The synthesis and evaluation of multivalent glycopeptoids as inhibitors of the adhesion of candida albicans

Article abstract of DOI:10.3390/pathogens10050572

Multivalency is a strategy commonly used by medicinal carbohydrate chemists to increase the affinity of carbohydrate-based small molecules for their protein targets. Although this approach has been very successful in enhancing binding to isolated carbohyd

Full text of DOI:10.3390/pathogens10050572

pathogens
Article
The Synthesis and Evaluation of Multivalent Glycopeptoids as
Inhibitors of the Adhesion of Candida albicans
Harlei Martin 1,† , Hannah Masterson 2, Kevin Kavanagh 2,3,* and Trinidad Velasco-Torrijos 1,3,
*
1
Department of Chemistry, Maynooth University, Maynooth, W23VP22 Co. Kildare, Ireland;
harlei.martin.2012@mumail.ie
Department of Biology, Maynooth University, Maynooth, W23VP22 Co. Kildare, Ireland;
hannah.masterson.2017@mumail.ie
The Kathleen Lonsdale Institute for Human Health Research, Maynooth University, Maynooth,
W23VP22 Co. Kildare, Ireland
2
3
*
†
Correspondence: Kevin.Kavanagh@mu.ie (K.K.); trinidad.velascotorrijos@mu.ie (T.V.-T.)
Present address: Centre de Biophysique Moléculaire, Université d’Orléans, Rue Charles Sadron,
F-45071 Orléans 2, France.
Abstract: Multivalency is a strategy commonly used by medicinal carbohydrate chemists to increase
the affinity of carbohydrate-based small molecules for their protein targets. Although this approach
has been very successful in enhancing binding to isolated carbohydrate-binding proteins, anticipat-
ing the multivalent presentations that will improve biological activity in cellular assays remains
challenging. In this work we investigate linear molecular scaffolds for the synthesis of a low valency
presentation of a divalent galactoside
opportunistic fungal pathogen Candida albicans to buccal epithelial cells (BECs). Adhesion inhibition
assays revealed that multivalent glycoconjugate is more effective at blocking C. albicans adherence
to BECs upon initial exposure to epithelial cells. Interestingly, did not seem to have any effect when
it was pre-incubated with yeast cells, in contrast to the original lead compound , which caused a
25% reduction of adhesion. In competition assays, where yeast cells and BECs were co-incubated,
multivalent glycoconjugate inhibited up to 49% C. albicans adherence in a dose-dependent manner.
The combined effect of compound towards both yeast cells and BECs allowed it to achieve over
1, previously identified by us as an inhibitor of the adhesion of
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ꢀꢁꢂꢃꢄꢅꢆ
3
Citation: Martin, H.; Masterson, H.;
Kavanagh, K.; Velasco-Torrijos, T. The
Synthesis and Evaluation of
3
1
Multivalent Glycopeptoids as
Inhibitors of the Adhesion of Candida
albicans. Pathogens 2021, 10, 572.
https://doi.org/10.3390/
3
1
60% inhibition of the adhesion of C. albicans to BECs in competition assays.
pathogens10050572
Keywords: multivalency; anti-adhesion glycoconjugates; antifungal agents; Candida albicans; gly-
comimetics
Academic Editor: Jonathan
Richardson
Received: 1 April 2021
Accepted: 5 May 2021
Published: 8 May 2021
1. Introduction
Protein-carbohydrate recognition is the first step in the initiation of many human
diseases. In many cases, for microorganisms to infect their hosts, microbe proteins initially
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
adhere to carbohydrate epitopes displayed on the host cell surface [
inhibitors of the adhesion process have been successfully developed and studied for many
years [ ]. The use of glycoconjugates as anti-adhesion ligands is desirable since they
1]. Small-molecule
2
can mimic the host cell surface glycans and block pathogen attachment, thus preventing
infection. However, carbohydrates interact with their protein receptors (lectins) with low
affinity (generally millimolar to micromolar dissociation constants) [
the development of strategies for increasing the lectin–ligand binding affinities to levels
required for therapeutic use has received much attention in glycoscience research [ ]. In
nature, carbohydrate epitopes are expressed multiple times on the cell surface: many
lectins have more than one binding site to counteract the low-affinity problem, leading
to stronger interactions. This phenomenon has become known as the “multivalent effect”
or “cluster glycoside effect”, which was first reported by Lee and co-workers in 1995 [5].
Numerous multivalent glycoconjugates with various valencies and spatial arrangement of
carbohydrate ligands have been developed to enhance carbohydrate–lectin interactions.
3]. Consequently,
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The multivalent glycoconjugates can have well-defined molecular structures and display
a specific number of carbohydrate ligands when they are built around scaffolds such as
calixarenes [
6], dendrimers [7], cyclodextrins [8], cyclopeptides [9] and fullerenes [7]; they
can also have higher valencies such as those provided by polymers [10], nanoparticles [11
]
and quantum dots [12]. There are many reports of multivalent glycoconjugates with
increased affinity for isolated lectins, in comparison to their monovalent counterparts [13].
Most of these studies involve lectins whose structures have been well characterized by
X-ray crystallography, and the strength of lectin–ligand binding interactions is measured
using techniques such as haemagglutination inhibition assays, enzyme-linked lectin assays
(ELLA) and isothermal titration calorimetry [14
protein interactions has also been investigated with ligands other than carbohydrates, to
potentiate different types of biological effects [16 17]. Despite the many achievements
,15]. The multivalent effect in ligand–
,
in this field, the design of high-affinity multivalent presentations is still a challenging
task, as it strongly depends on multiple factors: these include structural parameters of
the glycoconjugates such as linker length, density of the ligands in the glycoconstruct
and heterogeneity of the carbohydrate epitopes presented [18]. Moreover, the impact of
multivalency on biological activity is particularly difficult to anticipate in cellular assays,
given the structural complexity encountered by the ligands at the cell surface [19,20].
C. albicans is an opportunistic pathogenic yeast and the most prevalent cause of
hospital-acquired fungal infections worldwide. It is therefore hugely important that new
treatments are developed for these infections, which are now becoming resistant to conven-
tional antifungal medicines [21]. The molecular mechanisms of the adherence processes
of C. albicans to host cells and abiotic surfaces are complex, albeit essential for infection
and biofilm formation [22]. Hence, targeting adhesion in C. albicans offers an interesting
approach towards antifungal therapies as alternatives to conventional drugs [23]. Early re-
ports indicate that C. albicans adhesins recognise and bind to many cell surface glycans and
carbohydrates, including mono- and disaccharides (e.g., L-fucose,
and N-acetyl-D-glucosamine) [24 25]. Glycosphingolipids have also been shown to act as
adhesion receptors for yeasts. C. albicans bound specifically to lactosylceramide (Gal (1-
4)Glc (1-1)Cer), with the terminal galactose essential for binding [26], and to asialo-GM1
(gangliotetraosylceramide: Gal(1-3) GalNAc(1-4) Gal(1-4) Glc(1-1)Cer), with the mini-
mal carbohydrate sequence required for binding being GalNAc(1-4) Gal [27]. Previous
α-D-methyl mannoside
,
β
β
β
β
β
β
β
β
research in our group hence screened a small library of synthetic glycoconjugates with
various sugars and valencies, and tested their ability to inhibit the adhesion of C. albicans to
human BEC. This study showed that the divalent galactoside
1 (Figure 1) was a successful
inhibitor of the adhesion of C. albicans to BECs [28]. Ongoing research in the group is
aiming to identify the adhesin on the cell surface of the C. albicans with which this galacto-
sylated compound
derivative of the original lead compound
inhibition of the adhesion of C. albicans to BECs. Recently, we reported the multivalent pre-
sentation of lead compound on RAFT cyclopeptide- and polylysine-based scaffolds [29].
Tetra-, hexa- and hexadecavalent displays of compound were thus evaluated as inhibitors
1
is interacting. In this work, we developed a low valency-multivalent
1
to assess the effect of multivalent displays in the
1
1
of C. albicans adhesion, revealing a better performance for the lower valency glycoconju-
gates. In our investigations towards an optimal multivalent architecture to enhance the
activity of lead compound
30] to prepare glycoconjugate
C. albicans is not known, a tetravalent display of compound
as could provide a very useful comparison with the results observed when more rigid
and cyclic scaffolds were used. Peptoid scaffolds have been successfully investigated by
Taillefumier and co-workers in the design of defined valency glycoconjugates [31 32]. Thus,
lead compound was modified with a linker to facilitate connection with this scaffold using
Copper(I)-catalyzed Azide–Alkyne Cycloaddition (CuAAC) methodology. The tetravalent
display of compound as presented in glycoconjugate may significantly affect how
it interacts with carbohydrate-binding proteins in C. albicans and BECs surface, which,
1
, in the current study we opted for linear peptoid scaffold
(Figure 1). Since the structure of the target lectin in
in a flexible scaffold such
2
[
3
1
2
,
1
1
3

Pathogens 2021, 10, 572
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in turn, should allow for identification of the most suitable structural features to inhibit
pathogen–cell interaction.
Figure 1. Shows the structure of lead compound 1, peptoid scaffold 2 and multivalent glycoconjugate 3.
2. Results and Discussion
2.1. Chemical Synthesis
In order to install lead compound 1 onto the alkyne-featuring scaffold 2 via the CuAAC
reaction, it had to be functionalized with an azido group in a manner that would not com-
promise its interaction with target proteins in the C. albicans cell wall. Our previous work
showed that functionalization of 5-aminobenzene position in compound 1 with a fluores-
cent label still allowed for the recognition of the digalactoside motifs and localization of the
compound at the cell wall in C. albicans [28]. Thus, 5-amino-isophthalic acid was protected
using tert-butoxycabonyl (Boc) anhydride, followed by reaction with propargylamine and
TBTU to give compound
per-acetylated-1- -azido galactose [33] moieties to dialkyne
was then treated with TFA to remove the N-Boc-protecting group yielding compound
28]. Reaction with bromoacetyl bromide to give compound and subsequent bromide
displacement by treatment with sodium azide gave key azido-functionalized compound
29]. Attempts of direct reaction of this compound with alkynated scaffolds using CuAAC
4
(Scheme 1). CuAAC chemistry was used to conjugate the
β
4
to give compound , which
5
6
[
7
8
[
reaction conditions failed to yield any of the desired product, likely due to steric effects.
Oligo(ethyleneglycol)s are commonly used as linkers between the carbohydrate moieties
and the scaffold in multivalent glycoconjugates, because of their flexibility, water-solubility,
the availability of various lengths and the presence of functionalizable hydroxy groups [34].
Therefore, it was decided to use a triethylene glycol (TEG) derivative as a linker to join the
divalent galactoside 8 to the alkyne scaffolds 2.
Using well-documented procedures, TEG was reacted with propargyl bromide to
give the mono-propargylated linker
was carried out in excess of TEG. Compound
10 36]. This was then reacted with the azido di-galactoside
9
[
35]. To ensure mono-propargylation, the reaction
was then tosylated, yielding compound
using microwave (MW)
9
[
8
assisted CuAAC methodology to give compound 11. The tosyl group was then replaced
by an azide upon treatment with sodium azide to give compound 12. This key synthetic
intermediate presents the acetylated divalent galactoside with a TEG linker functionalized
with a terminal azide, available for CuAAC conjugation to various propargylated scaffolds.

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Scheme 1. Synthesis of azido-TEG divalent galactoside 12 (inset shows the synthesis of TEG
◦
linker): 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/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) CuAAC methodology, propargylated scaffolds, no product isolated; (viii) propargyl bromide,
◦
NaH, anhydrous THF, N2, 16 h, 75%; (ix) TsCl, KOH, DCM, 0 C, 2 h, 87%; (x) 10, CuSO4
·
5H2O/Na
◦
◦
Asc, CH3CN/H2O, MW (microwave), 100 C, 30 min, 74%; (xi) NaN3, CH3CN, DMF, 80 C, 24 h, 95%.
β
-peptoid scaffolds designed by Faure, Taillefumier and coworkers have been used to cre-
ate linear and cyclic displays of various recognition motifs, including carbohydrates [30–32]
Linear scaffold 30] was reacted with compound 12 using, once again, microwave-assisted
.
2
[
CuAAC conditions (Scheme 2); acetylated glycocluster 13 was thus obtained and subjected
to mild base hydrolysis that resulted in the removal of the tert-butyl ester and acetyl
protecting groups, yielding glycoconjugate
lead divalent galactoside . In addition, CuAAC reaction of linear scaffold
acetylated-1- -azido galactose [33] gave tetragalacoside 14, which was deprotected under
mild basic conditions to give 15 (Scheme 2). This compound, lacking structural features of
lead compound other than terminal -triazolyl-galactosides, would serve as a control in
3, which presents a tetravalent display of the
1
2
with per-
β
1
β
subsequent biological evaluation of anti-adhesion activity against C. albicans.
2.2. Biological Evaluation
In our previous work [28
not toxic to C. albicans yeast cells at the concentrations used in the current study. Glyco-
conjugates , featuring multivalent displays of the original lead compound , along with
tetra- -triazolyl-galactoside 15, were then evaluated for their anti-adhesive properties
,29] we established that compound 1 and derivatives were
3
1
β
against C. albicans using several assays.
2.2.1. Exclusion Assay
The initial adherence assay was performed by treating the C. albicans with compounds
3
and 15. After an incubation period of 90 min, the treated yeast cells were exposed to
exfoliated BECs. The average number of yeast cells attached to each BEC was calculated
(Figure 2a), and the percentage increase or decrease of the number of C. albicans cells
adhering to the BECs was compared to a control (untreated yeast) and the lead compound
1
(Figure 2b). In this assay, compound 15 only slightly reduced the adhesion of the yeast
to the BECs, whereas compound did not have any appreciable effect. Compound
3
1
showed moderate inhibition of adhesion at this concentration (up to 25% reduction at
10 mg/mL). On the other hand, when the BECs were treated with the above compounds,
allowing the incubation period and followed by exposure to C. albicans cells, the average

Pathogens 2021, 10, 572
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number of yeasts attached per BEC was reduced (Figure 2c). The tetra-galactoside 15
caused similar reduction of yeast adhesion as in the previous assay, while multivalent
glycoconjugate
by lead compound
compound disfavours significantly the interactions with structural components of the cell
wall in C. albicans, which had been previously proposed for compound 28]. However,
3
inhibited C. albicans adhesion by 35%, similarly to the inhibition produced
1
(Figure 2d). These results suggest that the multivalent presentation in
3
1
[
this effect was not observed upon preliminary exposure to BECs, since both compounds
1
and 3 caused a similar decrease in yeast adhesion.
Scheme 2. Synthesis of glycoconjugate
CH3CN/H2O, MW, 100 C, 30 min, 58–68%; (ii) MeOH, NEt3, H2O, 45 C, 6 h, 82–92%.
3
.
Reagents and conditions:◦ (i) CuSO4
·
5H2O/Na Asc,
◦
2.2.2. Competition Assay
The glycoconjugates were then evaluated in a competition assay, in which their anti-
adhesion abilities were tested in the presence of both C. albicans and BECs. Co-incubation
with the compounds resulted in a reduction in yeast adhesion in all cases (Figure 3a), with
compound
3 causing up to a 49% decrease. Interestingly, lead compound 1, which showed
activity in both exclusion assays discussed above (i.e., when pre-incubated with C. albicans
and BECs, independently), was able to inhibit up to 64% the adhesion of C. albicans to BECs
in the competition assay.
The compounds were then tested at lower concentrations. Glycoconjugate
3 exhib-
ited a typical dose–response pattern, with ca. 50% inhibition of adhesion at the highest
concentration (Figure 3b). Compound 15 (Figure 3c) did not show a linear relationship
between concentration and activity, where the best inhibition of adhesion was observed at
1 mg/mL.

Pathogens 2021, 10, 572
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Figure 2. Exclusion assays of glycoconjugates
3
and 15: (
a) Shows the average number of yeast
attached per BEC (buccal epithelial cells), after the yeast cells were pre-treated with glycoconjugates;
) Shows the percentage change in the adherence of yeast to the BEC versus the control and the
lead compound after the yeast were pre-treated; ( ) Shows the average number of yeast attached
per BEC, after the BEC were pre-treated with compounds; ( ) Shows the percentage change in the
adherence of yeast to the BEC versus the control and the lead compound after the BEC were
(b
1
c
d
1
pre-treated. All assays were performed at a glycoconjugate concentration of 10 mg/mL.
Overall, this study shows that the tetravalent presentation of lead compound
1
gener-
ated in glycoconjugate 3 significantly impaired its ability to interact with C. albicans cells,
although it maintained anti-adhesion activity when pre-incubated with BECs. While no
significant reduction of adhesion was observed when C. albicans cells were pre-treated with
glycoconjugate 3, this compound was able to inhibit adhesion of the yeast when BECs were
pre-treated or in competition assays. This clearly indicates that the multivalent compounds
in this study do not interact effectively with C. albicans cell wall components; the observed
reduction in adhesion appears to be due to preferential binding of the compounds to BECs
over the yeast cells. Tetra-galactoside 15, which does not display the structural features of
lead compound
although at lower concentration it showed better activity; however, this compound did
not follow a dose–response pattern. The original lead compound was the most effective
inhibitor of yeast adhesion. Interestingly, the exclusion assays show that compound pro-
duces a decrease in adhesion not only when pre-exposed to C. albicans cells, as our previous
work has shown, but also there is a significant contribution to the activity of compound
arising from interaction with BECs. These results highlight that the optimization of activity
of derivatives of compound should consider also interactions with BECs as a potential
approach to broad-spectrum anti-adhesive glycoconjugates.
1 nor a TEG linker, generally showed the lowest inhibition of adhesion,
1
1
1
1

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Figure 3. Competition assays of glycoconjugates
1,
3
and 15: (
a) Shows the change in adherence
compared to the control and the lead compound
1
at 10 mg/mL; (b) Shows the average number
of yeast attached to BEC in the presence of compound
3
(10, 1, 0.1 mg/mL); (c) Shows the average
number of yeast attached to BEC in the presence of compound 15 (10, 1, 0.1 mg/mL).
3. Materials and Methods
3.1. Chemistry
3.1.1. General Methods
All reagents for synthesis were bought commercially and used without further purifi-
cation. Tetrahydrofuran (THF) was freshly distilled over sodium wire and benzophenone.
Dichloromethane (DCM) was freshly distilled over CaH2 prior to use. 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 spectrometer at 293K. All chemical shifts
were referenced relative to the relevant deuterated solvent residual peaks. Assignments of
the NMR spectra were deduced using 1H 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 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 −1. High-performance liquid chro-
g
·
matography 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
obtained as a film on NaCl plates or as KBr disks in the region 4000–400 cm−1 on a Perkin

Pathogens 2021, 10, 572
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Elmer Spectrum 100 FT-IR spectrophotometer. Spectroscopic data for all compounds are
provided in the Supplementary Materials.
3.1.2. Synthetic Procedures
Synthesis of 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 (7)
6 [21] (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
7 without
further purification as a brown, sticky solid (1.056 g, 83%). Rf = 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, 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-60), 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.
Synthesis of 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 (8)
7
(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 re-dissolved in DCM (20 mL)
and was washed with brine (20 mL
solvent was removed in vacuo to obtain the pure product
as a yellow solid (1.056 g, 83%). Rf = 0.41 (DCM:MeOH 9:1).[α]2D2 -5.6 (c 0.9, DCM). 1H
NMR (500 MHz, CDCl3) 9.10 (s, 1H, NHCOCH2N3), 8.18 (s, 2H, NHCH2CCH), 8.02 (s,
×
3). The organic phase was dried over MgSO4 and the
8
without further purification
δ
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-60), 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.
Synthesis of 2-[2-(2-Propargyloxyethoxy)ethoxy]ethanol (9)
Procedure adapted from Weil et al. [30]. Triethylene glycol (1 mL, 7.48 mmol, 3 equiv)
was diluted with dry THF (10 mL) under N2. The solution was cooled to 0 ◦C and NaH
(60% oil dispersion) (0.1 g, 2.49 mmol) was added portion-wise. The reaction was allowed
to warm up to rt and was stirred for 20 min. Propargyl bromide (0.27 mL, 2.49 mmol)
was added dropwise. The reaction mixture was allowed to stir overnight. Column chro-

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matography (100% EtOAc) eluted the pure product
EtOAc) 1H NMR (500 MHz, CDCl3)
9
as a clear oil (0.292 g, 75%). (Rf = 0.42:
δ
4.09–4.08 (m, 2H, CH2CCH), 3.63–3.53 (m, 10H, CH2
×
5), 3.50–3.46 (m, 2H, CH2-OH), 3.11 (s, 1H, OH), 2.39 (t, J = 2.8 Hz, 1H, CH2CCH). The
NMR data is in agreement with the data reported in the literature [30].
Synthesis of 2-(2-(2-Propargyloxyethoxy)ethoxy)ethyl-4-methylbenzenesulfonate (10)
This procedure was adapted from Ramström et al. [31].
9 (0.288 g, 1.53 mmol) was
dissolved in DCM (5 mL). TsCl (0.321 g, 1.68 mmol, 1.1 equiv) was added and the mixture
◦
was cooled to 0 C on ice. KOH (0.343 g, 6.12 mmol, 4 equiv) was added slowly after
grinding. The mixture was vigorously stirred for 2 h. The mixture was poured onto ice-
water and extracted with DCM (3
×
20 mL). The combined organic layers were dried over
MgSO4, filtered and concentrated in vacuo to give the pure product 10 as a clear oil (0.457 g,
1
87%). H NMR (500 MHz, CDCl3)
δ
7.79 (d, J = 8.3 Hz, 2H, Ar-H), 7.33 (d, J = 8.2 Hz, 2H,
Ar-H), 4.18 (d, J = 2.4 Hz, 2H, CH2CCH), 4.16–4.13 (m, 2H, CH2OTs), 3.69–3.65 (m, 4H, CH2
2), 3.65–3.61 (m, 2H, CH2), 3.58 (s, 4H, CH2 2), 2.43 (s, 3H, CH3-Ar), 2.42 (t, J = 2.4 Hz,
1H, CH2CCH). The NMR data are in agreement with the data reported in the literature [31].
×
×
Synthesis of 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-(4-methylbenzenesulfonate)ethoxy)ethoxy)ethoxy) methyl)-1H-
1,2,3-triazol-1-yl)acetamido)-5-aminobenzene-1,3-dicarboxamide (11)
Copper sulphate pentahydrate (20 mg) and sodium ascorbate (40 mg) were added to a
solution
8
(0.516 g, 0.476 mmol) and 10 (0.163 g, 0.476 mmol) in CH3CN/H2O (4 mL/2 mL).
◦
The reaction was allowed to stir in the MW at 100 C until deemed complete by TLC
analysis (15 min). The solvent was removed in vacuo. The residue was dissolved in DCM
(30 mL), washed with brine (20 mL
×
3), and dried (MgSO4). The mixture was filtered
and the solvent was removed in vacuo to yield the crude product, which was purified
by silica gel column chromatography (DCM:MeOH 98:2–95:5) to give the pure product
11 as a yellow solid (0.307 g, 74%). Rf = 0.45 (DCM:MeOH 9:1). [α]2D2-5 (c 1, DCM). 1H
NMR (500 MHz, CDCl3)
CH-triaz), 7.84 (appd, J = 2.8 Hz, 3H, Ar-H
J = 8.3 Hz, 2H, Ar-H of OTs), 7.26 (d, J = 8.1 Hz, 2H, Ar-H of OTs), 5.89 (d, J = 9.2 Hz, 2H,
H-1), 5.60 (t, J = 9.7 Hz, 2H, H-2) 5.50 (d, J = 2.9 Hz, 2H, H-4), 5.26 (dd, J = 10.3, 3.3 Hz,
2H, H-3), 5.21 (s, 2H, CH2), 4.67–4.55 (m, 6H, CH2-triaz and CH2CN3), 4.28 (t, J = 6.6 Hz,
2H, H-5), 4.11 (qd, J = 11.5, 6.6 Hz, 2H, H-6 and H-60), 4.06–4.04 (m, 2H, CH2), 3.65–3.59
δ
9.82 (s, 1H, NHCH2N3), 8.16 (s, 2H, NHCH2CCH), 7.97 (s, 2H,
2 and CH-triaz), 7.76 (s, 1H, Ar-H), 7.69 (d,
×
(m, 2H, CH2), 3.58–3.54 (m, 4H, CH2
(s, 6H, CH3 of OAc), 1.96 (s, 6H, CH3 of OAc), 1.95 (s, 6H, CH3 of OAc), 1.75 (s, 6H, CH3
of OAc). 13C NMR (125 MHz, CDCl3)
170.4 (CO of OAc), 170.1 (CO of OAc), 169.9 (CO
×
2), 3.48 (s, 2H, CH2), 2.37 (s, 3H, CH3 of OTs), 2.14
δ
of OAc), 169.3 (CO of OAc), 166.6 (CONHCH2CCH), 164.5 (COCH2N3), 145.4 (C-triaz),
144.9 (Ar-C of OTs), 144.8 (CHCN3), 138.1 (Ar-C), 134.8 (Ar-C), 132.7 (Ar-C of OTs), 129.9
(Ar-CH of OTs), 127.9 (Ar-CH of OTs), 125.3 (CHCN3), 121.9 (CH-triaz), 121.6 (Ar-CH),
121.3 (Ar-CH), 86.0 (C-1), 73.8 (C-5), 70.9 (C-3), 70.5 (CH2), 70.4 (CH2), 70.3 (CH2), 69.7
(CH2), 69.4 (CH2), 68.6 (CH2), 68.0 (C-2), 66.9 (C-4), 64.3 (NHCOCH2N3), 61.1 (C-6), 52.8
(CH2), 35.4 (CH2-triaz), 21.6 (CH3 of OAc), 20.6 (CH3 of OAc), 20.5 (CH3 of OAc), 20.2 (CH3
of OAc). IR (film on NaCl): 3344, 3091, 2939, 1754, 1657, 1599, 1535, 1448, 1370, 1223, 1176,
1095, 1054, 924 cm−1. HRMS (ESI+): m/z calculated for C60H74N12O27S + Na+ [M+Na+]:
1449.4405, found 1449.4332.
Synthesis of N,N’-di-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-1,2,3-triazol-4-
ylmethylamide)-N”-(2-4-((2-(2-(2-azidoethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-
yl)acetamido)-5-aminobenzene-1,3-dicarboxamide (12)
Compound 11 (183 mg, 0.128 mmol) and NaN3 (17 mg, 0.256 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 dissolved
in DCM (20 mL) and was washed with brine (20 mL
(MgSO4) and the solvent was removed in vacuo to obtain the pure product 12 without
×
3). The organic phase was dried

Pathogens 2021, 10, 572
10 of 14
further purification as a yellow solid (167 g, 100%). Rf = 0.42 (DCM:MeOH 9:1). [α]2D2-3 (c 1,
DCM). 1H NMR (500 MHz, CDCl3)
δ 9.79 (s, 1H, NHCH2N3), 8.13 (s, 2H, NHCH2-triaz),
7.94 (s, 2H, CH-triaz), 7.82 (s, 1H, CH-triaz), 7.77 (s, 2H, Ar-H), 7.70 (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), 5.25–5.15 (m,
4H, H-3 and CH2), 4.66–4.48 (m, 6H, CH2-triaz
×
3), 4.24 (t, J = 6.3 Hz, 2H, H-5), 4.13–4.04
4), 3.27–3.24 (m, 2H, CH2), 2.12 (s, 6H, CH3
0
(m, 4H, H-6 and H-6 ), 3.67–3.47 (m, 8H, CH2
×
of OAc), 1.94 (s, 6H, CH3 of OAc), 1.93 (s, 6H, CH3 of OAc), 1.73 (s, 6H, CH3 of OAc). 13C
NMR (125 MHz, CDCl3)
δ 170.5 (CO of OAc), 170.2 (CO of OAc), 170.0 (CO of OAc), 169.4
(CO of OAc), 166.7 (CONHCH2-triaz), 164.5 (COCH2N3), 145.6 (C-triaz), 145.0 (CHCN3),
134.9 (Ar-C), 125.4 (CHCN3), 121.9 (CH-triaz), 121.7 (Ar-CH), 121.3 (Ar-CH), 86.2 (C-1),
74.0 (C-5), 71.0 (C-3), 70.6 (CH2
(NHCOCH2N3), 61.2 (C-6), 50.7 (CH2), 35.5 (CH2-triaz), 20.7 (CH3 of OAc
×
2), 70.0 (CH2), 69.9 (CH2), 68.1 (C-2), 67.0 (C-4), 64.5
×
2), 20.6 (CH3
of OAc), 20.4 (CH3 of OAc). IR (film on NaCl): 3335, 3088, 2924, 2109, 1754, 1658, 1600,
1534, 1447, 1370, 1222, 1054, 924 cm−1. HRMS (ESI+): m/z calculated for C53H67N15O24
+
Na+ [M+Na+]: 1320.4381, found 1320.4375.
Synthesis of tert-butyl-(4,8,12,16-tetra-aza)(5,9,13,17-tetra-oxo)(4,8,12,16-tetra-N-propargyl)
octadeconoate (2)
The method outlined by Faure, Taillefumier and coworkers was adapted to synthesise
the tetrameric propargylated scaffold [25]. The secondary amine was then acetylated using
the following procedure:
Tetrameric propargylated scaffold (0.490 g, 0.96 mmol) was dissolved in DCM (20 mL).
Acetic anhydride (0.91 mL, 9.6 mmol) was added to the solution. The reaction mixture was
allowed to stir for 6 h at rt and was concentrated under reduced pressure. The crude mixture
was dissolved in ethyl acetate (20 mL) and was washed with sat NaHCO3 (20 mL
×
2) and
brine (20 mL 2). The organic layer was dried (MgSO4), filtered and then concentrated in
×
vacuo. The crude product was purified by silica gel column chromatography (DCM:MeOH
1
9:1) to give the pure product as a pale-yellow oil (515 mg, 97%). H NMR (500 MHz, CDCl3)
δ
4.24–4.06 (m, 8H), 3.83–3.70 (m, 3H), 3.72–3.60 (m, 5H), 2.91–2.68 (m, 6H), 2.55 (m, 2H),
2.37–2.23 (m, 2H), 2.24–2.10 (m, 4H), 1.43 (s, 9H, C(CH3)3). 13C NMR (125 MHz, CDCl3)
171.5, 171.2, 170.9, 170.1, 81.7, 81.6, 81.4, 80.9, 78.9, 78.9, 78.6, 72.9, 72.8, 72.6, 72.5, 72.5,
δ
72.1, 71.9, 71.7, 44.4, 44.2, 43.9, 43.7, 43.6, 43.5, 43.0, 42.8, 39.8, 39.1, 39.0, 38.9, 38.6, 38.4,
34.8, 34.5, 34.4, 34.3, 34.1, 32.2, 31.8, 29.7, 28.1, 21.8, 21.5. HRMS (ESI+): m/z calculated for
1
C30H40N4O6 + H+ [M+H+]:553.3026, found 553.3015. H NMR and 13C NMR spectroscopic
data corresponded to that found in the literature [25].
Synthesis of Acetylated Tetravalent β-Peptoid Glycocluster (13)
Copper sulphate pentahydrate (40 mg) and sodium ascorbate (80 mg) were added to
a solution of 10 (30 mg, 0.0231 mmol) and 14 (13 mg, 0.0231 mmol) in CH3CN/H2O (4 mL/
2 mL). The reaction was allowed to stir in the MW at 100 ◦C for 10 min
×
2. The solvent
was removed in vacuo. The residue was dissolved in DCM (30 mL), washed with brine
(20 mL 3), and dried (MgSO4). The mixture was filtered and the solvent was removed in
×
vacuo to yield the crude product, which was purified by silica gel column chromatography
(DCM:MeOH 98:2–95:5) to give the pure product 13 as a yellow sticky solid (77 mg, 58%).
Rf = 0.48 (DCM:MeOH 9:1). [α]2D4-7 (c 1, DCM). H NMR (500 MHz, CDCl3)
δ 10.08 (s,
1
4H, NHCOCH2N3), 8.56–7.59 (m, 36H, NHCH2CCH × 8, CH2CCH × 16 and Ar-H × 12),
5.91 (s, 8H, H-1), 5.63 (t, J = 10.2 Hz, 8H, H-2), 5.53 (s, 8H, H-4), 5.28 (d, J = 10.2 Hz, 16H,
H-3 and CH2
CH20 s), 3.86–3.31 (m, 13H, CH20 s), 3.11–2.31 (m, 4H, CH20 s), 2.18 (s, 7H, OAc and NAc),
1.99 (s, 15H, OAc), 1.79 (s, 6H, OAc), 1.43 (s, 3H, tert-butyl). 13C NMR (125 MHz, CDCl3)
×
4), 4.80–4.39 (m, 6H, CH2-triaz, CH20 s), 4.38–4.05 (m, H, H-5 and H-6,
δ
170.59, 170.35, 170.09, 122.1, 121.6, 120.7, 86.3 (C-1), 74.1 (C-5), 71.1 (C-3), 71.0 (CH2), 70.7
(CH2), 70.6 (CH2), 70.5 (CH2), 69.8 (CH2), 69.3 (CH2), 68.2 (C-2), 67.1 (C-4), 64.7 (CH2), 61.3
(C-6), 52.9 (CH2), 50.3 (CH2), 44.2 (CH2), 43.5 (CH2), 43.2 (CH2), 42.8 (CH2), 40.0 (CH2),
39.8 (CH2), 38.7 (CH2), 35.5 (CH2-triaz), 34.3 (CH2), 33.8 (CH2), 32.1 (CH2), 29.89, 28.3

Pathogens 2021, 10, 572
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(C(CH3)), 21.7 (CH3 of NHAc), 20.84 (CH3 of OAc), 20.72 (CH3 of OAc), 20.45 (CH3 of
OAc), 1.21. IR (film on NaCl): 3392, 2927, 1753, 1647, 1536, 1448, 1370, 1223, 1092, 1060, 923,
732 cm−1. MALDI-TOF-MS [M+H]+: m/z calculated for C242H309N64O102 +H+: 5744.104,
found 5744.346.
Synthesis of Tetravalent β-Peptoid Glycocluster (3)
Compound 13 (70 mg, 0.0122 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 the pure
1
product
3
as a white fluffy solid (44 mg, 82%). H NMR (500 MHz, D2O)
δ
8.62–7.72 (m,
28H, Ar-H and triaz-H), 5.69 (s, 10H, H-1 and CH2s), 5.47 (s, 6H, CH2s), 4.74–4.44 (m, 24H,
CH2-triaz and CH2s), 4.22 (s, 14H, H-2 and CH2s), 4.11 (d, J = 20.3 Hz, 14H, H-4 and CH2s),
3.99 (s, 8H, H-5), 3.88 (d, J = 9.8 Hz, 14H, H-3 and CH2s), 3.84–3.36 (m, 60H, H-6, H-60 and
CH2s), 2.99–2.31 (m, 24H, CH2s), 1.99–1.86 (m, 3H CH3). 13C NMR (125 MHz, DMSO)
δ
171.1, 170.9, 166.1, 166.0, 165.1, 162.8, 145.5, 144.3, 139.1, 135.6, 126.2, 124.0, 122.3, 121.7,
88.5, 78.9, 74.2, 70.2, 70.1, 70.0, 69.8, 69.5, 69.4, 69.1, 68.9, 63.9, 60.9, 60.7, 52.6, 49.9, 49.8, 45.9,
40.5, 40.4, 40.2, 40.0, 39.9, 39.7, 39.5, 36.3, 35.4, 31.3, 28.2, 9.1. IR (ATR): 3301, 2925, 1650,
1540, 1443, 1388, 1253, 1092, 1055, 891 cm−1. MALDI-TOF-MS [M+H]+: m/z calculated for
C242H309N64O102 +Na+: 4422.7465, found 4422.807.
Synthesis of 2,6,10,14-tetraoxo-3,7,11,15-tetrakis((1-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl
-1H-1,2,3-triazol-4-yl)methyl)-3,7,11,15-tetraazaoctadecan-18-oic acid (14)
Copper sulphate pentahydrate (40 mg) and sodium ascorbate (80 mg) were added to a
solution of per-acetylated-1- -azido galactose [28] (567 mg, 1.520 mmol) and 25] (200 mg,
0.362 mmol) in CH3CN/H2O (4 mL/2 mL). The reaction was stirred in the MW at 100 ◦C
for 20 min (10 min 2). The solvent was removed in vacuo. The residue was dissolved
in DCM (30 mL), washed with brine (20 mL 3), and dried (MgSO4). The mixture was
β
2 [
×
×
filtered and the solvent was removed in vacuo to yield the crude product, which was
purified by silica gel column chromatography (DCM:MeOH 98:2–95:5) to give the pure
product 14 as a yellow sticky solid (503 mg, 68%). Rf = 0.47 (DCM:MeOH 9:1). [α]2D7-4.71
(c 0.85, DCM). 1H NMR (500 MHz, CDCl3)
H-1), 5.59–5.42 (m, 8H, H-2 and H-4), 5.37–5.18 (m, 4H, H-3), 4.81–4.49 (m, 8H, CH2
4.35–4.08 (m, 12H, H-5, H-6 and H-60), 3.83–3.53 (m, 8H, CH2
4), 3.02–2.68 (m, 6H, CH2
3), 2.65–2.47 (m, 2H, CH2), 2.27–2.21 (m, 12H, OAc), 2.20–2.15 (m, 3H, NAc), 2.06–1.98
(m, 24H, OAc), 1.92–1.80 (m, 12H, OAc), 1.47–1.40 (m, 9H, C(CH3)3). 13C NMR (125 MHz,
CDCl3) 171.3, 170.3, 170.1, 169.8, 168.8, 144.8, 144.6, 122.3, 122.2, 86.3, 77.3, 77.0, 76.8, 74.0,
δ
7.98–7.77 (m, 4H, triaz-H), 5.98–5.74 (m, 4H,
4),
×
×
×
δ
70.8, 68.0, 66.8, 61.1, 45.2, 44.2, 40.8, 34.6, 31.9, 31.8, 28.1, 22.0, 21.5, 20.7, 20.6, 20.5, 20.2. IR
(ATR): 2938, 1746, 1638, 1423, 1368, 1211, 1157, 1091, 1044, 952, 922, 841, 733 cm−1. HRMS
(ESI+): m/z calculated for C86H116N16O42 + Na+ [M+Na+]: 2067.7331, found 2067.7223.
Synthesis of 2,6,10,14-tetraoxo-3,7,11,15-tetrakis((1-(β-D-galactopyranosyl-1H-1,2,3-triazol-
4-yl)methyl)-3,7,11,15-tetraazaoctadecan-18-oic acid (15)
Compound 14 (452 mg, 0.221 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 the
pure product 15 as a white fluffy solid (267 mg, 92%). [α]2D4+14.0 (c 0.5, H2O). 1H NMR
(500 MHz, D2O)
8H, CH2-triaz and CH2), 4.19–4.06 (m, 4H, H-2), 4.01 (appdd, J = 5.2, 3.2 Hz, 4H, H-4),
3.97–3.86 (m, 4H, H-5), 3.85–3.76 (m, 4H, H-3), 3.75–3.61 (m, 12H, H-6, H-60 and CH2
2),
3.58–3.51 (m, 4H), 2.80–2.46 (m, 8H, CH2
4), 2.13–2.04 (m, 1H), 1.16 (s, 3H, CH3). 13C
δ 8.23–8.16 (m, 2H), 8.08 (s, 2H), 5.64–5.54 (m, 4H, H-1), 4.65–4.42 (m,
×
×

Pathogens 2021, 10, 572
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NMR (126 MHz, DMSO)
δ
173.1, 170.5, 170.3, 144.6, 122.5, 122.3, 88.6, 88.5, 78.9, 75.6, 74.2,
69.9, 68.9, 60.9, 58.6, 45.7, 44.7, 43.8, 43.0, 42.9, 39.9, 22.1, 21.7, 9.0. IR (film on NaCl): 3299,
2916, 1719, 1620, 1451, 1421, 1365, 1224, 1087, 1053, 885 cm−1. HRMS (ESI+): m/z calculated
for C50H76N16O26 + Na+ [M+Na+]: 1339.5014, found 1339.5032.
3.2. Biological Evaluation
Fungal Strain: C. albicans (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 volun-
teers 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.
Adherence assays: Yeast cells were mixed with BECs in a ratio of 50:1 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 cells or BECs were incubated
for 90 min in the presence of each compound at the given concentration. After this time,
the cells were harvested and washed twice with PBS before being resuspended in 1 mL
PBS before being mixed with BECs or yeast cells (as described). In the competition assay
format yeast cells, BECs and compound (at the given concentration) were co-incubated for
90 min prior to harvesting.
Statistics: All experiments were performed on three independent occasions. In each
assay, the number of yeast cells adhering to 200 randomly chosen BECs was determined.
Results are mean ± SEM.
4. Conclusions
In summary, we report the synthesis of a multivalent display of divalent galactoside
1
, a potent inhibitor of the adhesion of C. albicans to BECSs. Glycoconjugate
upon a linear peptoid scaffold, to access distinct spatial presentations of the recognition
motifs provided by lead compound . This compound, together with control compound 15
3 was built
1
,
were successfully synthesised using a series of sequential CuAAC reactions. The ability of
these compounds to inhibit adhesion of C. albicans to BECs was then evaluated. Although
multivalent presentations of carbohydrate epitopes are commonly used to increase their
biological activity, we did not observe this effect for the compounds studied herein. In
fact, the exclusion assays indicated that the multivalent glycoconjugates did not bind
effectively to C. albicans cell wall, in contrast to their parent compound
1. Since the
fungal target for lead compound is not known, the results highlight the importance
1
of detailed structural knowledge of the target proteins when considering the design of
multivalent presentation. Interestingly, the inhibition of C. albicans adhesion observed for
the compounds in this study is then likely due to interactions with BECs. Further studies
are currently underway in our laboratory to establish if these are specific interactions
and to optimize structural parameters, as this could extend the scope of activity of these
compounds to other pathogens in the oral cavity.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/
10.3390/pathogens10050572/s1; it includes spectroscopic data for the synthetic compounds de-
scribed herein.

Pathogens 2021, 10, 572
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Author Contributions: Conceptualization, K.K. and T.V.-T.; methodology, H.M. (Harlei Martin) and
H.M. (Hannah Masterson); formal analysis, H.M. (Harlei Martin); investigation, H.M. (Harlei Martin);
resources, K.K. and T.V.-T.; data curation, all authors; writing—original draft preparation, H.M.
(Harlei Martin); writing—review and editing, H.M. (Harlei Martin), K.K. and T.V.-T.; supervision,
K.K. and T.V.-T. All authors have read and agreed to the published version of the manuscript.
Funding: We would like to acknowledge Maynooth University for the provision of the John and Pat
Hume Scholarship to H. Martin.
Institutional Review Board Statement: All experiments were performed in accordance with the
Guidelines stipulated in Directive 2004/23/EC of the European Parliament and of the Council (31
March 2004). Experiments were conducted according to the guidelines by the Ethics Committee at
Maynooth University.
Informed Consent Statement: Informed consent was not necessary for this study.
Data Availability Statement: The data presented in this study are available within the article and in
the supplementary material.
Acknowledgments: We would like to acknowledge Maynooth University for the provision of the
John and Pat Hume Scholarship to H. Martin.
Conflicts of Interest: The authors declare no conflict of interest.
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Varki, A. Biological roles of glycans. Glycobiology 2012, 27, 3–49. [CrossRef]
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1457–1468. [CrossRef]
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