Ruthenium-centred btp glycoclusters as inhibitors for: Pseudomonas aeruginosa biofilm formation

Article abstract of DOI:10.1039/d0ra05107a

Carbohydrate-decorated clusters (glycoclusters) centred on a Ru(ii) ion were synthesised and tested for their activity against Pseudomonas aeruginosa biofilm formation. These clusters were designed by conjugating a range of carbohydrate motifs (galactose, glucose, mannose and lactose, as well as galactose with a triethylene glycol spacer) to a btp (2,6-bis(1,2,3-triazol-4-yl)pyridine) scaffold. This scaffold, which possesses a C2 symmetry, is an excellent ligand for d-metal ions, and thus the formation of the Ru(ii)-centred glycoclusters 7 and 8Gal was achieved from 5 and 6Gal; each possessing four deprotected carbohydrates. Glycocluster 8Gal, which has a flexible spacer between the btp and galactose moieties, showed significant inhibition of P. aeruginosa bacterial biofilm formation. By contrast, glycocluster 7, which lacked the flexible linker, didn't show significant antimicrobial effects and neither does the ligand 6Gal alone. These results are proposed to arise from carbohydrate-lectin interactions with LecA, which are possible for the flexible metal-centred multivalent glycocluster. Metal-centred glycoclusters present a structurally versatile class of antimicrobial agent for P. aeruginosa, of which this is, to the best of our knowledge, the first example.

Full text of DOI:10.1039/d0ra05107a

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Ruthenium-centred btp glycoclusters as inhibitors
for Pseudomonas aeruginosa biofilm formation†
Cite this: RSC Adv., 2021, 11, 16318
a
c
Ciaran O'Reilly,ab Salvador Blasco,
Gordon Cooke,
Bina Parekh,b Helen Collins,
bc
a
d
*
Thorfinnur Gunnlaugsson
and Joseph P. Byrne
Carbohydrate-decorated clusters (glycoclusters) centred on a Ru(II) ion were synthesised and tested for
their activity against Pseudomonas aeruginosa biofilm formation. These clusters were designed by
conjugating a range of carbohydrate motifs (galactose, glucose, mannose and lactose, as well as
galactose with a triethylene glycol spacer) to a btp (2,6-bis(1,2,3-triazol-4-yl)pyridine) scaffold. This
scaffold, which possesses a C2 symmetry, is an excellent ligand for d-metal ions, and thus the formation
of the Ru(II)-centred glycoclusters 7 and 8Gal was achieved from 5 and 6Gal; each possessing four
deprotected carbohydrates. Glycocluster 8Gal, which has a flexible spacer between the btp and
galactose moieties, showed significant inhibition of P. aeruginosa bacterial biofilm formation. By
contrast, glycocluster 7, which lacked the flexible linker, didn't show significant antimicrobial effects and
neither does the ligand 6Gal alone. These results are proposed to arise from carbohydrate–lectin
interactions with LecA, which are possible for the flexible metal-centred multivalent glycocluster. Metal-
centred glycoclusters present a structurally versatile class of antimicrobial agent for P. aeruginosa, of
which this is, to the best of our knowledge, the first example.
Received 9th June 2020
Accepted 26th April 2021
DOI: 10.1039/d0ra05107a
rsc.li/rsc-advances
together by these lectins and encapsulated in a complex extracel-
lular matrix,2,7 and their formation is implicated in chronic P.
Introduction
aeruginosa infections, which exhibit marked resistance to antibi-
otics and the capacity to evade host defences.8 The physical barrier
of the biolm may be sufficient to prevent or slow penetration by
conventional antimicrobial agents, which are effective on the
bacteria in its planktonic form. Novel compounds that prevent
biolm-based infection without directly killing the bacteria could
restore sensitivity to established antibiotics.
These surface lectins, LecA and LecB, show specic binding
for galactosides and fucosides respectively and thus targeting
these proteins with carbohydrate-appended therapeutic mole-
cules is a valuable strategy (e.g. for disrupting biolm forma-
tion).9–12 In 2008, Hauber et al. showed that inhalation of
galactose and fucose monosaccharides was a safe treatment,
and subsequently research has focussed on developing more
selective or potent antimicrobial agents.9,13–15 Carbohydrate
multivalency has been shown to be very important for providing
high-affinity interactions, represented by a range glycopeptide
dendrimers, glyco-nanoparticles and glycoclusters, including
compounds built upon macrocyclic scaffolds.11,15–22
Pseudomonas aeruginosa is a ubiquitous Gram-negative patho-
genic bacteria, which is of great medical signicance as a result
of its multidrug resistance and effects on immune-
compromised patients, such as those suffering from cystic
brosis. It has been identied as one of the ‘ESKAPE’ patho-
gens, which are the cause of many healthcare-acquired infec-
tions, and for which development of new antimicrobial agents
is vital.1 Biolm formation is a key part of pathogenic behaviour
for many bacteria, and its inhibition is an important target.2–4 P.
aeruginosa expresses two carbohydrate-binding proteins on its
surface which have been identied as essential to its patho-
genesis, namely lectins LecA and LecB (also known as PA-IL and
PA-IIL).5,6 These lectins are pivotal in processes including
adhesion to epithelial and endothelial cells, as well as biolm
formation. Biolms are structured aggregates of bacteria linked
aSchool of Chemistry, Trinity Biomedical Sciences Institute, Trinity College Dublin,
Ireland
bSchool of Medicine, University College Dublin, Beleld, Dublin 4, Ireland
A recent example of such an antiadhesive glycocluster
system, described by Vidal and co-workers, was based on a calix
[4]arene scaffold, that was derivatised with galactose and fucose
monosaccharide units.19 These carbohydrates selectively bind
LecA and LecB and, as a result, this macrocyclic system was
shown to inhibit biolm formation, and was also studied in an
in vivo mouse model where it was shown to demonstrate
cDepartment of Applied Science, Tallaght Campus, Technological University Dublin,
Ireland
dSchool of Chemistry, National University of Ireland Galway, University Road, Galway,
Ireland. E-mail: joseph.byrne@nuigalway.ie
† Electronic supplementary information (ESI) available: NMR spectra, biological
testing data & supplementary crystallographic data. CCDC 2004944. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/d0ra05107a
16318 | RSC Adv., 2021, 11, 16318–16325
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
protection against P. aeruginosa lung infection. The topology of
carbohydrate presentation from the calix[4]arene scaffold was
shown to play an important role, with tetravalent clusters pre-
senting two pairs of epitopes from opposite faces of the calix[4]
arene scaffold proving most potent.23,24 In this example, tri-
ethylene glycol was used successfully as a exible spacer,
however different spacers, both rigid and exible, have also
been utilised to allow for tuning of avidity of lectin inhibi-
tors.10,14,25,26 In order to present the saccharide units in a three-
dimensional way, various different scaffold-types have been
utilised to design ligands for LecA, including glycopeptide
dendrimer, cyclodextrins and carbohydrate-based scaffolds. To
the best of our knowledge, coordination of a metal ion has yet to
be exploited in design of inhibitors targeting the P. aeruginosa
lectins. Such a strategy would facilitate access to three-
dimensional geometries of carbohydrate epitopes dened by
the metal coordination geometry, and allow for immediate increase
of multivalency of a ligand system, upon coordinating multiple
ligands to the same metal centre, providing complexes with poten-
tial to inhibit biolm formation by the bacteria. Cationic charge has
also been shown to be a characteristic of many antimicrobial den-
drimers, which can disrupt bacterial membranes; this presents
another possible added advantage to introducing metal ions into
glycocluster structures for targeting bacteria.27–29
Ligands containing the glycosyl-triazolyl-pyridyl motif have
come to light in recent years, mostly applied as ligands for
catalysis,30–32 and enzyme inhibitors.33,34 The only metal
complexes reported with such promising ligands are Pd(II)-
containing catalysts for C–C coupling reactions35,36 and Ru(II)
complexes.37 We have an interest in triazole ligands, such as
those based on the 2,6-bis(1,2,3-triazol-4-yl)pyridine (btp)
motif.38 Having exploited such ligands to coordinate transition
metal ions including Ru(II), Ni(II), Ir(III) and Pt(II)39 as well as
forming luminescent self-assemblies and MOFs with lantha-
nide ions,40–44 and mechanically interlocked molecules,45 we set
out to investigate the use of btp systems as potential antimi-
crobial agents. In previous studies, we and others have crystal-
lographically demonstrated that Ru(II) forms 1 : 2 complexes
with btp ligands, which would allow for the incorporation of
a minimum of four glycans into a single complex.38,39,46–48 In this
article, we describe a series of Ru(II)-centred tetravalent glyco-
clusters, synthesised from carbohydrate-derived btp ligands,
and assess their suitability as inhibitors for P. aeruginosa bio-
lm formation.
Scheme 1 Synthesis of Ru(II)-centred glycoclusters 7 and 8Gal.
calix[4]arene-based inhibitors for LecA, and as such were also
included in the family of new compounds designed here.19,23
These carbohydrate azides were then used as substrates for the
CuAAC reaction with 2,6-bis(TMS-ethynyl)pyridine, yielding
protected btp compounds 3 in good yields (63–82%) and 4Gal in
79% yield. The 1H NMR spectra of triazole compounds each
displayed a single set of resonances, corresponding to a single
anomer, for instance with doublets observed at 6.40 ppm (J ¼
9.2 Hz) for b-anomer 3Gal,32 and 4.48 ppm (J ¼ 8.0 Hz) for b-
anomer 4Gal. HRMS analysis further supported the formation
of the proposed structures (see Experimental and ESI† for full
characterisation).
In addition to the above characterisation of the acetyl pro-
tected ligands, single crystals of 3Gal were grown by slow
diffusion of (CH3)2CHOH into a DMSO-d6 solution as colourless
hexagonal plates, which were found to be suitable for X-ray
diffraction analysis. The ligand crystallised in the trigonal
space group P3221. The asymmetric unit contains two distinct
residues, each being half a molecule of 3Gal, the other half of
each molecule being generated by symmetry. These two
complete molecules are shown in Fig. 1(a). The btp motifs of the
two crystallographically distinct molecules were also shown to
intertwine by non-classical hydrogen bonding interactions
between the triazolyl C–H and the pyridyl nitrogen atoms of the
other btp motif, with hydrogen bond lengths of C–H/N ¼
Results and discussion
Synthesis of ligands and Ru(II) complexes
˚
3.511(10) and 3.482(9) A, Fig. 1(b). This dimeric interaction
matched our previous observations, which have been exploited
to form interlocked supramolecular architectures.40,45
Crystallographic packing analysis also showed weak supra-
molecular interactions between the carbohydrate moieties of
the adjacent molecules, causing them to stack in the manner
The synthesis of acetyl-protected btp compounds, hydrolysed
ligands, and target Ru(II) complexes are shown in Scheme 1. The
CuAAC reaction is typically used to prepare btp ligands, and as
such azide functionality was introduced to monosaccharides.
Stereoselective azidation of peracetylated glycosides via treat-
ment with TMS-N3 under Lewis acid catalysed conditions gave
access to azide precursors 1,49 while 2Gal was synthesised from
a chloro-precursor, as described in the literature.50 Triethylene
glycol spaced galactosides have previously been included in
˚
seen in Fig. 1(c); all C–H/O distances were of the order of 3.5 A
and are shown in a table in ESI.† The triazole rings of btp were
found to be slightly twisted out of the plane of the pyridyl rings
by ca. 13ꢀ in this elegant structure. The unit cell of this crystal
© 2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 16318–16325 | 16319

View Article Online
RSC Advances
Paper
Fig. 2 1H NMR spectra (D2O) of btp ligand 5Man (top) and the
resulting Ru(II) complex 7Man (bottom).
Fig. 1 Projections of the X-ray crystal structure of 3Gal,‡1 showing
various features of the structure (hydrogen atoms omitted for clarity,
CCDC deposition number 2004944†): (a) capped stick model showing
the two distinct molecules in the structure packing due to weak
supramolecular interactions between the adjacent sugar moieties; (b)
thermal ellipsoid model showing the non-classical hydrogen bonding
interactions between the two btp motifs; the carbohydrate arms are
omitted for clarity (represented as orange spheres); (c) a space-filling
model, showing the packing of the molecules with a C32 screw-axis.
aeruginosa,52–55 with these usually being bimetallic complexes.
However in general Ru(II)–polypyridyl complexes have shown lower
activity towards Gram-negative species (particularly P. aeruginosa)
when compared to Gram-positive bacteria (such as MRSA).55–57
In order to determine the potential of Ru(II) glycoclusters 7
and 8Gal to inhibit bacterial biolm formation, samples of P.
aeruginosa (PAO1) were incubated with 5 mM of Ru(II)
complexes for 24 hours and the biolm biomass determined by
staining with crystal violet. Inhibition was characterised by
decrease in absorbance at 590 nm, compared to control experi-
ments in the absence of any glycocluster (see Fig. 3). None of the
tested complexes were either bacteriostatic nor bactericidal to
PAO1 at these concentrations (see ESI† for MIC and MBC data).
When complexes 7, where the carbohydrates are directly
bonded to the triazole moieties, were incubated with P.
structure contained 6 molecules (Z ¼ 6); the two molecules
shown in Fig. 1(a) repeat in a le-handed 32 screw axis, Fig. 1(c).
´
O-Deacetylation under Zemplen conditions gave access to
the fully deprotected carbohydrate derivatives 5 and 6Gal in
good yields (63–78%), which were soluble in aqueous media. 1H
NMR spectra conrmed the removal of acetyl groups (note the
absence of proton resonances at ca. 2 ppm in Fig. 2) and HRMS
analysis was also consistent with the formation of btp structures
decorated with unprotected saccharide moieties.
The corresponding Ru(II) complexes 7 and 8Gal were prepared
upon heating 2 equivalents of the ligand (5 or 6Gal) with RuCl3-
$3H2O in aqueous ethanol solution under microwave irradiation at
120 ꢀC for 40 minutes. Complexes 7 and 8Gal, of the general
structure [Ru$(L)2]Cl2, were formed as a single species. Changes
1
observed in the H NMR spectrum (400 MHz, D2O) of 7Man, for
instance (Fig. 2), including a shi in the resonance arising from the
triazolyl CH from 8.35 to 8.91 ppm, and the coalescence of the
proton resonances from the pyridyl ring into a multiplet centred at
8.2 ppm, are indicative of coordination of Ru(II) by the btp ligand.
HRMS analysis gave signals corresponding to the doubly charged
[Mꢁ2Cl]2+ ion in all cases. Single crystals suitable for X-ray diffrac-
tion were not obtained for these complexes, but based on known
structures of related [Ru(btp)2]X2 complexes, it is anticipated that the
two tridentate btp ligands coordinate Ru(II) in an approximately
octahedral geometry,38,39,47,51 and as such the carbohydrate epitopes
from each divalent ligand would be presented orthogonally.
Fig. 3 Percentage biofilm formation determined by crystal violet staining of
biofilms upon incubation of P. aeruginosa with 5 mM of a range of Ru(II)
glycoclusters. Decreased absorbance at 590 nm indicated inhibition of biofilm
Biolm inhibition
To the best of our knowledge, only a few ruthenium complexes
have reported signicant inhibition of biolm formation by P. formation. One way ANOVA on ranks performed, *p < 0.05 versus control.
16320 | RSC Adv., 2021, 11, 16318–16325
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
aeruginosa, none of the Ru(II) complexes had statistically
signicant impact on biolm formation, regardless of the
identity of the carbohydrate. This would suggest that the
saccharide motifs were not able to interact with the target lectin
in this conguration, either due to lack of exibility, or due to
the distances between carbohydrate units not matching the
distance between lectin carbohydrate-binding sites. When
designing LecA inhibitors, Pieters and co-workers describe
Conclusions
We have successfully synthesised
a
range of protected
carbohydrate-derived tridentate btp ligands and characterised
them, including by single crystal X-ray diffraction in the case of
3Gal. These ligands were conveniently deprotected under
´
Zemplen conditions and formed Ru(II) complexes of the form
[Ru$(L)2]Cl2, which were tetravalent glycoclusters.
˚
a separation of 26 A between the carbohydrates in neighbouring
Complex 8Gal, containing exible triethylene glycol spacers
between the btp and galactose moieties, showed signicant
inhibition of P. aeruginosa biolm formation at 5 mM concen-
tration, while neither complexes 7 nor ligand 6Gal had an
impact on biolm formation under the same conditions. None
of the complexes were bacteriostatic or bactericidal. This
behaviour is rationalised in terms of the known affinity of
soluble lectin LecA for galactose and the advantages of multi-
valency reported in the literature for inhibitors of this protein
that are able to span adjacent carbohydrate-binding sites. As
such, it is clear that the topology of presentation of carbohy-
drate units, templated by coordination chemistry plays an
important role in antimicrobial activity of such metal-centred
glycoclusters.
These results indicate that coordination chemistry may be
used to conveniently direct the topology of multivalent glyco-
clusters, built up from simpler lower-valency building block
ligands. As the Ru(II) centre does not appear to play a signicant
role in any antimicrobial properties of 7 or 8Gal, it is conceivable
that other metal ions could easily be used, either for increased
economy, or to exploit complementary properties like magnetism
or luminescence (e.g. for use in applications such as imaging of
biolms). We will further develop on this work by investigating
the biolm inhibition properties, and impact on biolm-related
antibiotic resistance, of related systems with differing geome-
tries, metal centres, and spacers in future studies.
binding sites of the lectin,14,25,58 and while X-ray crystal struc-
tures of Ru(II) complexes 7 were not obtained, the analogous
galactose–galactose distance in ligand 3Gal, measured between
˚
the anomeric carbons, was signicantly shorter (ca. 8 A). Based
on analogy to previously-reported octahedral Ru(II) btp
complexes, the distance between anomeric carbons in
˚
complexes 7 would be expected to be 8–11 A. Hence, it is
possible that this signicant deviation from the ideal multiva-
lent binding geometry for LecA explains the lack of interaction,
even with a carbohydrate epitope known to interact with the
lectin.
Complex 8Gal, on the other hand, where triethylene glycol
chains confer the structure with further exibility,19,25,59
demonstrated signicant inhibition of biolm formation, with ca.
80% decrease versus the control (p < 0.05). Although highly-
multivalent glyconanoparticles have been able to achieve these
results at much lower concentrations (mM),22 the activity of 8Gal
compares favourably with other similar-valency calix[4]arene and
triazine-based glycoclusters, which also showed statistically signif-
icant biolm reduction at 5 mM concentration.13,19 The ligand 6Gal
alone does not lead to biolm inhibition under analogous condi-
tions (see ESI, Fig. S28†), suggesting that the multivalent glyco-
cluster structure is required for this effect to be observed.
The striking difference in behaviour between complexes 7
and 8Gal, all of which are cationic Ru(II)-containing
compounds, suggests that antimicrobial properties of this
complex do not arise simply from the presence of the cationic
metal ion. Nor is the ligand structure alone responsible for the
effect on P. aeruginosa biolm formation. Rather, the combination
Experimental
Materials and methods
of the exible ligand and the mode of presentation of the carbo- All chemicals and reagents were purchased from commercial
hydrate units in the tetravalent cluster structure is proposed to be sources and used without further purication. Electrospray
the origin of the observed effect. This result conrms the potential mass spectra (ESI) were acquired using a Micromass time of
of using metal ions as scaffolds for the construction of antimi- ight mass spectrometer (TOF), interfaced to a Waters 2690
crobial multivalent glycoclusters from ligands of lower valency, HPLC or a Waters LCT Premiere XE (with leucine enkephalin
giving rise to biolm inhibition activity which is not primarily used as an internal lock mass). MALDI Q-Tof mass spectra were
caused by the metal ion. This will form a basis for future devel- carried out on a MALDI Q-Tof Premier (Waters Corporation,
opments, which could build on the variety of geometric structures Micromass MS technologies, Manchester, UK) and high-
available through coordination chemistry of alternative d- and f- resolution mass spectrometry was performed using Glu-Fib as
metal ions to explore the relationship between complex topology an internal reference. NMR spectra were recorded on a 400 MHz
and biolm inhibition in detail.
Bruker Avance III spectrometer or a 500 MHz Agilent spec-
trometer. Chemical shis expressed in parts per million (ppm/
d) are reported relative to internal tetramethylsilane in CDCl3 or
CD3OD or relative to HOD in D2O. Coupling constants (J) are
expressed in Hz. Infrared spectra were recored on a PerkinElmer
Spectrum 100 or 400 FT-IR spectrometer with universal ATR
sampling accessory. All microwave reactions were carried out
Biotage Microwave Vials in a Biotage Initiator Eight EXP
microwave reactor.
˚
‡ Selected crystallographic and renement data for crystal of 3Gal: a, b, c (A):
3
ꢀ
˚
13.033(15), 13.033(15), 44.95(5); a, b, g ( ): 90, 90, 120; V (A ): 6613(17); Z: 6;
F(000): 2748; Dc (Mg mꢁ3): 1.316; m (mmꢁ1): 0.91; GOF, R1, wR2, ack: 1.075,
0.062, 0.151, 0.03(9).
© 2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 16318–16325 | 16321

View Article Online
RSC Advances
Paper
X-ray diffraction data for 3Gal was measured on a Bruker calculated for C37H43N7O18Na+ m/z ¼ 896.2556 [M + Na]+. Found
1
Apex2 Duo using a high intensity Cu-Ka radiation source (l ¼ m/z ¼ 896.2532; H NMR (CDCl3, 500 MHz): d ¼ 2.05–2.11 (m,
˚
1.54178 A). The datasets were collected and processed using 18H, 3 OAc CH3), 2.21 (s, 6H, OAc CH3), 3.93–4.00 (m, 2H, Man
Bruker APEX3 suite of programs. All structures were solved by CH), 3.99–4.15 (m, 2H, Man CHH), 4.39 (dd, 2H, J ¼ 12.6, 5.1 Hz,
direct methods (SHELXS-2018/3) and rened against all F2 data Man CHH), 5.42 (t, 2H, J ¼ 8.9 Hz, Man CH), 5.98 (dd, 2H, J ¼
(SHELXL-2018/3).60 All H-atoms, were positioned geometrically 8.9, 3.5 Hz, Man CH), 6.02 (t, 2H, J ¼ 3.5 Hz, Man CH), 6.10 (d,
and rened using a riding model.
2H, J ¼ 2.5 Hz, anomeric Man CH), 7.93 (t, 1H, J ¼ 7.8 Hz, pyr
CH), 8.16 (d, 2H, J ¼ 7.8 Hz, pyr CH), 8.37 (s, 2H, triazolyl CH);
13C NMR (CDCl3, 101 MHz): d ¼ 20.6, 20.65, 20.66, 20.71 (4 OAc
CH3), 61.5 (Man CH2), 66.0, 68.3, 68.7, 72.3 (4 Man CH), 83.8
(Man anomeric CH), 120.0 (pyr CH), 122.4 (triazolyl CH), 138.0
(pyr CH), 148.7 (qt), 149.4 (qt), 169.3, 169.6, 169.7, 170.4; FT-IR
(ATR, cmꢁ1): 2981, 2258, 1744 (s), 1611, 1576, 1430, 1369, 1214
(s), 1130, 1051, 1028, 907, 808, 727.
Biolm formation assay
Biolm formation assays were used based on the methodology
from previous published work.61 In brief, starting from an
overnight liquid culture, a dilution containing approximately
108 CFUs mLꢁ1 was made of Pseudomonas aeruginosa, PAO1
strain (a kind donation from Prof. Seamas Donnelly, School of
Medicine, Trinity College Dublin). For each biolm experiment,
8 wells of a round-bottomed polypropylene 96-well micro plate
(Corning® Costar® purchased from Sigma Aldrich, Dublin
Cat#CLS3596) were inoculated with 100 ml of this dilution, 8
wells were inoculated with the dilution and treated with 5 mM
of the Ru(II) glycoclusters and 8 control wells were lled with
sterile medium (bacteria alone). Following 4 hours of adhesion,
the supernatant, containing non-adhered cells, was removed
from each well and plates rinsed using phosphate buffered
saline (PBS) solution. Following this 100 ml of fresh media was
added to the control wells and fresh media with 5 mM of the
Ru(II) glycoclusters was added to the appropriate wells, the plate
was then incubated for a further 24 hours. Aer 24 hours bio-
lm formation, the supernatants were again removed, and the
wells rinsed with PBS again. Once the wells were washed, 100 ml
of a 0.5% crystal violet (CV) solution was added to all wells. Aer
20 min, the excess CV was removed by washing the plates under
running tap water. Finally, bound CV was released by adding
150 ml of 33% acetic acid (Sigma). The absorbance was
measured at 590 nm. All steps were carried out at room
temperature.
3Lac. Yield: 0.210 g, 0.145, 63%. Product decomposed above
200 C. HRMS (m/z) (MALDI+): calculated for C61H75N7O34Na+
ꢀ
m/z ¼ 1472.4253. Found m/z ¼ 1472.4188; 1H NMR (CDCl3, 400
MHz): d ¼ 1.85 (s, 6H, OAc CH3), 1.94 (s, 6H, OAc CH3), 1.98–
2.09 (m, 24H, OAc CH3), 2.13 (s, 6H, OAc CH3), 4.01–4.25 (m, 6H,
3 Lac CH), 3.85–4.02 (m, 6H, Lac CH2 and Lac CHH), 4.46 (app d,
2H, Lac CHH), 4.54 (d, 2H, J ¼ 7.8 Hz, anomeric Gal CH), 4.97
(dd, 2H, J ¼ 10.4, 3.3 Hz, Lac CH), 5.15–5.04 (m, 2H, Lac CH),
5.34 (d, 2H, J ¼ 2.8 Hz, Lac CH), 5.42 (dd, 2H, J ¼ 17.1, 8.7 Hz,
Lac CH), 5.52 (t, 2H, J ¼ 9.4 Hz, Lac CH), 5.89 (d, 1H, J ¼ 9.3 Hz,
anomeric Glc CH), 7.83 (t, 1H, J ¼ 7.8 Hz, pyr CH), 8.04 (d, 2H, J
¼ 7.8 Hz, pyr CH), 8.32 (s, 2H, triazolyl CH); 13C NMR (CDCl3,
101 MHz): d ¼ 20.2, 20.5, 20.58, 20.58, 20.6, 20.70, 20.73 (7 Ac
CH3), 60.7 (Lac CH2), 61.9 (Lac CH2), 66.5, 69.0, 70.6, 70.8, 70.9,
72.8, 75.7, 75.9 (8 Lac CH), 85.6 (anomeric Glc CH), 101.1
(anomeric Gal CH), 119.8 (pyr CH), 120.8 (triazolyl CH), 137.7
(pyr CH), 148.7 (qt), 149.4 (qt), 169.0, 169.2, 169.5, 169.98,
170.03, 170.2, 170.3 (7 Ac C]O qt); FT-IR (ATR, cmꢁ1): 2943,
1743 (s), 1612, 1576, 1435, 1368, 1212 (s), 1045, 915, 810, 732.
4Gal. Synthesised according to the general procedure from
azide 2Gal (0.152 g, 0.30 mmol), yielding the desired product as
a brown oil. Concentration in vacuo yielded a very hygroscopic
off-white solid. Yield: 0.135 g, 0.12 mmol, 79%. HRMS (m/z)
General synthesis of protected ligands 3 and 4Gal
(ESI+): calculated for C49H67N7O24Na+ m/z ¼ 1160.4130 [M +
1
The relevant peracetylated carbohydrate was treated with TMS- Na]+. Found m/z ¼ 1160.4137; H NMR (CDCl3, 500 MHz): d ¼
N3 and SnCl4 according to an established protocol to introduce 1.98 (s, 6H, Ac CH3), 2.02 (s, 6H, Ac CH3), 2.04 (s, 6H, Ac CH3),
azide functionality into the anomeric position.49 This carbohy- 2.14 (s, 6H, Ac CH3),3.56–3.72 (m, 14H, 3 ꢂ CH2 and Gal C6HH),
drate azide (2 equiv.), CuI (0.5 equiv.), DIPEA (3 equiv.) and 2,6- 3.85–3.95 (m, 4H, Gal C6HH and Gal CH), 3.98 (t, 4H, J ¼ 5.3 Hz,
bis(trimethylsilylethynyl)pyridine (1 equiv.) were suspꢀended in CH2), 4.07–4.20 (m, 4H, CH2), 4.48 (d, 2H, J ¼ 8.0 Hz, Gal
DMF and heated under microwave irradiation at 100 C for 20 anomeric CH), 4.40–4.69 (m, 4H, CH2), 5.00 (dd, 2H, J ¼ 3.4,
minutes. The reaction mixture was poured into an aqueous 10.5 Hz, Gal C3H), 5.18 (dd, 2H, J ¼ 8.0, 10.5 Hz, Gal C2H), 5.38
solution of EDTA/NH4OH, extracted three times into CH2Cl2, (d, 2H, J ¼ 2.6 Hz, Gal CH), 7.85–7.91 (m, 1H, 4-pyr CH), 8.09 (d,
washed with water and brine, dried over MgSO4, ltered and 2H, J ¼ 7.8 Hz, 3- and 5-pyr CH), 8.31 (s, 2H, triazolyl CH); 13C
concentrated under reduced pressure; the product was puried NMR (CDCl3, 126 MHz): d ¼ 20.57, 20.65, 20.67, 20.7 (4 ꢂ OAc
by ash chromatography (CH2Cl2–CH3OH gradient) or tritura- CH3), 50.5 (CH2), 61.2 (CH2), 67.0 (Gal CH), 68.8 (Gal CH), 69.1
tion with cold CH3OH, yielding 3 or 4Gal as an off-white solid, (Gal C6H2), 69.5 (CH2), 70.2 (CH2), 70.4 (CH2), 70.61 (CH2), 70.62
which was used without further purication (3Gal and 3Glc (Gal CH), 70.8 (Gal CH), 101.3 (Gal anomeric CH), 119.3 (3- and
were previously reported32).
5-pyr CH), 123.3 (triazolyl CH), 137.7 (4-pyr CH), 148.2 (qt),
3Man. Synthesised according to the general procedure with 150.1 (qt), 169.4, 170.1, 170.2, 170.4 (4 OAc qt C]O); FT-IR
azide 1Man (0.330 g, 0.88 mmol), CuI (0.042 g, 0.22 mmol), (ATR, cmꢁ1): 2884, 1742 (s), 1608, 1575, 1435, 136, 1216 (s),
DIPEA (0.24 mL, 1.32 mmol) and 2,6-bis(trimethylsilylethynyl) 1174, 1133, 1042 (s), 915, 813, 730.
pyridine (0.120 g, 0.44 mmol). Yield: 0.270 g, 0.31 mmol, 68%.
Product decomposed above 200 ꢀC. HRMS (m/z) (ESI+):
16322 | RSC Adv., 2021, 11, 16318–16325
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
7.6 Hz, anomeric Gal CH), 5.67 (d, 2H, J ¼ 9.4 Hz, anomeric Glc
CH), 7.42 (d, 2H, J ¼ 7.3 Hz, pyr CH), 7.56–7.63 (m, 1H, pyr CH),
8.38 (s, 2H, triazolyl CH); 13C NMR (D2O, 101 MHz): d ¼ 59.9
(Lac CH2), 61.0 (Lac CH2), 68.5, 70.9, 72.1, 72.5, 74.5, 75.4, 77.4,
77.7 (8 Lac CH), 87.4 (anomeric Glc CH), 102.9 (anomeric Gal
CH), 120.1 (pyr CH), 123.3 (triazolyl CH), 138.8 (pyr CH), 146.8
(qt), 147.9 (qt); FT-IR (ATR, cmꢁ1): 3326 (br), 2885, 1610, 1578,
1375, 1231, 1032 (s), 894, 806.
6Gal. Yield: 0.023 g, 0.023 mmol, 66%. HRMS (m/z) (ESI+):
calculated for C33H51N7O16Na+ m/z ¼ 824.3290 [M + Na]+. Found
m/z ¼ 824.3248; 1H NMR (CD3OD, 500 MHz): d ¼ 3.41–3.48 (m,
4H, 2 ꢂ Gal CH), 3.49–3.54 (m, 2H, Gal CH), 3.59–3.69 (m, 14H,
3 ꢂ CH2 and Gal C6HH), 3.71 (t, 4H, J ¼ 5.6 Hz, CH2), 3.90–3.96
(m, 2H, Gal CH), 4.00 (t, 4H, J ¼ 5.0 Hz, CH2), 4.17 (d, 2H, J ¼
7.64 Hz, anomeric Gal CH), 4.68–4.73 (m, 4H, CH2), 7.95–8.01
(m, 3H, 4-pyr CH and triazolyl CH), 8.64–8.69 (m, 2H, 3- and 5-
pyr CH); 13C NMR (CD3OD, 126 Mz): d ¼ 50.3 (CH2), 61.1 (CH2),
68.2 (Gal C6H2), 68.8 (CH2), 68.9 (Gal CH), 70.01 (CH2), 70.05
(CH2), 71.1 (Gal CH), 73.5 (Gal CH), 75.2 (Gal CH), 103.6
(anomeric Gal CH), 118.7 (pyr CH), 124.2 (pyr CH), 137.9 (tri-
azolyl CH), 147.5 (qt), 149.9 (qt); FT-IR (ATR, cmꢁ1): 3367 (br),
2885, 2496, 1612, 1350, 1035 (s), 808, 776.
General synthesis of deprotected ligands 5 and 6
To a suspension of the relevant protected ligand 3 or 4 in
CH3OH (10 mL) was added a 1 M solution of NaOCH3 in CH3OH
(0.1 mL). The reaction mixture was stirred at room temperature.
Upon completion, the solution was neutralised by addition of
DOWEX® 50X8 H+ resin, ltered and concentrated under
reduced pressure, yielding the free sugar ligands as off-white
solids (5Glc and 5Gal), or amber oils (5Man, 5Lac and 6Gal).
5Gal. Synthesised according to the general procedure from
3Gal (0.090 g, 0.10 mmol) and 1 M NaOCH3 solution (0.1 mL).
ꢀ
Yield: 0.035 g, 0.065 mmol, 63%. m.p. 238–246 C. Anal. calcd
for C21H27N7O10$2.5(H2O) (582.524 g molꢁ1), C 43.29, H 5.53, N
16.83%; found C 42.90, H 5.04, 16.38%. HRMS (m/z) (ESIꢁ):
ꢁ
calculated for C21H26N7O10 m/z ¼ 536.1741 [MꢁH]ꢁ. Found m/z
¼ 536.1740; calculated for C21H27N7O10Clꢁ m/z ¼ 572.1507 [M +
Cl]ꢁ. Found m/z ¼ 572.1526; 1H NMR (D2O, 500 MHz): d ¼ 3.63–
3.79 (m, 4H, Gal CH2), 3.82 (dd, 2H, J ¼ 9.8, 3.3 Hz, Gal CH), 3.92–
3.97 (m, 2H, Gal CH), 4.02 (d, 2H, J ¼ 3.3 Hz, Gal CH), 4.20 (t, 2H, J
¼ 9.5 Hz, Gal C2H), 5.67 (d, 2H, J ¼ 9.2 Hz, anomeric CH), 7.69 (d,
2H, J ¼ 7.8 Hz, pyr CH), 7.82 (t, 1H, J ¼ 7.8 Hz, pyr CH), 8.60 (s, 2H,
triazolyl CH); 13C NMR (D2O, 126 MHz): d ¼ 60.9 (Gal CH2), 68.6,
69.8, 72.9, 78.4 (4 Gal CH), 88.2 (anomeric Gal CH), 120.5 (pyr CH),
123.2 (triazolyl CH), 139.2 (pyr CH), 147.1 (qt), 148.3 (qt); FT-IR
(ATR, cmꢁ1): 3288, 2895, 2115, 1647, 1577, 1389, 1318, 1230,
1086, 1049, 877, 809, 672, 610, 572, 561.
Preparation of Ru(II) complexes of btp ligands
Carbohydrate ligands, 5 or 6 (2 equiv.) were dissolved in 7 mL
70% aqueous ethanol solution and RuCl3$3H2O (1 equiv.)
added. The reaction mixture was heated under microwave
irradiation to 120 ꢀC for 40 minutes and concentrated under
reduced pressure to yield the product as very hygroscopic
orange or red solids 7 or 8.
5Glc. Yield: 0.050 g, 0.09 mmol, 78%. m.p. 242–248 ꢀC. Anal.
calcd for C21H27N7O10$2.5(H2O) (582.524 g molꢁ1), C 43.29, H
5.53, N 16.83%; found C 43.56, H 5.05, 16.63%. HRMS (m/z)
(ESI+): calculated for C21H28N7O10 m/z ¼ 538.1898 [M + H]+.
+
Found m/z ¼ 538.1902; calculated for C21H27N7O10Na+ m/z ¼
1
560.1717 [M + Na]+. Found m/z ¼ 560.1710; H NMR (CD3OD,
7Gal. Synthesised according to the general procedures from
5Gal (0.020 g, 0.037 mmol) and RuCl3$3H2O (0.005, 0.021
mmol). Residue was dissolved in CH3OH and ltered, concen-
trating the ltrate to give the product as a hygroscopic red solid.
Yield: 0.018 g, 0.014 mmol, 75%. Product decomposed above
180 ꢀC. Anal. calcd for C42H54N14O20$10(H2O)$2NaCl
(1544.821 g molꢁ1), C 32.66, H 4.82, N 12.69%; found C 32.81, H
4.33, 12.57%. HRMS (m/z) (ESI+): calculated for
500 MHz): d ¼ 3.49–3.67 (m, 6H, CH2 and Glc CH), 3.75 (dd, 2H,
J ¼ 12.3, 5.5 Hz, Glc CH), 3.84–4.00 (m, 4H, Glc CH), 5.70 (d, 2H,
anomeric Glc CH, J ¼ 9.1 Hz), 7.98 (app br s, 3H, pyr CH), 8.86
(s, 2H, triazolyl CH); 13C NMR (D2O, 126 MHz): d ¼ 60.5 (Glc
CH2), 68.9, 72.4, 75.8, 78.9 (4 Glc CH), 87.6 (anomeric Glc CH),
120.3 (pyr CH), 123.3 (triazolyl CH), 139.0 (pyr CH), 146.9 (qt),
148.2 (qt); FT-IR (ATR, cmꢁ1): 3266, 2883, 1611, 1577, 1437,
1313, 1227, 1095, 1073, 1036, 899, 807, 675, 600, 577, 562.
5Man. Yield: 0.012 g, 0.022 mmol, 69%. HRMS (m/z) (ESI+):
calculated for C21H27N7O10Na+ m/z ¼ 560.1717 [M + Na]+. Found
m/z ¼ 560.1689; 1H NMR (D2O, 500 MHz): d ¼ 3.31–3.45 (m, 2H,
Man CH), 3.61–3.80 (m, 6H, Man CH and Man CH2), 4.10 (dd, 2H, J
¼ 3.5, 9.0 Hz, Man CH), 4.77 (dd, 2H, J ¼ 3.5, 2.5 Hz, Man CH), 6.12
(d, 2H, J ¼ 2.6 Hz, anomeric Man CH), 7.87 (d, 2H, J ¼ 7.8 Hz, pyr
C
42H54N14O20Ru2+ m/z ¼ 588.1341 [Mꢁ2Cl]2+. Found m/z ¼
588.1350; 1H NMR (D2O, 500 MHz): d ¼ 3.48–3.58 (m, 4H, 2 Gal
CH), 3.63 (dd, 2H, J ¼ 10.0, 3.0 Hz, Gal CHH), 3.68–3.77 (m, 2H,
Gal CHH), 3.78–3.93 (m, 4H, 2 Gal CH), 5.36 (d, 2H, J ¼ 9.2 Hz,
anomeric Gal CH), 8.20 (app s, 3H, pyr CH), 8.97 (s, 2H, triazolyl
CH); 13C NMR (D2O, 126 MHz): d ¼ 60.7 (Gal CH2), 68.3, 69.3,
72.5, 78.4 (4 Gal CH), 88.9 (anomeric Gal CH), 120.8 (pyr CH),
124.6 (triazolyl CH), 137.9 (pyr CH), 150.1 (qt), 150.4 (qt); FT-IR
(ATR, cmꢁ1): 3288 (s, br), 1622, 1389, 1209, 1089 (s), 887, 805.
7Glc. Yield: 0.024 g, 0.013 mmol, 66%. Product decomposed
above 180 ꢀC. HRMS (m/z) (ESI+): calculated for
C42H54N14O20Ru2+ m/z ¼ 588.1341 [Mꢁ2Cl]2+. Found m/z ¼
588.1335; 1H NMR (D2O, 500 MHz): d ¼ 3.35 (t, 2H, J ¼ 9.3 Hz, Glc
CH), 3.40–3.57 (m, 8H, 2 Glc CH and CH2), 3.58–3.70 (m, 4H, 2 Glc
CH), 5.43 (d, 2H, J ¼ 9.3 Hz, anomeric Glc CH), 8.20 (app s, 3H, pyr
CH), 8.95 (s, 2H, triazolyl CH); 13C NMR (D2O, 126 MHz): d ¼ 62.7
(Glc CH2), 71.1, 74.4, 78.0, 81.4 (4 Glc CH), 90.8 (anomeric Glc CH),
123.4 (pyr CH), 127.3 (triazolyl CH), 151.2 (pyr CH), 152.7 (qt), 153.0
CH), 7.96 (t, 1H, J ¼ 7.8 Hz, pyr CH), 8.64 (s, 2H, triazolyl CH); 13
C
NMR (D2O, 126 MHz): d ¼ 60.4 (Man CH2), 66.5, 68.3, 70.5, 76.2 (4
Man CH), 86.9 (anomeric Man CH), 120.7 (pyr CH), 123.8 (triazolyl
CH), 139.3 (pyr CH), 147.2 (qt), 148.6 (qt); FT-IR (ATR, cmꢁ1): 3306
(br), 2457, 1577, 1438, 1215, 1033 (s), 794.
5Lac. Yield: 0.085 g, 0.10 mmol, 77%. m.p. 207–212 ꢀC.
HRMS (m/z) (ESI+): calculated for C33H47N7O20Na+ m/z ¼
1
884.2774 [M + Na]+. Found m/z ¼ 884.2783; H NMR (D2O, 400
MHz): d ¼ 3.44–3.52 (m, 2H, Lac CH), 3.57 (dd, 2H, J ¼ 10.0,
3.2 Hz, Lac CH), 3.62–3.86 (m, 16H, 5 Lac CH, Lac CH2, Lac
CHH), 3.93 (m, 4H, Lac CH and Lac CHH), 4.40 (d, 2H, J ¼
© 2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 16318–16325 | 16323

View Article Online
RSC Advances
Paper
(qt); FT-IR (ATR, cmꢁ1): 3286 (s, br), 2927, 1624, 1591, 1447, 1389
(s), 1335, 1263, 1208, 1095, 1051, 1015 (s), 895, 806, 611.
Notes and references
7Man. Yield: 0.010 g, 0.008 mmol, 80%. HRMS (m/z) (ESI+):
calculated for C42H54N14O20Ru2+ m/z ¼ 588.1341 [Mꢁ2Cl]2+.
Found m/z ¼ 588.1341; 1H NMR (D2O, 400 MHz); d ¼ 2.80–3.09
(m, 2H, Man CH), 3.38 (dd, 2H, J ¼ 8.3, 3.1 Hz, Man CHH), 3.49–
3.66 (m, 6H, 2 Man CH and Man CHH), 4.27 (br s, 2H, Man CH),
5.84 (d, 2H, J ¼ 2.1 Hz, anomeric CH), 8.20 (m, 3H, pyr CH), 8.91
(s, 2H, triazolyl CH); 13C NMR (D2O, 101 MHz): d ¼ 60.1 (Man
CH2), 66.1, 67.5, 70.2, 77.1 (4 Man CH), 87.6 (anomeric CH),
120.6, 120.8, 125.0, 150.0 (qt), 150.5 (qt); FT-IR (ATR, cmꢁ1):
3264 (s, br), 1618, 1447, 1068, 1051, 799.
1 L. B. Rice, J. Infect. Dis., 2008, 197, 1079–1081.
2 M. N. Hurley, Drugs Future, 2010, 35, 1007–1014.
´
3 M. N. Hurley, M. Camara and A. R. Smyth, Eur. Respir. J.,
2012, 40, 1014–1023.
4 A. Tynan, L. Mawhinney, M. E. Armstrong, C. O'Reilly,
¨
S. Kennedy, E. Caraher, K. Julicher, D. O'Dwyer, L. Maher,
K. Schaffer, A. Fabre, E. F. McKone, L. Leng, R. Bucala,
J. Bernhagen, G. Cooke and S. C. Donnelly, FASEB J., 2017,
31, 5102–5110.
´
5 A. Imberty, M. Wimmerova, E. P. Mitchell and N. Gilboa-
7Lac. Yield: 0.011 g, 0.0058 mmol, 67%. HRMS (m/z) (ESI+):
calculated for C66H94N14O40Ru2+ m/z ¼ 912.2402 [Mꢁ2Cl]2+.
Garber, Microbes Infect., 2004, 6, 221–228.
6 N. Gilboa-Garber, Methods Enzymol., 1982, 83, 378–385.
1
Found m/z ¼ 912.2392; H NMR (D2O, 500 MHz): d ¼ 3.39 (dd,
´
7 S. P. Diggle, R. E. Stacey, C. Dodd, M. Camara, P. Williams
2H, J ¼ 9.9, 7.8 Hz, Lac CH), 3.51 (dd, 2H, J ¼ 10.2, 3.2 Hz, Lac
CH), 3.54–3.74 (m, 18H, 3 ꢂ Lac CH and 2 ꢂ Lac CH2), 3.75–
3.85 (m, 4H, 2 ꢂ Lac CH), 4.29 (d, 2H, J ¼ 7.8 Hz, anomeric Gal
CH), 5.47 (d, 2H, J ¼ 9.0 Hz, anomeric Glc CH), 8.17–8.21 (m,
3H, pyr CH), 8.96 (s, 2H, triazolyl CH); 13C NMR (D2O, 126 MHz)
d ¼ 59.8 (Lac CH2) 61.0 (Lac CH2), 68.4, 70.8, 71.5, 72.4, 74.1,
75.3, 76.9, 77.7 (8 Lac CH), 88.1 (anomeric Glc CH, assigned by
HSQC), 102.8 (anomeric Gal CH), 120.9 (pyr CH, assigned by
HSQC), 137.9 (pyr CH, assigned by HSQC), 150.1 (qt), 150.4 (qt);
FT-IR (ATR, cmꢁ1): 3269 (s, br), 1627, 1400, 1027, 894.
and K. Winzer, Environ. Microbiol., 2006, 8, 1095–1104.
8 T. Bjarnsholt, O. Ciofu, S. Molin, M. Givskov and N. Høiby,
Nat. Rev. Drug Discovery, 2013, 12, 791–808.
9 H. P. Hauber, M. Schulz, A. Pforte, D. Mack, P. Zabel and
U. Schumacher, Int. J. Med. Sci., 2008, 5, 371–376.
10 A. V. Grishin, M. S. Krivozubov, A. S. Karyagina and
A. L. Gintsburg, Acta Naturae, 2015, 7, 29–41.
11 R. Visini, X. Jin, M. Bergmann, G. Michaud, F. Pertici, O. Fu,
A. Pukin, T. R. Branson, D. M. E. Thies-Weesie, J. Kemmink,
E. Gillon, A. Imberty, A. Stocker, T. Darbre, R. J. Pieters and
J. L. Reymond, ACS Chem. Biol., 2015, 10, 2455–2462.
12 G. Yu, A. C. Vicini and R. J. Pieters, J. Org. Chem., 2019, 84,
2470–2488.
13 M. Smadhi, S. de Bentzmann, A. Imberty, M. Gingras,
R. Abderrahim and P. G. Goekjian, Beilstein J. Org. Chem.,
2014, 10, 1981–1990.
8Gal. Yield: 0.025 g, 0.014 mmol, 88%. Product decomposed
above 180 ꢀC. HRMS (m/z) (ESI+): calculated for
C
66H102N14O32Ru2+ m/z ¼ 852.2918 [Mꢁ2Cl]2+. Found m/z ¼
852.2890; 1H NMR (D2O, 600 MHz): d ¼ d ¼ 3.30–3.37 (m, 8H, 2
ꢂ CH2), 3.39 (dd, 2H, J ¼ 10.1, 7.8 Hz, Gal CH), 3.43–3.46 (m,
4H, CH2), 3.50–3.56 (m, 4H, 2 ꢂ Gal CH), 3.57–3.69 (m, 10H, 2 ꢂ
CH2, Gal CHH), 3.80 (app d, 2H, J ¼ 4.1 Hz, Gal CH), 3.83–3.93
(m, 2H, Gal CHH), 4.25 (d, 2H, J ¼ 7.8 Hz, anomeric Gal CH),
4.36 (br t, 4H, CH2), 8.17–8.21 (m, 3H, pyr CH), 8.80 (s, 2H,
triazolyl CH); 13C NMR (D2O, 101 MHz): d ¼ 51.6 (CH2), 61.0
(CH2), 68.1 (CH2), 68.5 (CH2), 68.6 (Gal CH), 69.4 (CH2), 69.7
(Gal CH), 70.7 (Gal CH), 72.7 (Gal CH), 75.2 (Gal CH), 102.8
(anomeric Gal CH), 120.3 (pyr CH), 126.0 (pyr CH), 137.9 (tri-
azolyl CH), 150.1 (qt), 150.5 (qt); FT-IR (ATR, cmꢁ1): 3345 (s, br),
2923, 1622, 1404, 1060 (s), 809.
14 F. Pertici, N. J. De Mol, J. Kemmink and R. J. Pieters, Chem.–
Eur. J., 2013, 19, 16923–16927.
15 E. Kolomiets, M. A. Swiderska, R. U. Kadam,
E. M. V. Johansson, K. E. Jaeger, T. Darbre and
J. L. Reymond, ChemMedChem, 2009, 4, 562–569.
16 C. Ligeour, O. Vidal, L. Dupin, F. Casoni, E. Gillon, A. Meyer,
S. Vidal, G. Vergoten, J. M. Lacroix, E. Souteyrand,
A. Imberty, J. J. Vasseur, Y. Chevolot and F. Morvan, Org.
Biomol. Chem., 2015, 13, 8433–8444.
´
17 M. Reynolds, M. Marradi, A. Imberty, S. Penades and
´
S. Perez, Chem.–Eur. J., 2012, 18, 4264–4273.
Conflicts of interest
´
18 A. Bernardi, J. Jimenez-Barbero, A. Casnati, C. De Castro,
T. Darbre, F. Fieschi, J. Finne, H. Funken, K.-E. Jaeger,
M. Lahmann, T. K. Lindhorst, M. Marradi, P. Messner,
A. Molinaro, P. V. Murphy, C. Nativi, S. Oscarson,
There are no conicts to declare.
´
S. Penades, F. Peri, R. J. Pieters, O. Renaudet,
Acknowledgements
¨
J.-L. Reymond, B. Richichi, J. Rojo, F. Sansone, C. Schaffer,
The authors acknowledge nancial support from Science
Foundation Ireland: JPB (Starting Investigator Research Grant
18/SIRG/5501), and TG (PI Award, 13/IA/1895). Preliminary work
was carried out as part of a UCD School of Medicine Student
Summer Research Award (GC, BP, COR, JPB). We thank Dr
W. B. Turnbull, T. Velasco-Torrijos, S. Vidal, S. Vincent,
T. Wennekes, H. Zuilhof and A. Imberty, Chem. Soc. Rev.,
2013, 42, 4709–4727.
´
19 A. M. Boukerb, A. Rousset, N. Galanos, J.-B. Mear,
´
M. Thepaut, T. Grandjean, E. Gillon, S. Cecioni,
´
Samuel Bradberry, Dr Sandra Bright, Karolina Wojtczak, Sean
C. Abderrahmen, K. Faure, D. Redelberger, E. Kipnis,
R. Dessein, S. Havet, B. Darblade, S. E. Matthews, S. de
Hennessey and June Lovitt for assistance. JPB thanks Prof.
Paul V. Murphy for support and mentoring, and Prof. Eoin
Scanlan for helpful advice and discussion.
16324 | RSC Adv., 2021, 11, 16318–16325
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
´
´
Bentzmann, B. Guery, B. Cournoyer, A. Imberty and S. Vidal, 40 J. P. Byrne, M. Martınez-Calvo, R. D. Peacock and
J. Med. Chem., 2014, 57, 10275–10289. T. Gunnlaugsson, Chem.–Eur. J., 2016, 22, 486–490.
¨
´
20 S. Wang, L. Dupin, M. Noel, C. J. Carroux, L. Renaud, 41 E. P. McCarney, J. P. Byrne, B. Twamley, M. Martınez-Calvo,
´
¨
T. Gehin, A. Meyer, E. Souteyrand, J. J. Vasseur,
G. Ryan, M. E. Mobius and T. Gunnlaugsson, Chem.
G. Vergoten, Y. Chevolot, F. Morvan and S. Vidal, Chem.–
Eur. J., 2016, 22, 11785–11794.
Commun., 2015, 51, 14123–14126.
42 S. J. Bradberry, J. P. Byrne, C. P. McCoy and
T. Gunnlaugsson, Chem. Commun., 2015, 51, 16565–16568.
˜
21 K. V. Marino, M. L. Uhrig, J. M. Benito, A. Imberty,
O. Renaudet, D. Goyard, B. Thomas and E. Gillon, Front. 43 J. P. Byrne, J. A. Kitchen, J. E. O'Brien, R. D. Peacock and
Chem., 2019, 7, 666. T. Gunnlaugsson, Inorg. Chem., 2015, 54, 1426–1439.
22 T. R. Flockton, L. Schnorbus, A. Araujo, J. Adams, 44 E. P. McCarney, C. S. Hawes, J. A. Kitchen, K. Byrne,
M. Hammel and L. J. Perez, Pathogens, 2019, 8, 55.
23 S. Cecioni, R. Lalor, B. Blanchard, J. P. Praly, A. Imberty,
W. Schmitt and T. Gunnlaugsson, Inorg. Chem., 2018, 57,
3920–3930.
S. E. Matthews and S. Vidal, Chem.–Eur. J., 2009, 15, 45 J. P. Byrne, S. Blasco, A. B. Aletti, G. Hessman and
13232–13240.
T. Gunnlaugsson, Angew. Chem., Int. Ed., 2016, 55, 8938–
24 D. Sicard, S. Cecioni, M. Iazykov, Y. Chevolot, S. E. Matthews,
8943.
J. P. Praly, E. Souteyrand, A. Imberty, S. Vidal and M. Phaner- 46 R. M. Meudtner, M. Ostermeier, R. Goddard, C. Limberg and
Goutorbe, Chem. Commun., 2011, 47, 9483–9485. S. Hecht, Chem.–Eur. J., 2007, 13, 9834–9840.
25 F. Pertici and R. J. Pieters, Chem. Commun., 2012, 48, 4008– 47 M. Ostermeier, M.-A. Berlin, R. M. Meudtner, S. Demeshko,
4010.
F. Meyer, C. Limberg and S. Hecht, Chem.–Eur. J., 2010, 16,
10202–10213.
26 C. Ligeour, O. Vidal, L. Dupin, F. Casoni, E. Gillon, A. Meyer,
S. Vidal, G. Vergoten, J. M. Lacroix, E. Souteyrand, 48 B. Zhang, H. Wang, X. Yan, Y. A. Duan, S. Guo and F. X. Luo,
A. Imberty, J. J. Vasseur, Y. Chevolot and F. Morvan, Org.
Biomol. Chem., 2015, 13, 8433–8444.
Transition Met. Chem., 2020, 45, 99–110.
¨
´
49 H. Paulsen, Z. Gyorgydeak and M. Friedmann, Chem. Ber.,
27 S. Alfei and A. M. Schito, Nanomaterials, 2020, 10, 1–50.
1974, 107, 1568–1578.
28 G. Michaud, R. Visini, M. Bergmann, G. Salerno, R. Bosco, 50 C. Bouillon, A. Meyer, S. Vidal, A. Jochum, Y. Chevolot,
E. Gillon, B. Richichi, C. Nativi, A. Imberty, A. Stocker,
T. Darbre and J. L. Reymond, Chem. Sci., 2016, 7, 166–182.
J. P. Cloarec, J. P. Praly, J. J. Vasseur and F. Morvan, J. Org.
Chem., 2006, 71, 4700–4702.
29 D. H. Simpson, A. Hapeshi, N. J. Rogers, V. Brabec, 51 Y. Li, J. C. Huffman and A. H. Flood, Chem. Commun., 2007,
G. J. Clarkson, D. J. Fox, O. Hrabina, G. L. Kay, A. K. King, 2692–2694.
J. Malina, A. D. Millard, J. Moat, D. I. Roper, H. Song, 52 S. Li, C. Wu, X. Tang, S. Gao, X. Zhao, H. Yan and X. Wang,
N. R. Watereld and P. Scott, Chem. Sci., 2019, 10, 9708–
Sci. China: Chem., 2013, 56, 595–603.
9720.
53 P. Rogala, G. Czerwonka, S. Michałkiewicz, M. Hodorowicz,
´
30 C. Eidamshaus and H. U. Reissig, Tetrahedron: Asymmetry,
2011, 22, 1644–1652.
B. Barszcz and A. Jabłonska-Wawrzycka, Chem. Biodiversity,
2019, 16, e1900403.
´
31 C. Shen, P. F. Zhang and X. Z. Chen, Helv. Chim. Acta, 2010, 54 G. Czerwonka, D. Gmiter, A. Guzy, P. Rogala, A. Jabłonska-
93, 2433–2438.
Wawrzycka, A. Borkowski, T. Cłapa, D. Naro˙zna,
´
32 J. Kra, D. Schmollinger, J. Maudrich and T. Ziegler, Synth,
2015, 47, 199–208.
P. Kowalczyk, M. Syczewski, M. Drabik, M. Danczuk and
W. Kaca, Biofouling, 2019, 35, 59–74.
33 S. Wang, J. A. Cuesta-Seijo, D. Lafont, M. M. Palcic and 55 X. Li, K. Heimann, F. Li, J. M. Warner, F. Richard Keene and
S. Vidal, Chem.–Eur. J., 2013, 19, 15346–15357. J. Grant Collins, Dalton Trans., 2016, 45, 4017–4029.
34 I. Carvalho, P. Andrade, V. L. Campo, P. M. M. Guedes, 56 F. Li, J. G. Collins and F. R. Keene, Chem. Soc. Rev., 2015, 44,
R. Sesti-Costa, J. S. Silva, S. Schenkman, S. Dedola, L. Hill, 2529–2542.
M. Rejzek, S. A. Nepogodiev and R. A. Field, Bioorg. Med. 57 S. V. Kumar, S. O. Scottwell, E. Waugh, C. J. McAdam,
Chem., 2010, 18, 2412–2427.
35 H. Shen, C. Shen, C. Chen, A. Wang and P. Zhang, Catal. Sci.
Technol., 2015, 5, 2065–2071.
L. R. Hanton, H. J. L. Brooks and J. D. Crowley, Inorg.
Chem., 2016, 55, 9767–9777.
´
58 A. Imberty, M. Wimmerova, E. P. Mitchell and N. Gilboa-
36 K. J. Kilpin, E. L. Gavey, C. J. McAdam, C. B. Anderson,
Garber, Microbes Infect., 2004, 6, 221–228.
¨
S. J. Lind, C. C. Keep, K. C. Gordon and J. D. Crowley, 59 P. Braun, B. Nagele, V. Wittmann and M. Drescher, Angew.
Inorg. Chem., 2011, 50, 6334–6346. Chem., Int. Ed., 2011, 50, 8428–8431.
37 D. Schmollinger, J. Kra, C. Ewald and T. Ziegler, 60 G. M. Sheldrick and IUCr, Acta Crystallogr., Sect. C: Struct.
Tetrahedron Lett., 2017, 58, 3643–3645. Chem., 2015, 71, 3–8.
38 J. P. Byrne, J. A. Kitchen and T. Gunnlaugsson, Chem. Soc. 61 E. Peeters, H. J. Nelis and T. Coenye, J. Microbiol. Methods,
Rev., 2014, 43, 5302–5325.
2008, 72, 157–165.
39 J. P. Byrne, J. A. Kitchen, O. Kotova, V. Leigh, A. P. Bell,
J. J. Boland, M. Albrecht and T. Gunnlaugsson, Dalton
Trans., 2014, 43, 196–209.
© 2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 16318–16325 | 16325