhesion processes may be advantageous to prevent morpho-
genesis and biofilm formation, which are major virulence
factors in C. albicans infections.47
matrix which forms during the maturation stage of biofilm
development, encompasses a complex network of yeast,
pseudohyphal, and hyphal cells, forming densely packed
groups of cells adhered to a surface. These biofilms are
inherently resistant to conventional antifungal drugs and the
host immune system, causing a huge problem in clinical
infections.49 Hence, the discovery of a treatment that can
inhibit the initial adhesion process of the yeast and prevent the
formation of biofilms is hugely desirable.
Before the biofilm assays, the compounds were tested for
their fungicidal properties, showing no toxicity toward C.
albicans (Figure S24). Under biofilm growing conditions, C.
albicans cultures were treated with D-galactose as a positive
control,50 lead compound 1 and glycodendrimer 16. As shown
in Figure 4, D-galactose promoted the formation of the biofilm,
while glycomimetic 1 decreased the formation of the biofilm by
over 30%, highlighting the potential antivirulence activity of
Biofilm Assays. Biofilm assays were then carried out using
D-galactose, lead compound 1, and multivalent glycodendrimer
16. C. albicans has the ability to form biofilms, a major
virulence characteristic of this pathogen.48 The extracellular
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Figure 2. Left: Exclusion assays where C. albicans yeast cells were pretreated with the glycoconjugates before incubation with BECs: (a) and (b)
show the average number of yeast cells attached per BEC; (c) shows the percentage reduction in adhesion compared to the control (Phosphate
Buffer solution, PBS). Right: Competition assays where the C. albicans yeast cells, BECs, and glycoconjugates were coincubated: (a) and (b) show
the average number of yeast cells attached per BEC; (c) shows the percentage reduction in adhesion compared to the control (PBS). All
compounds were tested at 1 mg/mL (see also ESITable 1).
was functionalized with a triethylene glycol linker appended
with a terminal alkyne group. Using CuAAC chemistry, this
was connected to azide-containing scaffolds to form tetra-,
hexa-, and hexadecavalent RAFT cyclopeptides (R4 11, R6 14,
RR16 18) and dendrimeric polylysine (D4 16, DD16 19) based
multivalent displays of compound 1. Anti-adhesion assays on
these compounds (Table 1) showed that the core peptidic
Table 1. Summary of Anti-Adhesion Activity of Lead
Compound 1 Glycomimetic 1, the Lower Valency
Glycoclusters 11, 14, and 16, and the Higher Valency
Glycoclusters 18 and 19
number of copies exclusion assay: % competition assay: %
of the
glycomimetic
reduction in
adhesion
reduction in
adhesion
compound
Figure 3. SEM images of C. albicans yeast cells at different
magnifications after treatment with PBS, lead compound 1, tetravalent
glycodendrimer 16, and hexavalent glycocluster 14.
1
1
4
6
4
16
16
45
30
47
64
33
34
48
33
42
62
38
40
11 (R4)
14 (R6)
16 (D4)
18 (RR16)
19 (DD16)
scaffold strongly influences the presentation of the glycomi-
metic structure in the multivalent glycoclusters, and hence
their ability to inhibit adhesion of C. albicans to human BECs:
tetravalent glycodendrimer 16 outperformed the original
compound 1, inhibiting over 60% of yeast adhesion to BECs.
In contrast, tetravalent counterpart 11, built onto a RAFT
cyclopeptide scaffold, showed only approximately half the
activity (30% reduction in adhesion), being a less effective
inhibitor than monomer 1 (with 45% reduction). Interestingly,
an increase in the number of copies of glycomimetic 1 did not
result in an improvement of anti-adhesion activity, with
hexavalent 14 and hexadecavalent compounds 18 and 19
performing comparably or slightly worse than original inhibitor
1, regardless of the chemical structure of the core scaffolds. As
discussed earlier, remarkable increases in affinity toward
isolated bacterial and fungal lectins have been reported upon
multivalent display of carbohydrate epitopes. However, it is
Figure 4. Biofilm inhibition showing the percentage change in the
biofilm formation. Control: PBS. (Error: Standard deviation).
this compound. Surprisingly, even though compound 16
preformed best in the adhesion assays, it was slightly less
effective than 1 at reducing biofilm formation (26%).
In summary, five novel multivalent displays of anti-adhesion
glycomimetic 1 were successfully synthesized. The core
divalent galactosyl structure required for biological activity
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possible that whole bacterial or fungal cells, with the inherent
complexity of the cell wall, present a more challenging
environment for large glycoclusters to be able to access
optimal three-dimensional presentations. A glycomimetic
approach, guided by structural knowledge of the target
proteins, may promote more effective interactions. In fact,
the SEM images of C. albicans yeast cells treated with
glycoclusters 14 and 16 showed distinctly different yeast
adhesion patterns when compared to original compound 1,
even if these compounds were all effective inhibitors of yeast
adhesion to BECs. Overall, this work highlights that increasing
the number of glycomimetic epitopes presented by multivalent
scaffolds does not necessarily result in an increase of biological
activity. In particular, for the inhibitors of the adhesion of C.
albicans discussed herein, lower valency dendrimeric presenta-
tions yielded the most promising results.
Ar−H), 5.93 (d, J = 9.2 Hz, 2H, H-1), 5.60 (t, J = 9.7 Hz, 2H,
H-2), 5.54 (d, J = 2.9 Hz, 2H, H-4), 5.32−5.26 (m, 2H, H-3),
4.67 (ddd, J = 39.3, 15.1, 5.4 Hz, 4H, CH2−triaz), 4.31 (t, J =
6.4 Hz, 2H, H-5), 4.22−4.11 (m, 4H, H-6 and H-6′), 3.98 (s,
2H, CH2-Br), 2.21 (s, 6H, OAc), 2.00 (app d, J = 2.7 Hz, 12H,
OAc × 2), 1.82 (s, 6H, OAc). 13C NMR (125 MHz, CDCl3) δ
170.4 (CO of OAc), 170.1 (CO of OAc), 169.8 (CO of OAc),
169.4 (CO of OAc), 166.5 (CONHCH2−triaz), 165.0
(COCH2Br), 145.6 (C-triaz), 138.3 (Ar-C), 135.0 (Ar-C),
121.6 (CH-triaz), 121.4 (Ar-CH), 121.2 (Ar-CH), 86.2 (C-1),
74.0 (C-5), 70.8 (C-3), 68.1 (C-2), 66.8 (C-4), 61.2 (C-6),
35.5 (CH2-triaz), 29.6 (NHCOCH2Br), 20.7 (CH3 of OAc),
20.6 (CH3 of OAc), 20.5 (CH3 of OAc), 20.3 (CH3 of OAc).
IR (film on NaCl): 3345, 3087, 2975, 1752, 1651, 1536, 1446,
1371, 1227, 1063, 924 732 cm−1. HRMS (ESI+): m/z
calculated for C44H52BrN12O21 + H+ [M+H+]: 1122.2539,
found 1122.2545.
N,N′-Di(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-
1,2,3-triazol-4-ylmethyl amide)-N″-(2-azidoacetamido)-5-
aminobenzene-1,3-dicarboxamide (6). Compound 5 (231
mg, 0.206 mmol) and NaN3 (30 mg, 0.412 mmol) were
dissolved in anhydrous DMF (10 mL) and heated to 80 °C.
The reaction mixture was allowed to stir for 16 h. The solvent
was removed in vacuo, and the resulting residue was
redissolved in DCM (20 mL) and washed with brine (20
mL × 3). The organic phase was dried over MgSO4 and the
solvent was removed in vacuo to obtain the pure product 6
without further purification as a yellow solid (1.056 g, 83%). Rf
EXPERIMENTAL PROCEDURES
■
Synthesis. General Methods. All reagents for synthesis
were bought commercially and used without further
purification. Reactions were monitored with thin layer
chromatography (TLC) on Merck Silica Gel F254 plates.
Detection was effected by UV (λ = 254 nm) or charring in a
mixture of 5% sulfuric acid−ethanol. NMR spectra were
recorded using Bruker Ascend 500 and Avance III
spectrometers at 293 K. All chemical shifts were referenced
relative to the relevant deuterated solvent residual peaks.
1
Assignments of the NMR spectra were deduced using H
= 0.41 (DCM:MeOH 9:1). [α]2D2−5.6 (c 0.9, DCM). 1H NMR
(500 MHz, CDCl3) δ 9.10 (s, 1H, NHCOCH2N3), 8.18 (s,
2H, NHCH2CCH), 8.02 (s, 2H, Ar−H), 7.97 (s, 2H, CH-
triaz), 7.82 (s, 1H, Ar−H), 5.95 (d, J = 9.2 Hz, 2H, H-1), 5.61
(t, J = 9.7 Hz, 2H, H-2), 5.56 (d, J = 3.1 Hz, 2H, H-4), 5.32
(dd, J = 10.1, 3.5 Hz, 2H, H-3), 4.67 (ddd, J = 20.4, 15.4, 5.5
Hz, 4H, CH2-triaz), 4.34 (t, J = 6.6 Hz, 2H, H-5), 4.23−4.13
(m, 4H, H-6 and H-6′), 4.06 (s, 2H, CH2-N3), 2.21 (s, 6H,
OAc), 2.01 (s, 12H, OAc × 2), 1.82 (s, 6H, OAc). 13C NMR
(125 MHz, CDCl3) δ 170.4 (CO of OAc), 170.1 (CO of
OAc), 169.9 (CO of OAc), 169.3 (CO of OAc), 166.5
(CONHCH2CCH), 166.3 (COCH2N3), 145.5 (C-triaz), 138.0
(Ar-C), 134.9 (Ar-C), 121.6 (Ar-CH and CH-triaz), 121.4 (Ar-
CH), 86.1 (C-1), 73.9 (C-5), 70.8 (C-3), 68.1 (C-2), 66.9 (C-
4), 61.2 (C-6), 52.5 (CH2N3), 35.4 (CH2-triaz), 20.7 (CH3 of
OAc), 20.6 (CH3 of OAc), 20.5 (CH3 of OAc), 20.2 (CH3 of
OAc). IR (film on NaCl): 3342, 2942, 2110, 1747, 1655, 1528,
1427, 1368, 1211, 1046, 923, 733 cm−1. HRMS (ESI+): m/z
calculated for C44H52N12O21 + Na+ [M+Na+]: 1107.3268,
found 1107.3303.
NMR and 13C 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 (μW) reactions were carried out using a CEM
Discover Microwave Synthesizer. Optical rotations were
obtained from an AA-100 polarimeter and [α]D values are
given in 10−1 cm2·g−1. High resolution mass spectrometry
(HRMS) was performed on an Agilent-LC 1200 Series
coupled to a 6210 Agilent Time-Of-Flight (TOF) and a
Waters Xevo G2-S QToF mass spectrometers equipped with
an electrospray source in both positive and negative (ESI )
modes. SEM images were taken on a HITACHI S-3200N
Scanning Electron Microscope. FT-IR spectra were recorded
on a PerkinElmer Spectrum 100 spectrophotometer, via ATR
as a solid on a zinc selenide crystal or as a film on NaCl plates
in the region 4000−400 cm−1. Spectroscopic data for all
compounds are provided in the Supporting Information.
N,N′-Di(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-
1,2,3-triazol-4-ylmethylamide)-N″-(2-bromoacetamido)-5-
aminobenzene-1,3-dicarboxamide (5). Compound 442
(1.128 g, 1.13 mmol) was dissolved in dry DCM (20 mL).
NEt3 (0.19 mL, 1.35 mmol) was added to this solution.
Bromoacetyl bromide (0.12 mL, 1.35 mmol) was dissolved in
dry DCM (5 mL) in a separate round-bottom flask. The first
solution was added to the second dropwise via a cannula, and
the resulting reaction mixture was allowed to stir for 16 h. The
reaction mixture was washed with water (20 mL), HCl (1 N,
20 mL), sat. NaHCO3 solution (20 mL), followed by brine (20
mL). The organic phase was dried (MgSO4), and the solvent
was removed in vacuo to obtain the pure product 5 without
further purification as a brown, sticky solid (1.056 g, 83%). Rf
Triethylene Glycol Dipropargyl Ether (7). (Adapted from
Feng et al.45) Triethylene glycol (1 mL, 7.33 mmol) was
dissolved in dry THF under N2. NaH (60% dispersion in oil,
1.172 g, 29.3 mmol) was added, and the reaction mixture was
allowed to stir for 1 h. Propargyl bromide (4 mL, 44 mmol)
was added, and the reaction mixture was allowed to stir for at
rt for 48 h. The solvent was removed in vacuo, and the resulting
residue was dissolved in DCM (20 mL) and washed with water
(10 mL × 2), dried (MgSO4), filtered, and concentrated in
vacuo. The crude product was purified by silica gel column
chromatography (EtOAc:Pet Ether 2:1) to give the pure
= 0.65 (DCM, 5% MeOH). [α]2D4−4.0 (c 1.0, DCM). 1H NMR
(500 MHz, CDCl3) δ 9.10 (s, 1H, NHCOCH2Br), 8.09−7.90
(m, 6H, triaz-H, CONHCH2−triaz and Ar−H), 7.75 (s, 1H,
1
product as a yellow oil (1.526 g, 92%). H NMR (500 MHz,
CDCl3) δ 4.20 (d, J = 2.4 Hz, 4H, CH2CCH), 3.72−3.51 (m,
1
12H, OCH2 × 6), 2.42 (t, J = 2.4 Hz, 2H, CH2CCH). H
G
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NMR and 13C NMR spectroscopic data corresponded to that
found in the literature.23
7.78 (s, 1H, Ar−H), 5.58 (d, J = 9.3 Hz, 2H, H-1), 5.34 (s, 2H,
CH2), 4.58 (s, 2H, CH2-triaz), 4.58 (s, 2H, CH2-triaz), 4.55 (s,
4H, CH2-triaz), 4.13 (t, J = 9.8 Hz, 2H, H-2), 4.02 (s, 2H,
OCH2CCH), 3.99 (s, 2H, H-4), 3.89 (m, 2H, H-5), 3.79−3.77
(m, 2H, H-3), 3.68−3.63 (m. 4H, H-6 and H-6′), 3.61 (bs,
2H, OCH2), 3.57−3.53 (m, 10H, 3 × CH2), 2.67 (s, 1H,
CCH). 13C NMR (125 MHz, CDCl3) δ 168.4 (CONHCH2−
triaz), 166.1 (COCH2−triaz), 144.9 (C-triaz), 144.2 (C-triaz),
137.6 (Ar-C), 134.6 (Ar-C), 126.6 (CH-triaz), 123.1 (CH-
triaz), 123.4 (Ar-CH × 2), 88.1 (C-1), 78.3 (C-5), 73.1 (C-3),
69.8 (C-2), 69.5 (CH2), 69.3 (CH2), 68.6 (C-4), 68.6, 68.5 (2
× CH2), 63.1 (NHCOCH2N3), 60.1 (C-6), 57.8
(OCH2CCH), 52.5 (CH2), 34.1 (CH2-triaz). IR (ATR):
3269, 2927, 2491, 1704, 1645, 1598, 1538, 1447, 1347, 1227,
1090, 1052, 890 cm−1. HRMS (ESI+): m/z calculated for
C40H54N12O17 + Na+ [M+Na+]: 997.3628, found 997.3615.
General Procedure for CuAAC Ligation for Glyco-
N,N′-Di(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-
1,2,3-triazol-4-ylmethyl amide)-N″-(2-4-((2-(2-(2-(prop-2-
yn-1-yloxy)ethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-
1-yl)acetamido)-5-aminobenzene-1,3-dicarboxamide (8). 5
mL of a solution of compound 6 (prepared by dissolving 293
mg in 22 mL CH3CN and 5 mL of water, [1 mM]) was
combined with 5 mL of a solution of compound 7 (prepared
by dissolving 223 mg in 22 mL CH3CN and 5 mL of water,
[36 mM]) in a microwave flask. To this solution, 0.5 mL of a
solution of sodium ascorbate (prepared by dissolving 180 mg
in 2 mL of water) was added, followed by 0.5 mL of a solution
of copper sulfate pentahydrate (prepared by dissolving 70 mg
in 2 mL of water). The mixture was allowed to react in the
MW at 100 °C for 10 min. TLC was used to monitor the
reaction; staining the TLC using potassium permanganate
solution displayed the product as a bright yellow spot, whereas
the starting compound 6 was a white spot and the diclick
product was a brown spot on the TLC plate after staining. This
procedure was repeated until all the stock solutions were used.
The reaction mixture was evaporated and the crude product
was purified by silicia gel column chromatography
(DCM:MeOH 100:0-90:10) to give the product as a pale
yellow solid (160 mg, 45%). Rf = 0.65 (DCM:MeOH 9:1).
clusters.
A solution of CuSO4 ·5H2 O, tris(3-
hydroxypropyltriazolylmethyl)amine (THPTA) and sodium
ascorbate in phosphate-buffered saline (PBS, pH 7.5, 10 mM)
was added to a solution of the azido-containing compound and
the alkyne-containing compound (1.5 equiv per azide) in DMF
at room temperature. The mixture was degassed under argon
and stirred at room temperature for 1 h under argon. UPLC
analysis was used to determine the end point of the reaction.
Chelex resin was added to the reaction mixture, which was
stirred for an additional 30 min to remove excess Cu2+ ions.
The reaction mixture was then purified by semipreparative RP-
HPLC to afford pure compounds as white fluffy solids after
lyophilization.
23
1
[α]D −1 (c 1, DCM). H NMR (500 MHz, CDCl3) δ 9.63 (s,
1H, NHCH2N3), 8.10 (bs, 2H, NHCH2−triaz), 7.91 (s, 2H,
CH-triaz), 7.80 (s, 1H, CH-triaz), 7.78 (s, 2H, Ar−H), 7.73 (s,
1H, Ar−H), 5.85 (d, J = 9.2 Hz, 2H, H-1), 5.57 (t, J = 9.8 Hz,
2H, H-2), 5.47 (d, J = 2.7 Hz, 2H, H-4), 5.23−5.19 (m, 4H, H-
3 and CH2), 4.65−4.56 (m, 6H, CH2-triaz), 4.24 (t, J = 6.3 Hz,
2H, H-5), 4.13−4.04 (m, 6H, H-6, H-6′ and OCH2CCH),
3.61−3.53 (m, 12H, 3 × OCH2CH2O), 2.32 (s, 1H, CCH),
2.12 (s, 6H, CH3 of OAc), 1.94 (s, 12H, CH3 of OAc), 1.73 (s,
6H, CH3 of OAc). 13C NMR (125 MHz, CDCl3) δ 169.3 (CO
of OAc), 169.1 (CO of OAc), 168.9 (CO of OAc), 168.4 (CO
of OAc), 165.6 (CONHCH2−triaz), 163.3 (COCH2−triaz),
144.5 (C-triaz), 143.9 (C-triaz), 137.1 (Ar-C), 133.9 (Ar-C),
124.2 (CH-triaz), 120.7 (CH-triaz and Ar-CH), 120.4 (Ar-
CH), 85.1 (C-1), 78.5 (CCH), 73.9 (C-5), 72.8 (CCH), 69.7
(C-3), 69.4 (CH2 × 2), 69.2 (CH2), 68.8 (CH2), 68.0 (C-2),
67.0 (C-4), 65.9 (NHCOCH2N3), 60.1 (C-6), 57.3
(OCH2CCH), 52.4 (CH2), 34.5 (CH2-triaz), 19.6 (CH3 of
OAc × 2), 19.5 (CH3 of OAc), 19.2 (CH3 of OAc). IR (film
on NaCl): 3290, 3145, 2917, 2115, 1752, 1657, 1535, 1447,
1370, 1225, 1093, 1054, 924 cm−1. HRMS (ESI+): m/z
calculated for C56H70N12O25 + Na+ [M+Na+]: 1333.4473,
found 1333.4456.
Compound 11. A solution of CuSO4·5H2O (0.79 mg, 3.2
μmol), THPTA (2.8 mg, 6.4 μmol), and sodium ascorbate (3.8
mg, 19.2 μmol) in PBS buffer (400 μL, pH 7.5) was added to a
solution of compound 10 (7.2 mg, 6.4 μmol) and 9 (2.74 mL
of 10 mg/mL solution in PBS, 28.2 μmol) in 500 μL of DMF.
The mixture was degassed under argon and stirred at room
temperature for 1 h. UPLC analysis showed the reaction was
not complete. A further 2 equiv of 9 was added (1.24 mL of 10
mg/mL solution in PBS, 13.0 μmol). The mixture was
degassed under argon and stirred at room temperature for 1 h.
UPLC analysis showed complete coupling. Chelex resin was
added to the reaction mixture, which was stirred for an
additional 30 min and purified by semipreparative RP-HPLC
(5−40% CH3CN in 15 min) to afford the desired compound
11 as a white fluffy solid after lyophilization (21 mg, 65%). 1H
NMR (500 MHz, D2O) δ 8.47 (s, 1H), 8.22 (s, 8H), 8.05 (s,
4H), 7.95−7.85 (m, 8H), 7.87−7.71 (m, 8H), 5.64 (d, J = 9.2
Hz, 8H), 5.37 (s, 8H), 4.59 (s, 26H), 4.50 (s, 9H), 4.44−4.31
(m, 5H), 4.31−4.18 (m, 19H), 4.08 (d, J = 3.3 Hz, 8H), 4.07−
4.00 (m, 2H), 3.97 (t, J = 6.1 Hz, 8H), 3.86 (dd, J = 9.8, 3.3
Hz, 10H), 3.75 (d, J = 6.0 Hz, 21H), 3.67−3.48 (m, 53H),
2.96 (t, J = 7.6 Hz, 2H), 2.25 (s, 3H), 2.09−1.47 (m, 29H),
1.43−1.11 (m, 15H). HRMS (ESI+): m/z calculated for
Synthesis of N,N′-di(β-D-galactopyranosyl-1,2,3-triazol-4-
ylmethylamide)-N″-(2-4-((2-(2-(2-(prop-2-yn-1-yloxy)-
ethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl) acet-
amido)-5-aminobenzene-1,3-dicarboxamide (9). Compound
8 (150 mg, 0.114 mmol) was dissolved in methanol/H2O (4
mL, 2 mL). NEt3 (0.1 mL) was added, and the reaction
mixture was allowed to stir at 45 °C for 6 h. The solution was
cooled, Amberlite H+ was added, and the mixture was allowed
to stir for 30 min. The solution was filtered, and the solvent
was removed in vacuo. Excess NEt3 was removed using the
Schlenk line. The product was freeze-dried overnight to yield
C
207H297N71O78 + 4H+ [M+4H]4+: 1256.28586, found
1256.28652.
Compound 12. Compound 11 (15.8 mg, 3.15 μmol) and
N-succinimidyl pentynoate (0.9 mg, 4.6 μmol) were dissolved
in dry DMF (1 mL). Diisopropylethylamine (2 μL × 3, mmol)
were added until the solution was at pH 9. The mixture was
stirred at room temperature for 1 h after which UPLC analysis
showed complete conversion. H2O (3 mL) was added to the
mixture, which was then purified by semipreparative RP-HPLC
the pure product 9 as a white solid (100 mg, 90%). [α]2D3+7 (c
1
1, MeOH:H2O 1:1). H NMR (500 MHz, D2O) δ 8.19 (s,
2H, CH-triaz), 8.02 (s, 1H, CH-triaz), 7.86 (s, 2H, Ar−H),
H
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1
(5−40% CH3CN in 15 min) to afford the desired compound
12 as a white fluffy solid after lyophilization (15.5 mg, 97%).
Compound 14. A solution of CuSO4·5H2O (0.6 mg, 2.4
μmol), THPTA (2.1 mg, 4.8 μmol), and sodium ascorbate (2.9
mg, 28.8 μmol) in PBS buffer (400 μL, pH 7.5) was added to a
solution of the 13 (4 mg, 2.4 μmol) and 9 (21 mg, 21.6 μmol)
in 500 μL of DMF. The mixture was degassed under argon and
stirred at room temperature for 1 h. UPLC analysis showed
complete coupling. Chelex resin was added to the reaction
mixture, which was stirred for an additional 30 min and
purified by semipreparative RP-HPLC (5−40% CH3CN in 15
min) to afford the desired compound 4.55 as a white fluffy
white fluffy solid after lyophilization (12 mg, 89%). H NMR
(500 MHz, DMSO) δ = 10.81 (s, 31H), 9.11 (s, 42H), 8.43 (s,
36H), 8.22 (s, 39H), 8.08 (m, 84H), 7.82 (m, 32H), 7.39 (s,
40H), 6.86 (s, 12H), 6.66 (m, 12H), 5.46 (d, J = 9.1, 32H, H-
1), 5.36 (s, 34H), 5.23 (s, 34H), 5.02 (s, 36H), 4.67 (m, 70H),
4.53 (m, 123H), 4.27 (s, 64H), 4.11 (s, 27H), 4.00 (d, J = 4.2,
40H), 3.75 (s, 42H), 3.69 (t, J = 5.9, 40H), 3.52 (m, 266H),
3.00 (s, 29H), 2.80 (s, 24H), 2.64 (s, 17H), 2.40 (m, 31H),
2.07 (m, 66H), 1.73 (m, 125H), 1.50 (m, 40H), 1.23 (m,
156H), 0.86 (m, 17H). MALDI-TOF-MS [M + H]+: m/z
calculated for C895H1263N307O326 + H+: 21539.474, found
21539.616.
1
Compound 19. A solution of CuSO4·5H2O (0.1 mg, 0.46
μmol), THPTA (0.41 mg, 0.93 μmol), and sodium ascorbate
(0.56 mg, 2.79 μmol) in PBS buffer (400 μL, pH 7.5) was
added to a solution of the 15 (0.76 mg, 0.93 μmol) and 17
(19.7 mg, 4.11 mmol) in 500 μL of DMF. The mixture was
degassed under argon and stirred at room temperature for 1 h.
UPLC analysis showed complete coupling. Chelex resin was
added to the reaction mixture, which was stirred for an
additional 30 min and purified by semipreparative RP-HPLC
(5−40% CH3CN in 15 min) to afford the desired compound
19 as a white fluffy solid after lyophilization (16.2 mg, 87%).
1H NMR (500 MHz, DMSO) δ = 10.86 (s, 24H), 9.17 (s,
32H), 8.59 (m, 26H), 8.28 (s, 34H), 8.13 (m, 80H), 7.87 (m,
28H), 7.34 (s, 10H), 7.07 (s, 10H), 5.52 (d, J = 9.1, 32H, H-
1), 5.42 (s, 32H), 5.29 (s, 30H), 5.20 (s, 22H), 5.07 (s, 30H),
4.73 (m, 60H), 4.57 (m, 110H), 4.25 (m, 96H), 3.80 (s, 32H),
3.75 (t, J = 5.9, 30H), 3.57 (m, 260H), 3.23 (s, 16H), 3.06 (s,
24H), 2.92 (m, 18H), 2.71 (m, 15H), 2.43 (m, 18H), 1.79 (m,
80H), 1.30 (m, 82H), 0.91 (m, 6H). MALDI-TOF-MS [M +
H]+: m/z calculated for C815H1151N292O311 + H+: 20015.637,
found 20015.456.
Biology. Fungal Strain. C. albicans (MEN, serotype B,
clinical isolate from a corneal infection) was maintained on
sabouraud dextrose agar, and cultures were grow to the
stationary phase ((1−2) × 108 cells/mL) overnight in YEPD
broth (1% (w/v) yeast extract, 2% (w/v) bacteriological
peptone, 2% (w/v) glucose) at 30 °C, and 200 rpm. Stationary
phase yeast cells were harvested, washed with PBS, and
resuspended at a density of 1 × 108 cells/mL in PBS.
Buccal Epithelial Cells. Buccal epithelial cells (BECs) were
harvested from healthy volunteers by gently scraping the inside
of the cheek with a sterile tongue depressor. Cells were washed
in PBS and resuspended at a density of 5 × 105 cells/mL.
Toxicity. The compounds were incubated with C. albicans
for 24 h; a dilution was performed (1/50) and 100 μL of the
cell suspension was spread on YEPD agar plates. The
compounds did not have a fungicidal effect on the yeast
(Figure SI-1).
solid after lyophilization (6.2 mg, 34%). H NMR (500 MHz,
DMSO) δ = 10.71 (s, 9H), 9.02 (m, 14H), 8.31−8.11 (m,
17H), 8.01 (m, 17H), 7.95−7.65 (m, 26H), 5.37 (d, J = 9.2,
12H, H-1), 5.26 (d, J = 8.1, 14H), 5.13 (d, J = 5.8, 16H), 4.94
(m, 20H), 4.60 (t, J = 5.6, 13H), 4.55 (d, J = 5.5, 9H), 4.45
(dd, J = 12.0, 7.6, 29H), 4.16 (bs, 26H), 3.91 (dd, J = 15.1, 9.2,
18H), 3.65 (m, 11H), 3.60 (m, 12H), 3.41 (m, 55H), 2.97 (bs,
20H), 2.58−2.53 (m, 7H), 2.37−2.25 (m, 6H), 2.1−1.5 (m,
54H), 1.5−1.0 (m, 65H), 0.81−0.73 (m, 9H). MALDI-TOF-
MS [M + H]+: m/z calculated for C314H442N110O122 + H+:
7706.184, found 7706.360.
Compound 16. A solution of CuSO4·5H2O (0.79 mg, 3.2
μmol), THPTA (2.8 mg, 6.4 μmol), and sodium ascorbate (3.8
mg, 19.2 μmol) in PBS buffer (400 μL, pH 7.5) was added to a
solution of the 15 (7 mg, 6.8 μmol) and 9 (3.9 mL of 10 mg/
mL solution in PBS, 40.7 μmol) in 500 μL of DMF. The
mixture was degassed under argon and stirred at room
temperature for 1 h. UPLC analysis showed complete
coupling. Chelex resin was added to the reaction mixture,
which was stirred for an additional 30 min and purified by
semipreparative RP-HPLC (5−40% CH3CN in 15 min) to
afford the desired compound 4.57 as a white fluffy solid after
1
lyophilization (25 mg, 78%). H NMR (500 MHz, D2O) δ
8.47 (s, 1H), 8.21 (s, 8H), 8.05 (d, J = 2.4 Hz, 4H), 7.97−7.88
(m, 10H), 7.85−7.78 (m, 6H), 5.65 (d, J = 9.1 Hz, 8H), 5.38
(s, 8H), 5.22 (d, J = 8.2 Hz, 4H), 4.68−4.55 (m, 24H), 4.52
(d, J = 5.2 Hz, 9H), 4.30−4.14 (m, 17H), 4.08 (d, J = 3.3 Hz,
8H), 3.97 (t, J = 6.1 Hz, 8H), 3.86 (dd, J = 9.8, 3.3 Hz, 9H),
3.75 (d, J = 6.0 Hz, 17H), 3.67−3.47 (m, 51H), 3.00 (s, 2H),
2.94 (t, J = 7.6 Hz, 2H), 1.88−1.52 (m, 15H), 1.48−1.06 (m,
14H). HRMS (ESI+): m/z calculated for C191H274N68O75
+
4H+ [M+4H]4+: 1179.99237, found 1179.99202.
Compound 17. Compound 16 (23.6 mg, 5.0 μmol) and N-
succinimidyl pentynoate (1.46 mg, 7.5 μmol) were dissolved in
dry DMF (1 mL). Diisopropylethylamine (2 μL × 3, mmol)
were added until the solution was pH 9. The mixture was
stirred at room temperature for 1 h after which UPLC analysis
showed complete conversion. H2O (3 mL) was added to the
mixture, which was then purified by semipreparative RP-HPLC
(5−40% CH3CN in 15 min) to afford the compound 17 as a
white fluffy solid after lyophilization (19.7 mg, 82%).
Compound 18. A solution of CuSO4·5H2O (0.08 mg, 0.32
μmol), THPTA (0.27 mg, 0.62 μmol), and sodium ascorbate
(0.37 mg, 1.9 μmol) in PBS buffer (400 μL, pH 7.5) was added
to a solution of the 10 (0.7 mg, 0.62 μmol) and 12 (14 mg,
2.74 mmol) in 500 μL of DMF. The mixture was degassed
under argon and stirred at room temperature for 1 h. UPLC
analysis showed complete coupling. Chelex resin was added to
the reaction mixture, which was stirred for an additional 30
min and purified by semipreparative RP-HPLC (5−40%
CH3CN in 15 min) to afford the desired compound 18 as a
Adherence Assays. Yeast cells were mixed with 50:1
(yeast:BEC) in a final volume of 2 mL and incubated at 30
°C and 200 rpm for 90 min. The BEC/yeast cell mixture was
harvested by passing through a polycarbonate membrane
containing 30 μm pores which trapped the BECs but allowed
unattached yeast cells to pass through. This was washed 2×
with 10 mL PBS, and cells remaining on the membrane were
collected and placed on glass slides which were left to air-dry
overnight. The cells were heat-fixed and stained using 0.5%
(w/v) crystal violet, rinsed using cold water to remove any
surplus stain, and left to air-dry for 30 min. The number of C.
albicans cells adhering to a sample of 200 BECs per treatment
was assessed microscopically. In the exclusion assay, the yeast
I
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cells were incubated for 90 min in the presence of each
compound at the given concentration. After this time, the yeast
cells were harvested and washed twice with PBS before being
resuspended in 1 mL PBS before being mixed with BECs (as
described). In the competition assay format yeast cells, BECs
and compound (at the given concentration) were coincubated
for 90 min prior to harvesting.
SEM Sample Preparation. First, the yeast cells were
incubated separately with compounds 1, 14, and 16, along
with a control of only yeast in PBS, for 24 h at 30 °C and 200
rpm. Sample preparation for SEM was then adapted from
Manefield et al.51 The cells were fixed to a microscopic cover
clip, with 2.5% (v/w) glutaraldehyde and kept at 4 °C for 12 h.
The cells were then washed with sterile PBS (2×) dehydrated
by sequential washing with ethanol series (70%, 95%, and
100%). The samples were then treated with hexamethyldisi-
lazane (Sigma-Aldrich) and air-dried overnight in a desiccator.
The cells were then sputtered with gold (6−12 nm) prior SEM
imaging.
AUTHOR INFORMATION
■
Corresponding Authors
Trinidad Velasco-Torrijos − Department of Chemistry,
Maynooth University, Maynooth W23VP22, Co. Kildare,
Ireland; The Kathleen Lonsdale Institute for Human Health
Research, Maynooth University, Maynooth W23VP22, Co.
Kildare, Ireland; orcid.org/0000-0002-0701-8268;
Email: trinidad.velascotorrijos@mu.ie
Olivier Renaudet − DCM, UMR 5250, Université Grenoble
Alpes, CNRS, 38000 Grenoble, France; orcid.org/0000-
0003-4963-3848; Email: olivier.renaudet@univ-grenoble-
alpes.fr
Kevin Kavanagh − Department of Biology and The Kathleen
Lonsdale Institute for Human Health Research, Maynooth
University, Maynooth W23VP22, Co. Kildare, Ireland;
Email: Kevin.Kavanagh@mu.ie
Authors
Harlei Martin − Department of Chemistry, Maynooth
University, Maynooth W23VP22, Co. Kildare, Ireland;
orcid.org/0000-0002-6109-9855
David Goyard − DCM, UMR 5250, Université Grenoble
Alpes, CNRS, 38000 Grenoble, France; orcid.org/0000-
0002-6410-0866
Anatte Margalit − Department of Biology, Maynooth
University, Maynooth W23VP22, Co. Kildare, Ireland
Kyle Doherty − Department of Chemistry, Maynooth
University, Maynooth W23VP22, Co. Kildare, Ireland
Biofilm. C. albicans cells were washed twice with PBS and
enumerated before being resuspended in RPMI to give a cell
density of (2 × 107 cells/mL). Compounds were resuspended
in PBS to give a concentration of 2 mg/mL. The compounds
(250 μL) were mixed with RPMI (250 μL) in a 1:1 ratio to
give a concentration of 1 mg/mL. PBS (250 μL) was mixed
with RPMI (250 μL).
C. albicans cells (500 μL, 2 × 107 cells/mL) were incubated
with compounds (1 mg/mL) in a 1:1 ratio to give a final cell
density to concentration ratio of 5 × 106 cells/mL. The
suspension was incubated for 3 h at rt and mixed by vortex
every 20 min to prevent cells from falling out of solution. Cell
suspension (100 μL) was added to wells of a 96-well flat
bottom plate. The plate was incubated at 37 °C for 24 h. After
incubation, the medium was removed from the wells with a
pipet, with care taken not to disrupt the biofilm lining the
bottom of the plate. Wells were washed twice with 100 μL
aliquots of PBS to remove unbound yeast cells. Crystal violet
(5% v/v) (100 μL) was added to each well and incubated at
room temperature for 15 min. The crystal violet was removed
by pipet as described before. Wells were washed twice with
PBS (100 μL). The plates were incubated at room temperature
for 2 h, with the lids left off and allowed to dry. Acetic acid
(30% v/v) (100 μL) was added to each well. The plate was
incubated for 10 min and a pipet was used to mix the contents
of each well to ensure the biofilm/crystal violet was fully
dissolved in the acetic acid. The plate was read at 550 nm in a
96-well plate reader. The percentage reduction in biofilm was
calculated by dividing the absorbance value of the treated cells
into that of the control.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.bioconjchem.1c00115
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Irish Research Council (IRC)- Ulysses
2018 for the provision of funding for H. Martin to travel to
Prof Renaudet’s laboratory. We would like to thank Maynooth
University for the provision of the John and Pat Hume
Scholarship for H. Martin. We thank the CNRS, Université
Grenoble Alpes, ICMG FR 2607, the French ANR project
Glyco@Alps (ANR-15-IDEX-02), Labex ARCANE and CBH-
EUR-GS (ANR-17-EURE-0003). O.R. acknowledge the Euro-
pean Research Council Consolidator Grant “LEGO” (647938)
for D.G.
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ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.bioconjchem.1c00115.
Structures and spectroscopic data; images and molecular
weights (PDF)
J
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