Inhibition of bacterial growth and galactosyltransferase activity of WbwC by α, ω-bis(3-alkyl-1H-imidazolium)alkane salts: Effect of varying carbon content

Article abstract of DOI:10.1016/j.bmc.2020.115494

A series of compounds was designed and synthesized having two imidazolium rings separated by a polymethylene spacer and having alkyl substituents on each of the imidazolium rings. The compounds were assayed for their effects on the activity of galactosyltransferase WbwC, and also on the growth of Gram-negative and Gram-positive bacteria, as well as human cells. The inhibition observed on enzyme activities and cell growth was dependent on the total number of carbons in the spacer and the alkyl substituents on the imidazolium rings. These readily synthesized, achiral compounds have potential as antimicrobial and antiseptic agents.

Full text of DOI:10.1016/j.bmc.2020.115494

Journal Pre-proofs
Inhibition of bacterial growth and galactosyltransferase activity of WbwC by
α, ω-bis(3-alkyl-1H-imidazolium)alkane salts: effect of varying carbon con-
tent
Alexander Kocev, Jacob Melamed, Shuo Wang, Xianqi Kong, Jason Z.
Vlahakis, Yaozu Xu, Walter A. Szarek, Inka Brockhausen
PII:
S0968-0896(20)30315-1
https://doi.org/10.1016/j.bmc.2020.115494
BMC 115494
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
30 July 2019
2 April 2020
5 April 2020
Please cite this article as: A. Kocev, J. Melamed, S. Wang, X. Kong, J.Z. Vlahakis, Y. Xu, W.A. Szarek, I.
Brockhausen, Inhibition of bacterial growth and galactosyltransferase activity of WbwC by α, ω-bis(3-alkyl-1H-
imidazolium)alkane salts: effect of varying carbon content, Bioorganic & Medicinal Chemistry (2020), doi:
https://doi.org/10.1016/j.bmc.2020.115494
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
©
2020 Published by Elsevier Ltd.

1
Bioorganic & Medicinal Chemistry
Inhibition of bacterial growth and galactosyltransferase activity of
WbwC by , -bis(3-alkyl-1H-imidazolium)alkane salts: effect of
varying carbon content
a
a
a
b
b
Alexander Kocev , Jacob Melamed , Shuo Wang , Xianqi Kong , Jason Z.Vlahakis ,
a,b
b*
a*
Yaozu Xu , Walter A. Szarek , Inka Brockhausen
Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario K7L
a
3
N6, Canada
b
Department of Chemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada
*Corresponding authors: Tel: 1 613 533 2643
E-mail addresses: szarekw@chem.queensu.ca (W. A. Szarek), brockhau@queensu.ca (I.
Brockhausen).
ABSTRACT
A series of compounds was designed and synthesized having two imidazolium rings separated by a
polymethylene spacer and having alkyl substituents on each of the imidazolium rings. The
compounds were assayed for their effects on the activity of galactosyltransferase WbwC, and also
on the growth of Gram-negative and Gram-positive bacteria, as well as human cells. The inhibition
observed on enzyme activities and cell growth was dependent on the total number of carbons in the
spacer and the alkyl substituents on the imidazolium rings. These readily synthesized, achiral
compounds have potential as antimicrobial and antiseptic agents.
Keywords:
Imidazolium salts
Glycosyltransferase inhibitors
Cell cultures
Bacterial cultures

2
1
. Introduction
The development of new classes of antibacterial drugs is urgent and important as resistance
1
2
to existing classes of antibiotics emerges. Electron-deficient tetrazolium, triazolium, and
imidazolium3 compounds were initially discovered as potent inhibitors of Plasmodium falciparum
,4
3
parasite replication. The positively charged imidazolium salts could potentially bind to phosphate
groups or other negatively charged groups found in proteins, DNA, membrane lipids, and
glycosyltransferase substrates. The latest generation of our compounds consists of bivalent
imidazolium salts in which the imidazolium rings are separated by a polymethylene spacer and have
alkyl substituents on each of the rings. We previously observed that imidazolium compounds with
at least 18 carbons in the aliphatic spacer were effective glycosyltransferase inhibitors and
4
-8
selectively inhibited a number of mammalian and bacterial glycosyltransferases. For example,
6
7
α2,3-sialyltransferase WbwA and β1,3-galactosyltransferase WbwC from Escherichia coli O104
were strongly inhibited by imidazolium compounds with spacers of 18 or more carbons. However,
8
another galactosyltransferase (WbwB) showed little inhibition. The structures of the bis-
imidazolium compounds are not related to structures of glycosyltransferase acceptor or donor
substrates and inhibition appears to mainly target the proteins.
We have now synthesized a new series of compounds, varying the spacer length and the size
of the alkyl substituents with the aim to improve their potency and possibly specificity. In Table 1
are given details of the structures of the new compounds (1–21). Here, we studied the inhibition of
7
bacterial β1,3-galactosyltransferase WbwC from highly pathogenic Escherichia coli O104 . This
enzyme binds UDP-Gal as the donor substrate and GalNAc-diphosphate-phenyl-undecyl acceptor
substrate. WbwC has been classified by the Carbohydrate-Active Enzymes (CAZy) databank into
the GT2 family, having a DDxD motif where Asp residues are critical for activity. The genes
encoding WbwC homologs are also found in several other bacteria. An inhibition of these enzymes
would reduce the synthesis of O antigens that are virulence factors.
The effects of compounds on the growth of two Gram-negative bacteria (Escherichia coli
and Pseudomonas aeruginosa) and two Gram-positive bacteria (Bacillus subtilis and
Staphylococcus epidermidis), and for comparison, on the proliferation of mammalian cell cultures,
were examined. We found that compounds with high carbon content were able to both, inhibit
glycosyltransferase WbwC, and block bacterial growth and mammalian cell proliferation. The
results suggest the potential of imidazolium compounds as antimicrobials or antiseptic agents.
2
2
. Results and discussion
.1. Chemical synthesis
The candidate bis-imidazolium salt compounds 1–21 were synthesized following a
straightforward synthetic approach, as shown in Figure 1. The general strategy involved treating

,ω-alkanediols with methanesulfonyl chloride/triethylamine in THF to afford the ,ω-
4
bis(methanesulfonyloxy)alkanes as described in a previous publication. 1,16-Hexadecanediol was
synthesized from 16-hexadecanolide by reduction using LiAlH in THF. The 1-alkylimidazoles
4
were obtained by treatment of 1-bromoalkanes with imidazole/tetraethylammonium iodide/sodium
9
hydroxide in toluene following a published procedure. The final bis-imidazolium compounds in the
dimesylate form (compounds 1–21) were then obtained by the reaction of the appropriate ,ω-
bis(methanesulfonyloxy)alkane with the appropriate 1-alkylimidazole in acetone, as described in
4
our previous publication for the synthesis of 1,22-bis(3-methyl-1H-imidazolium-1-yl)docosane
dimesylate.

3
Figure 1. The synthesis of bis-imidazolium salt compounds (dimesylate form).
Reaction of β1,3-galactosyltransferase WbwC from E. coli O104:
3
UDP-[ H]Gal + GalNAcα-PO
3
-PO
3
-(CH
2
)11-O-phenyl 
3
UDP + [ H]Galβ1-3GalNAcα-PO
3
-PO
3
-(CH )11-O-phenyl
2
Figure 2.
In vitro β1,3-galactosyltransferase reaction by WbwC utilizing a synthetic analog of the natural
diphosphate-undecaprenol-based acceptor substrate GalNAcα-PO -PO -(CH 11-O-phenyl (GalNAc-
3
3
2
)
PP-PhU). The reaction product can be isolated by C18 chromatography and quantified by scintillation
counting.

4
Table 1. Inhibition of β1,3- galactosyltransferase activity of WbwC by ,ω-bis(3-alkyl-1H-
imidazolium)alkane salts (at 0.5 mM concentration). The acceptor concentration was 0.1 mM and
inhibitory compounds were at 0.5 mM. All assay mixtures contained 10% methanol. Compounds
were tested in at least duplicate determinations. The IC50 values were determined of highly active
inhibitors at increasing inhibitor concentrations.
H2
m+1 m
C
C H
m 2m+1
N
N
CH2
N
N
n
2
X
Compound Number of Number of
Total
%
IC50 (μM)
carbon
atoms in
spacer
carbon
atoms in
each alkyl
substituent
number of
carbons
Inhibition at 0.5mM
(
spacer +
alkyl
substituents,
n+2m)
(
n)
(
m)
1
2
3
4
5
6
7
6
6
6
6
6
6
6
6
7
8
9
10
11
12
18
20
22
24
26
28
30
6
16
13
<10
92
99
263
117
43
90
8
9
10
10
10
10
10
10
10
6
7
8
22
24
26
28
30
32
34
<1
<1
64
95
98
98
91
1
0
1
2
3
4
123
23
152
62
1
1
1
1
9
10
11
12
59
1
1
1
1
1
2
2
5
6
7
8
9
0
1
16
16
16
16
16
16
16
6
7
8
28
30
32
34
28
38
40
88
95
93
93
92
92
92
48
11
18
9
35
10
11
12
147
123
121
2
2
2
2
2
2
2
2
3
2
3
4
5
6
7
8
9
0
4
6
8
14
15
16
18
20
22
1
1
1
1
1
1
1
1
1
6
8
9
7
10
16
17
18
20
22
24
21
10
11
<1
<1
91
91
230
159
The syntheses of compounds 22–30 were reported in ref. 4. Compounds 1–21, 29, and 30
were prepared as dimesylate salts, and compounds 22–28 were prepared as dichloride salts.

5
2
.2. Inhibition of β1,3-galactosyltransferase WbwC
The compounds 1–21 were soluble at 5 mM concentration in MeOH, EtOH and DMSO.
o
Assays containing candidate compounds were carried out at 37 C with 10 % MeOH present.
However, strong inhibition was also observed in control assays lacking MeOH, suggesting that
there is significant solubility of compounds in the aqueous assay solution.
In bacterial cells glycosyltransferases are thought to be located at the cytoplasmic face of the
inner membrane where they access the nucleotide sugar-donor substrates, as well as the
diphosphate–lipid acceptor substrates that are anchored in the membrane. Bis-imidazolium
compounds having spacers of 18 to 22 carbons and a methyl group on each of the imidazolium
4
rings were previously found to be strong inhibitors of selected glycosyltransferases while
compounds having shorter spacers and unsusbstituted imidazole rings were ineffective. In the
present study, the new compounds 1–21 were examined with β1,3-Gal-transferase WbwC from E.
7
coli O104 . Included also are results with compounds 22–30 that only have 1-carbon substituents on
3
the imidazolium rings. The donor substrate was UDP-[ H]Gal and the acceptor substrate was 0.1
mM GalNAc-PP-PhU (Figure 2). The results of the inhibition studies are summarized in Table 1.
The comparison of inhibition of purified enzyme with enzyme in supernatants revealed remarkable
similarity (data not shown). The length of the polymethylene spacer as well as that of the alkyl
substituents had a significant effect on the inhibitory properties of the compounds. High inhibition
(>90 %) of WbwC activity was achieved with compounds having spacers of 20 and 22 carbons, or
with compounds having a total number of carbons (spacer + alkyl substituents) of 26 to 40.
When comparing compounds having a 6-carbon spacer, the alkyl substituent had to have a
length of at least 10 carbons to yield effective inhibition. The IC50 values were 263 and 117 μM for
compounds 5 and 6 having 10 and 11 carbons in each alkyl substituent. The IC50 value was lower
(43 μM) for compound 7 having longer (12-carbon) substituents. Similarly, in the series of
compounds having 10-carbon spacers, compounds 8 and 9 having shorter substituents were not
inhibiting WbwC, while compounds having substituents of 8 to12 carbons were inhibitors with IC50
values between 23 and 152 μM. The entire series of compounds having 16-carbon spacers and 6- to
1
2-carbon substituents (compounds 15 to 21) were successful inhibitors of WbwC with IC50 values
of 11 to 147 μM (Table 1).
Compounds having only 1-carbon substitutents (22 to 30) and 4- to 18-carbon spacers
showed minimal inhibition while compounds 29 and 30, having 20- and 22-carbon spacers, strongly
inhibited with IC50 values of 230 and 159 μM, respectively.
The results indicate that the length of the spacers of the compounds control the inhibition,
and in addition, the alkyl substituents played a critical role in creating highly effective inhibitors. In
1
0
contrast, inhibitors of mammalian Gal-transferases (e.g., 1-thio-N-butyrylGlcNβ-(2-naphthyl) did
not inhibit WbwC activity. Interestingly, strong inhibitors (compounds 5, 7, 11, 14, 15, 21) had only
minor effects on the activity of bovine milk β1,4-galactosyltransferase using 0.5 mM GlcNAcβ-Bn
as the acceptor and 0.5 mM candidate compound.
In order to examine if the inhibition of the hydrophobic and positively charged compounds
was due to their interactions with glycosyltransferase substrates, the compounds were mixed with
3
the donor substrate UDP-[ H]Gal and buffers at different pH values and ratios, followed by
chromatography using a C18 Sep-Pak cartridge Plus (Waters). In the absence of buffer, compounds
3
6
2
and 12 caused a 50 % retention of UDP-[ H]Gal by the C18 Sep-Pak column at a molar ratio of
3
.5:1 (compound 6 or 12:UDP-[ H]Gal). This result was likely due to electrostatic interaction
between the phosphates of UDP and imidazolium groups, thereby increasing the hydrophobic
property of UDP-Gal when complexed with the compounds. However, in the presence of buffer at
3
pH 7, no retention of UDP-[ H]Gal was observed under these conditions.
3
3
11
Both UDP-[ H]Gal and [ H]Galβ1-4GlcNAc-PP-PhU , quantitatively bound to AG-1x8
anion-exchange columns due to the presence of phosphate groups, and inhibitor 7 had no effect on
this binding. It was concluded that binding of negatively charged enzyme substrates to positively

6
charged compounds was not the cause of glycosyltransferase inhibition. Therefore, the inhibition
appeared to be glycosyltransferase-protein specific and likely based on spatially defined charge and
hydrophobic interactions. This conclusion is corroborated by previous findings that only a selected
4
number of glycosyltransferases were inhibited although they shared donor and acceptor substrates.
2
.3. Effect of compounds on bacterial growth
To assess the effect of bis-imidazolium compounds on live bacteria in culture, the
compounds were added to bacterial culture media and overnight growth was estimated by counting
colony numbers. At 50 μM concentration in DMSO, uncharged compounds, namely, 1,16-bis(1H-
imidazol-1-yl)hexadecane, 1,18-bis(1H-imidazol-1-yl)octadecane , and 1,20-bis(1H-imidazol-1-
4
yl)eicosane , and positively charged compounds 22 and 23 with short spacers did not show any
effect on bacterial growth of P. aeruginosa PAO1 cells. A minor growth effect was seen with
compound 28 (having an 18-carbon spacer) at 50 µM and with compound 25 (having a 14-carbon
spacer) at 5 to 25 µM. In contrast, significant growth inhibition was seen by compound 29 at 50 µM
having a 20-carbon spacer. Compound 30 (having a 22-carbon spacer) at 50 µM concentration
showed no colonies, and at 25 µM only 5% of the colonies were visible (Table 2).
In contrast, compounds 10 and 14 at 5 to 25 µM strongly inhibited PAO1 bacterial growth
while compound 20 had a less potent effect but eliminated colonies at 25 µM (Table 2). The
5
mammalian galactosyltransferase inhibitor 1-thio-N-butyrylGlcNβ-(2-naphthyl) at 50 μM had no
effect on PAO1 bacterial growth.
In comparison, Gram-negative Escherichia coli BL21 showed strong inhibition by
compounds 10, 14 and 20 with 26, 34 and 38 aliphatic carbons, respectively, at 5, 10, and 25 µM
concentrations with no apparent colonies. Compound 25 (with a 14-carbon spacer) was much less
effective at the same concentrations. Thus, the inhibitory effects were slightly different between the
two Gram-negative bacteria. The effects of candidate compounds on the growth of Gram-positive
bacteria Staphylococcus epidermidis and Bacillus subtilis closely resembled those of E. coli. No or
few colonies were seen after treatment with compounds 10, 14, and 20 at 5 to 25 µM
concentrations, and compound 25 did not appear to affect S. epidermidis. In addition, compound 30
at 50 µM eliminated colonies of B. subtilis and S. epidermidis.
These four strains of bacteria have different glycosyltransferases and produce different
polysaccharides. Our results suggest that the candidate compounds having a total number of carbons
(spacer + alkyl substituents) of 26 to 38 may have a general effect on disrupting the functions of
bacterial membranes. This effect might be attributable to the possible detergent-like properties of
candidate compounds by making the membranes leaky, or affecting the structure of the membrane
that is a prerequisite for the functions of membrane-bound or membrane-associated enzymes and
proteins. It is unlikely that this growth inhibition is mediated through inhibition of the cytoplasmic
4
enzymes that assemble polysaccharides. However, of the previously synthesized compounds and
the newly synthesized compounds (1 to 21) tested here, the inhibition of Gal-transferases paralleled
the inhibition of bacterial growth, suggesting a common underlying mechanism. These possibilities
should be explored in future experiments.

7
a
Table 2. Effect of ,ω-bis-(3-alkyl-1H-imidazolium)alkanes on growth of bacteria.
P.a., Pseudomonas aeruginosa PAO1; E.c., Escherichia coli BL21; B.s., Bacillus subtilis; S.e.,
Staphylococcus epidermidis. ND, not determined. Bacteria were pre-incubated for 4 hours at 37 °C
o
a
with and without candidate compound in 1% MeOH and then incubated overnight at 37 C. In the
absence of candidate compound, but with 1% MeOH in the culture medium, the number of colonies
was set to 100%. All experiments were carried out in at least duplicate determinations. The average
percentages of colony numbers counted on each plate are shown as percentage of controls lacking
inhibitory compounds.
Compound Total
number of
carbons
Concentration
µM
Inhibition of colony number (%)
E.c. B.s.
P.a.
S.e
(spacer + alkyl
substituents)
2
2
2
2
3
5
6
50
50
5
0
ND
ND
ND
ND
8
0
ND
ND
16
0
4
22
0
32
51
8
20
51
0
0
0
1
2
0
5
2
3
9
0
22
24
50
>90
ND
ND
ND
25
>95
100
ND
ND
ND
ND
ND
ND
5
0
1
1
2
0
4
0
26
34
38
5
0
5
96
100
100
100
100
100
100
100
100
100
100
100
1
2
5
0
5
>98
100
>98
100
100
100
100
100
100
100
100
100
1
2
5
0
5
28
77
100
100
100
100
100
100
100
100
100
100
1
2

8
2
.4. Effect of candidate compounds on mammalian cells in culture
Human embryonic kidney (AD293), lung cancer (A549) and prostate cancer (PC-3) cells,
1
2
as well as human ovarian cancer cells, OC-3-VGH, showed inhibition of cell proliferation by
candidate compounds measured by the MTT assay. DMSO (0.1%) was used as the solvent for
compounds in cell cultures that were not compatible with MeOH. Uncharged compounds, namely,
1
,16-bis-(1H-imidazol-1-yl)hexadecane, 1,18-bis-(1H-imidazol-1-yl)octadecane, and 1,20-bis-(1H-
4
imidazol-1-yl)eicosane at 50 μM, as well as solvent controls did not affect cell growth. However,
compounds with longer spacers, namely, compound 28 (18 spacer carbons) and compound 29 (20
spacer carbons) showed up to 80% inhibition of cell proliferation with IC50 values varying between
5
and 12 μM for OC-3-VGH cells. The effects on PC-3, A549 and AD293 cells were similar. The
mammalian galactosyltransferase inhibitor 1-thio-N-butyrylGlcNβ-(2-naphthyl)5 had
a
comparatively minor effect on OC-3-VGH cells and at 1 mM reduced growth by 50 %. To further
examine the effect of 29, the scratch wound healing assay of ovarian cancer cells was employed.
The migration of OC-3-VGH cells was significantly inhibited after 18 h incubation with 500 μM
2
9, compared to vehicle-treated control cells containing the same amount of DMSO solvent (data
not shown). The effects of the candidate compounds were not cancer-cell specific or selective. As
with bacteria, it is possible that the inhibitory effects on whole cells are mediated through
interactions with components of cell membranes. It remains to be shown if compounds penetrate
membranes and act on internal structures such as phosphorylated lipids or DNA in order to block
cell growth.
3
. Conclusions
The work described showed that, in the case of the newly synthesized bis-imidazolium
compounds 1–21, those having a total number of non-imidazolium carbons (spacer + alkyl
substituents) of at least 26, afforded potent inhibitors of two bacterial glycosyltransferases (see
Table 1). Targeted delivery of these inhibitors into bacterial cells has the potential of blocking the
biosynthesis of outer polysaccharides which are virulence factors. It remains to be shown if the bis-
imidazolium salts induce inactive, glycosyltransferase-protein conformations. The structural
features determine the inhibitory potential of the compounds. It is noteworthy that compounds that
exhibited strong inhibition of glycosyltransferase activity (e.g. compounds 14 and 20, Table 1), also
inhibited the growth of bacteria (Table 2). and could act as adjuvant antibiotics but the relationship
between glycosyltransferase inhibition and growth inhibition has not yet been clarified. Several
mechanisms for inhibition are possible, for example, the compounds may disturb the structures and
functions of membrane components, thereby exhibiting cell-growth inhibition of both Gram-
negative and Gram-positive bacteria as well as human cells. It remains to be shown if compounds
penetrate through membranes and inactivate compounds critical for growth. Future work will be
directed towards the differential toxic effects in cells and animal models, and the effective
concentrations necessary for the design of bacteria-specific inhibitors.
4
4
. Experimental
.1. General
1
13
Melting points were measured on a Mel-Temp II apparatus and are uncorrected. H and C
NMR spectra were recorded on a Bruker Avance 400 spectrometer in DMSO-d or CD OD The
chemical shifts are reported in δ (ppm) relative to tetramethylsilane. The compounds synthesized
6
3
.
1
3
1
were deemed to be >95% pure by H NMR analysis. High-resolution ESI mass spectra were
performed by the AIMS Mass Spectrometry Laboratory (Department of Chemistry) at The
University of Toronto on an Agilent 6538 UHD instrument.
Compounds 1–21 were synthesized for the present study, and the experimental details are
4
reported herein; syntheses for compounds 22–30 are described in a previous publication. All other
chemical reagents were obtained from Sigma–Aldrich and used without prior purification.

9
4
.2. General procedure for the formation of ,ω-bis(methanesulfonyloxy)alkanes
Under an atmosphere of nitrogen, the ,ω-alkanediol (8.5 mmol, 1 equiv) was dissolved in
warm tetrahydrofuran (20 mL) and the solution was then cooled to 0 ºC. To this solution was added
triethylamine (2.5 equiv) and then methanesulfonyl chloride (2.2 equiv). The mixture was stirred at
room temperature overnight, concentrated, and then dried under high vacuum. Water was added,
and the resulting solid was collected by filtration and washed with water (250 mL). The solid
material was dried under high vacuum to give the product.
4
.3. General procedure for the formation of 1-alkylimidazoles
A mixture of imidazole (22 mmol, 1 equiv) and the alkyl bromide (1 equiv) in toluene (30
mL) in the presence of tetraethylammonium iodide (0.2 equiv) and sodium hydroxide (3 equiv) was
heated at reflux temperature for 10 h. The resulting mixture was cooled to room temperature, water
was added, and the mixture extracted twice with ethyl acetate. The combined organic extracts were
washed with brine, dried over sodium sulfate, and concentrated. Purification by flash-column
chromatography on silica gel using ethyl acetate as an eluent, followed by drying under high
vacuum gave the product.
4
.4. General procedure for the formation of bis-imidazolium compounds (dimesylate form)
Under an atmosphere of nitrogen, the ,ω-bis(methanesulfonyloxy)alkane (0.36 mmol, 1
equiv) was mixed with the appropriate 1-alkylimidazole (5 equiv) and the mixture was heated at
8
0–90 ºC for 1h; acetone (20 mL) was then added and the mixture was stirred at reflux temperature
overnight, concentrated, and dried under high vacuum. The resulting golden oil was dissolved in
methanol, the solution was concentrated, and the residual oil was washed with diethyl ether (10
mL). This dissolution/concentration/washing procedure was repeated two more times. The solid
material was dried under high vacuum to give the product.
4
.5. Characterization of bis-imidazolium compounds (dimesylate form) synthesized following
the general procedure outlined in Section 4.4
4
.5.1. 1,6-Bis(3-hexyl-1H-imidazolium-1-yl)hexane dimesylate (1)
Obtained in 82% yield from reaction of 1,6-bis(methanesulfonyloxy)hexane with 1-
1
hexylimidazole affording a yellow oil: H NMR (400.13 MHz, DMSO-d
6
): δ 0.86 (t, J = 7.0 Hz,
6
9
H), 1.16–1.33 (m, 16H), 1.71–1.85 (m, 8H), 2.32 (s, 6H), 4.17 (t, J = 7.2 Hz, 8H), 7.82 (s, 4H),
1
3
.26 (s, 2H); C NMR (100.61 MHz, DMSO-d ): δ 13.8 (2C), 21.8 (2C), 24.8 (2C), 25.1 (2C), 29.0
6
(
[
2C), 29.2 (2C), 30.4 (2C), 39.8 (2C), 48.6 (2C), 48.8 (2C), 122.5 (4C), 136.0 (2C); HRMS (ESI)
2
+
M-C
2
H
6
S
2
6
O ] calcd for C24
H
44
4
N : 194.1778. Found: 194.1778.
4
.5.2. 1,6-Bis(3-heptyl-1H-imidazolium-1-yl)hexane dimesylate (2)
Obtained in 85% yield from reaction of 1,6-bis(methanesulfonyloxy)hexane with 1-
1
heptylimidazole affording a golden oil: H NMR (400.13 MHz, DMSO-d
6
): δ 0.85 (t, J = 6.8 Hz,
6
9
H), 1.15–1.33 (m, 20H), 1.71–1.85 (m, 8H), 2.31 (s, 6H), 4.16 (t, J = 7.0 Hz, 8H), 7.81 (s, 4H),
1
3
.26 (s, 2H); C NMR (100.61 MHz, DMSO-d ): δ 13.9 (2C), 21.9 (2C), 24.8 (2C), 25.4 (2C), 28.0
6
(
2C), 29.0 (2C), 29.3 (2C), 31.0 (2C), 39.8 (2C), 48.7 (2C), 48.8 (2C), 122.5 (4C), 136.0 (2C);
2
+
HRMS (ESI) [M-C
2
H
6
S O
2 6
] calcd for C26
H
48
4
N : 208.1934. Found: 208.1939.
4
.5.3. 1,6-Bis(3-octyl-1H-imidazolium-1-yl)hexane dimesylate (3)
Obtained in 82% yield from reaction of 1,6-bis(methanesulfonyloxy)hexane with 1-
1
octylimidazole affording a golden oil: H NMR (400.13 MHz, DMSO-d
6
): δ 0.85 (t, J = 6.6 Hz,
6
9
H), 1.15–1.33 (m, 24H), 1.72–1.84 (m, 8H), 2.31 (s, 6H), 4.16 (t, J = 7.0 Hz, 8H), 7.81 (s, 4H),
1
3
.25 (s, 2H); C NMR (100.61 MHz, DMSO-d ): δ 13.9 (2C), 22.0 (2C), 24.8 (2C), 25.5 (2C), 28.3
6

1
0
(
2C), 28.4 (2C), 29.0 (2C), 29.3 (2C), 31.1 (2C), 39.8 (2C), 48.7 (2C), 48.8 (2C), 122.5 (4C), 136.0
2
+
(2C); HRMS (ESI) [M-C
2
H
6
S
2
O
6
] calcd for C28
H
52
4
N : 222.2091. Found: 222.2097.
4
.5.4. 1,6-Bis(3-nonyl-1H-imidazolium-1-yl)hexane dimesylate (4)
Obtained in 78% yield from reaction of 1,6-bis(methanesulfonyloxy)hexane with 1-
1
nonylimidazole affording a golden oil: H NMR (400.13 MHz, DMSO-d
6
): δ 0.85 (t, J = 6.6 Hz,
6
9
H), 1.14–1.34 (m, 28H), 1.72–1.85 (m, 8H), 2.31 (s, 6H), 4.16 (t, J = 6.8 Hz, 8H), 7.81 (s, 4H),
1
3
.26 (s, 2H); C NMR (100.61 MHz, DMSO-d ): δ 13.9 (2C), 22.1 (2C), 24.8 (2C), 25.5 (2C), 28.3
6
(
2C), 28.5 (2C), 28.8 (2C), 29.0 (2C), 29.3 (2C), 31.2 (2C), 39.8 (2C), 48.6 (2C), 48.8 (2C), 122.4
2
+
(4C), 136.0 (2C); HRMS (ESI) [M-C
2
6
H S
2
O
6
] calcd for C30
H
56
4
N : 236.2247. Found: 236.2252.
4
.5.5. 1,6-Bis(3-decyl-1H-imidazolium-1-yl)hexane dimesylate (5)
Obtained in 73% yield from reaction of 1,6-bis(methanesulfonyloxy)hexane with 1-
1
decylimidazole affording a golden oil: H NMR (400.13 MHz, DMSO-d
6
): δ 0.85 (t, J = 6.6 Hz,
6
H), 1.15–1.34 (m, 32H), 1.72–1.84 (m, 8H), 2.31 (s, 6H), 4.16 (t, J = 7.0 Hz, 4H), 4.16 (t, J = 7.0
1
3
Hz, 4H), 7.80 (s, 2H), 7.81 (s, 2H), 9.25 (s, 2H); C NMR (100.61 MHz, DMSO-d
6
): δ 13.9 (2C),
2
3
2.1 (2C), 24.8 (2C), 25.5 (2C), 28.3 (2C), 28.6 (2C), 28.8 (2C), 28.9 (2C), 29.0 (2C), 29.3 (2C),
2
+
1.3 (2C), 39.8 (2C), 48.6 (2C), 48.8 (2C), 122.4 (4C), 136.0 (2C); HRMS (ESI) [M-C
: 250.2404. Found: 250.2409.
2
H
6
S
2
6
O ]
calcd for C32
H
60
N
4
4
.5.6. 1,6-Bis(3-undecyl-1H-imidazolium-1-yl)hexane dimesylate (6)
Obtained in 68% yield from reaction of 1,6-bis(methanesulfonyloxy)hexane with 1-
1
undecylimidazole affording a golden oil: H NMR (400.13 MHz, DMSO-d
6
): δ 0.85 (t, J = 6.6 Hz,
6
H), 1.15–1.33 (m, 36H), 1.72–1.84 (m, 8H), 2.32 (s, 6H), 4.16 (t, J = 7.2 Hz, 4H), 4.17 (t, J = 7.0
1
3
Hz, 4H), 7.80 (s, 2H), 7.81 (s, 2H), 9.26 (s, 2H); C NMR (100.61 MHz, DMSO-d
2
2
C
6
): δ 13.9 (2C),
2.1 (2C), 24.8 (2C), 25.5 (2C), 28.3 (2C), 28.7 (2C), 28.8 (2C), 28.91 (2C), 28.94 (2C), 29.0 (2C),
9.3 (2C), 31.3 (2C), 39.8 (2C), 48.6 (2C), 48.8 (2C), 122.5 (4C), 136.0 (2C); HRMS (ESI) [M-
2+
2
6
H S
2
O
6
] calcd for C34
H
64
4
N : 264.2560. Found: 264.2568.
4
.5.7. 1,6-Bis(3-dodecyl-1H-imidazolium-1-yl)hexane dimesylate (7)
Obtained in 62% yield from reaction of 1,6-bis(methanesulfonyloxy)hexane with 1-
1
dodecylimidazole affording a beige waxy solid: mp 44–59 °C; H NMR (400.13 MHz, DMSO-d
6
):
δ 0.85 (t, J = 6.2 Hz, 6H), 1.13–1.34 (m, 40H), 1.72–1.84 (m, 8H), 2.32 (s, 6H), 4.16 (t, J = 7.2 Hz,
1
3
4
(
2
H), 4.17 (t, J = 7.0 Hz, 4H), 7.81 (s, 4H), 9.26 (s, 2H); C NMR (100.61 MHz, DMSO-d
2C), 22.1 (2C), 24.8 (2C), 25.5 (2C), 28.3 (2C), 28.7 (2C), 28.8 (2C), 28.90 (2C), 28.99 (4C),
9.03 (2C), 29.3 (2C), 31.3 (2C), 39.8 (2C), 48.6 (2C), 48.8 (2C), 122.4 (4C), 136.0 (2C); HRMS
6
): δ 13.9
2
+
(ESI) [M-C
2
6
H S
2
O
6
] calcd for C36
H
68
N : 278.2717. Found: 278.2720.
4
4
.5.8. 1,10-Bis(3-hexyl-1H-imidazolium-1-yl)decane dimesylate (8)
Obtained in 75% yield from reaction of 1,10-bis(methanesulfonyloxy)decane with 1-
1
hexylimidazole affording a yellow oil: H NMR (400.13 MHz, DMSO-d
6
): δ 0.85 (t, J = 6.6 Hz,
6
H), 1.13–1.33 (m, 24H), 1.71–1.84 (m, 8H), 2.31 (s, 6H), 4.16 (t, J = 7.0 Hz, 4H), 4.17 (t, J = 6.8
13
Hz, 4H), 7.81 (s, 4H), 9.25 (s, 2H); C NMR (100.61 MHz, DMSO-d
6
): δ 13.8 (2C), 21.8 (2C),
2
1
2
5.1 (2C), 25.5 (2C), 28.3 (2C), 28.7 (2C), 29.2 (2C), 29.3 (2C), 30.5 (2C), 39.8 (2C), 48.8 (4C),
2
+
22.5 (4C), 136.0 (2C); HRMS (ESI) [M-C
22.2097.
2
H
6
S
2
6
O ]
calcd for C28
H
52
4
N : 222.2091. Found:
4
.5.9. 1,10-Bis(3-heptyl-1H-imidazolium-1-yl)decane dimesylate (9)
Obtained in 77% yield from reaction of 1,10-bis(methanesulfonyloxy)decane with 1-
1
heptylimidazole affording a beige waxy solid: mp 58–62 °C; H NMR (400.13 MHz, DMSO-d
6
): δ
0
8
.86 (t, J = 6.6 Hz, 6H), 1.13–1.34 (m, 28H), 1.72–1.84 (m, 8H), 2.31 (s, 6H), 4.17 (t, J = 6.4 Hz,
13
H), 7.81 (s, 4H), 9.24 (s, 2H); C NMR (100.61 MHz, DMSO-d ): δ 13.9 (2C), 21.9 (2C), 25.4
6

1
1
(
2C), 25.5 (2C), 28.0 (2C), 28.3 (2C), 28.8 (2C), 29.25 (2C), 29.28 (2C), 31.0 (2C), 39.8 (2C), 48.8
2
+
(4C), 122.5 (4C), 136.0 (2C); HRMS (ESI) [M-C
2
H
6
S
2
O
6
] calcd for C30
H
56
4
N : 236.2247. Found:
2
36.2252.
4
.5.10. 1,10-Bis(3-octyl-1H-imidazolium-1-yl)decane dimesylate (10)
Obtained in 72% yield from reaction of 1,10-bis(methanesulfonyloxy)decane with 1-
1
octylimidazole affording a golden oil: H NMR (400.13 MHz, CD
3
OD): δ 0.90 (t, J = 6.8 Hz, 6H),
1
2
.22–1.43 (m, 32H), 1.82–1.95 (m, 8H), 2.70 (s, 6H), 4.23 (t, J = 7.4 Hz, 8H), 7.66 (s, 2H), 7.67 (s,
1
3
H), 9.05 (s, 2H); C NMR (100.61 MHz, CD OD): δ 14.4 (2C), 23.7 (2C), 27.27 (2C), 27.29
3
(
5
2
2C), 30.0 (2C), 30.1 (2C), 30.2 (2C), 30.5 (2C), 31.09 (2C), 31.13 (2C), 32.9 (2C), 39.6 (2C),
2
+
0.87 (2C), 50.89 (2C), 123.8 (4C), 137.2 (2C); HRMS (ESI) [M-C
50.2404. Found: 250.2412.
2
6
H S
O
2 6
] calcd for C32
H
60
4
N :
4
.5.11. 1,10-Bis(3-nonyl-1H-imidazolium-1-yl)decane dimesylate (11)
Obtained in 71% yield from reaction of 1,10-bis(methanesulfonyloxy)decane with 1-
1
nonylimidazole affording a beige waxy solid: mp 65–71 °C; H NMR (400.13 MHz, DMSO-d
6
): δ
0
8
.85 (t, J = 6.8 Hz, 6H), 1.13–1.32 (m, 36H), 1.71–1.83 (m, 8H), 2.31 (s, 6H), 4.16 (t, J = 6.8 Hz,
13
H), 7.81 (s, 4H), 9.24 (s, 2H); C NMR (100.61 MHz, DMSO-d ): δ 13.9 (2C), 22.0 (2C), 25.4
6
(
2C), 25.5 (2C), 28.31 (2C), 28.35 (2C), 28.5 (2C), 28.8 (4C), 29.2 (2C), 29.3 (2C), 31.2 (2C), 39.8
2
+
(2C), 48.8 (4C), 122.5 (4C), 136.0 (2C); HRMS (ESI) [M-C
2
6
H S
2
O
6
]
calcd for C34
H
64
4
N :
2
64.2560. Found: 264.2570.
4
.5.12. 1,10-Bis(3-decyl-1H-imidazolium-1-yl)decane dimesylate (12)
Obtained in 75% yield from reaction of 1,10-bis(methanesulfonyloxy)decane with 1-
1
decylimidazole affording a golden oil: H NMR (400.13 MHz, CD
3
OD): δ 0.90 (t, J = 6.8 Hz, 6H),
1
2
3
.20–1.45 (m, 40H), 1.81–1.98 (m, 8H), 2.70 (s, 6H), 4.22 (t, J = 7.2 Hz, 8H), 7.66 (s, 4H), 9.04 (s,
1
3
H); C NMR (100.61 MHz, CD
3
OD): δ 14.4 (2C), 23.7 (2C), 27.27 (2C), 27.31 (2C), 30.06 (2C),
0.08 (2C), 30.4 (2C), 30.5 (2C), 30.55 (2C), 30.59 (2C), 31.10 (2C), 31.14 (2C), 33.0 (2C), 39.6
2
+
(2C), 50.87 (2C), 50.90 (2C), 123.8 (4C), 137.2 (2C); HRMS (ESI) [M-C
2
H
6
S
2
O
6
]
calcd for
C
36
H
68
4
N : 278.2717. Found: 278.2722.
4
.5.13. 1,10-Bis(3-undecyl-1H-imidazolium-1-yl)decane dimesylate (13)
Obtained in 56% yield from reaction of 1,10-bis(methanesulfonyloxy)decane with 1-
1
undecylimidazole affording a brown oily residue; H NMR (400.13 MHz, DMSO-d
6
): δ 0.85 (t, J =
6
4
.8 Hz, 6H), 1.13–1.32 (m, 44H), 1.71–1.83 (m, 8H), 2.30 (s, 6H), 4.15 (t, J = 7.2 Hz, 8H), 7.80 (s,
1
3
H), 9.21 (s, 2H); C NMR (100.61 MHz, CD OD): δ 14.4 (2C), 23.7 (2C), 27.26 (2C), 27.31
3
(2C), 30.1 (4C), 30.4 (2C), 30.48 (2C), 30.54 (2C), 30.6 (2C), 30.7 (2C), 31.09 (2C), 31.12 (2C),
3
3.1 (2C), 39.6 (2C), 50.85 (2C), 50.87 (2C), 123.77 (2C), 123.81 (2C), 137.2 (2C); HRMS (ESI)
2
+
[
M-C
2
H
6
S
2
6
O ] calcd for C38
H
72
4
N : 292.2873. Found: 292.2878.
4
.5.14. 1,10-Bis(3-dodecyl-1H-imidazolium-1-yl)decane dimesylate (14)
Obtained in 51% yield from reaction of 1,10-bis(methanesulfonyloxy)decane with 1-
1
dodecylimidazole affording a light brown waxy solid: mp 46–47 °C; H NMR (400.13 MHz,
DMSO-d
6
): δ 0.85 (t, J = 6.8 Hz, 6H), 1.12–1.32 (m, 48H), 1.72–1.83 (m, 8H), 2.30 (s, 6H), 4.16 (t,
1
3
J = 7.0 Hz, 8H), 7.81 (s, 4H), 9.24 (s, 2H); C NMR (100.61 MHz, DMSO-d
6
): δ 13.9 (2C), 22.1
2C), 25.4 (2C), 25.5 (2C), 28.3 (2C), 28.4 (2C), 28.7 (2C), 28.79 (2C), 28.82 (2C), 28.9 (2C),
(
2
8.98 (2C), 29.00 (2C), 29.2 (2C), 29.3 (2C), 31.3 (2C), 39.8 (2C), 48.8 (4C), 122.5 (4C), 136.0
2+
(2C); HRMS (ESI) [M-C
2
H
6
S
2
6
O ] calcd for C40
H
76
4
N : 306.3030. Found: 306.3038.
4
.5.15. 1,16-Bis(3-hexyl-1H-imidazolium-1-yl)hexadecane dimesylate (15)
Obtained in 71% yield from reaction of 1,16-bis(methanesulfonyloxy)hexadecane with 1-
1
hexylimidazole affording a golden oil: H NMR (400.13 MHz, CD
3
OD): δ 0.91 (t, J = 6.2 Hz, 6H),

1
2
1
2
3
1
2
.20–1.42 (m, 36H), 1.81–1.96 (m, 8H), 2.70 (s, 6H), 4.23 (t, J = 7.0 Hz, 8H), 7.66 (s, 4H), 9.04 (s,
1
3
H); C NMR (100.61 MHz, CD
3
OD): δ 14.3 (2C), 23.5 (2C), 26.9 (2C), 27.3 (2C), 30.1 (2C),
0.6 (2C), 30.7 (2C), 30.78 (2C), 30.81 (2C), 31.0 (2C), 31.1 (2C), 32.2 (2C), 39.5 (2C), 50.9 (4C),
2
+
23.8 (4C), 137.2 (2C); HRMS (ESI) [M-C
64.2569.
2
H
6
S
2
6
O ]
calcd for C34
H
64
4
N : 264.2560. Found:
4
.5.16. 1,16-Bis(3-heptyl-1H-imidazolium-1-yl)hexadecane dimesylate (16)
Obtained in 75% yield from reaction of 1,16-bis(methanesulfonyloxy)hexadecane with 1-
1
heptylimidazole affording a golden oil: H NMR (400.13 MHz, CD
3
OD): δ 0.90 (t, J = 6.8 Hz, 6H),
1
2
3
1
2
.22–1.43 (m, 40H), 1.83–1.95 (m, 8H), 2.70 (s, 6H), 4.23 (t, J = 7.2 Hz, 8H), 7.66 (s, 4H), 9.04 (s,
1
3
H); C NMR (100.61 MHz, CD
3
OD): δ 14.4 (2C), 23.6 (2C), 21.2 (2C), 27.3 (2C), 29.7 (2C),
0.1 (2C), 30.6 (2C), 30.7 (2C), 30.78 (2C), 30.81 (2C), 31.1 (4C), 32.8 (2C), 39.5 (2C), 50.9 (4C),
2
+
23.8 (4C), 137.2 (2C); HRMS (ESI) [M-C
78.2723.
2
H
6
S
2
6
O ]
calcd for C36
H
68
4
N : 278.2717. Found:
4
.5.17. 1,16-Bis(3-octyl-1H-imidazolium-1-yl)hexadecane dimesylate (17)
Obtained in 68% yield from reaction of 1,16-bis(methanesulfonyloxy)hexadecane with 1-
1
octylimidazole affording a golden oil: H NMR (400.13 MHz, CD
3
OD): δ 0.90 (t, J = 6.8 Hz, 6H),
1
2
3
5
.22–1.42 (m, 44H), 1.82–1.95 (m, 8H), 2.70 (s, 6H), 4.23 (t, J = 7.2 Hz, 8H), 7.66 (s, 4H), 9.05 (s,
1
3
H); C NMR (100.61 MHz, CD
3
OD): δ 14.4 (2C), 23.7 (2C), 27.26 (2C), 27.28 (2C), 30.0 (2C),
0.1 (2C), 30.2 (2C), 30.6 (2C), 30.7 (2C), 30.78 (2C), 30.82 (2C), 31.1 (4C), 32.9 (2C), 39.5 (2C),
2+
0.9 (4C), 123.8 (4C), 137.2 (2C); HRMS (ESI) [M-C
2
6
H S
2
O
6
] calcd for C38
H
72
4
N : 292.2873.
Found: 292.2881.
4
.5.18. 1,16-Bis(3-nonyl-1H-imidazolium-1-yl)hexadecane dimesylate (18)
Obtained in 62% yield from reaction of 1,16-bis(methanesulfonyloxy)hexadecane with 1-
1
nonylimidazole affording a beige waxy solid: mp 66–69 °C; H NMR (400.13 MHz, CD
3
OD): δ
0
8
.90 (t, J = 6.8 Hz, 6H), 1.20–1.42 (m, 48H), 1.82–1.95 (m, 8H), 2.70 (s, 6H), 4.23 (t, J = 7.2 Hz,
1
3
H), 7.66 (s, 4H), 9.04 (s, 2H); C NMR (100.61 MHz, CD OD): δ 14.4 (2C), 23.7 (2C), 27.26
3
(2C), 27.29 (2C), 30.08 (2C), 30.11 (2C), 30.3 (2C), 30.5 (2C), 30.6 (2C), 30.7 (2C), 30.80 (2C),
3
C
0.83 (2C), 31.1 (4C), 33.0 (2C), 39.5 (2C), 50.9 (4C), 123.8 (4C), 137.2 (2C); HRMS (ESI) [M-
2+
2
6
H S
2
6
O ] calcd for C40
H
76
4
N : 306.3030. Found: 306.3035.
4
.5.19. 1,16-Bis(3-decyl-1H-imidazolium-1-yl)hexadecane dimesylate (19)
Obtained in 65% yield from reaction of 1,16-bis(methanesulfonyloxy)hexadecane with 1-
1
decylimidazole affording a beige waxy solid: mp 83–87 °C; H NMR (400.13 MHz, CD
3
OD): δ
0
8
.90 (t, J = 6.8 Hz, 6H), 1.20–1.42 (m, 52H), 1.83–1.95 (m, 8H), 2.70 (s, 6H), 4.23 (t, J = 7.2 Hz,
1
3
H), 7.66 (s, 4H), 9.04 (s, 2H); C NMR (100.61 MHz, CD OD): δ 14.4 (2C), 23.7 (2C), 27.25
3
(2C), 27.28 (2C), 30.07 (2C), 30.11 (2C), 30.4 (2C), 30.5 (2C), 30.6 (4C), 30.7 (2C), 30.80 (2C),
3
0.83 (2C), 31.08 (2C), 31.09 (2C), 33.0 (2C), 39.5 (2C), 50.9 (4C), 123.8 (4C), 137.2 (2C);
2
+
HRMS (ESI) [M-C
2
H
6
S O
2 6
] calcd for C42
H
80
4
N : 320.3186. Found: 320.3192.
4
.5.20. 1,16-Bis(3-undecyl-1H-imidazolium-1-yl)hexadecane dimesylate (20)
Obtained in 58% yield from reaction of 1,16-bis(methanesulfonyloxy)hexadecane with 1-
1
undecylimidazole affording a beige waxy solid: mp 82–85 °C; H NMR (400.13 MHz, DMSO-d
6
):
δ 0.85 (t, J = 6.4 Hz, 6H), 1.11–1.32 (m, 56H), 1.71–1.83 (m, 8H), 2.30 (s, 6H), 4.16 (t, J = 7.0 Hz,
1
3
8
H), 7.80 (s, 4H), 9.23 (s, 2H); C NMR (100.61 MHz, CD OD): δ 14.4 (2C), 23.7 (2C), 27.26
3
(2C), 27.29 (2C), 30.07 (2C), 30.12 (2C), 30.45 (2C), 30.56 (2C), 30.62 (4C), 30.64 (2C), 30.7
(
1
2C), 30.8 (2C), 30.9 (2C), 31.07 (2C), 31.09 (2C), 33.1 (2C), 39.5 (2C), 50.9 (4C), 123.8 (4C),
2
+
37.2 (2C); HRMS (ESI) [M-C
2
H
6
S
2
6
O ] calcd for C44
H
84
4
N : 334.3343. Found: 334.3341.
4
.5.21. 1,16-Bis(3-dodecyl-1H-imidazolium-1-yl)hexadecane dimesylate (21)

1
3
Obtained in 49% yield from reaction of 1,16-bis(methanesulfonyloxy)hexadecane with 1-
1
dodecylimidazole affording a beige waxy solid: mp 91–95 °C; H NMR (400.13 MHz, CD
3
OD): δ
0
8
.90 (t, J = 6.2 Hz, 6H), 1.21–1.43 (m, 60H), 1.83–1.95 (m, 8H), 2.70 (s, 6H), 4.23 (t, J = 7.0 Hz,
1
3
H), 7.66 (s, 4H), 9.05 (s, 2H); C NMR (100.61 MHz, CD OD): δ 14.5 (2C), 23.7 (2C), 27.25
3
(2C), 27.28 (2C), 30.07 (2C), 30.11 (2C), 30.46 (2C), 30.56 (2C), 30.61 (2C), 30.63 (2C), 30.70
(
1
3
2C), 30.74 (4C), 30.81 (2C), 30.84 (2C), 31.06 (2C), 31.09 (2C), 33.1 (2C), 39.5 (2C), 50.9 (4C),
2
+
23.8 (4C), 137.2 (2C); HRMS (ESI) [M-C
48.3506.
2
H
6
S
2
6
O ]
calcd for C46
H
88
N
4
: 348.3499. Found:
4
.6. Glycosyltransferase assays
7
The β1,3-Gal-transferase WbwC from Escherichia coli serotype O104: H4 was expressed
in BL21 bacteria and the gene in plasmids was re-sequenced. Bacterial suspensions in 10% glycerol
o
and 90 % PBS were stored at -20 C. For enzyme activity assays, bacterial suspensions were
sonicated 3 times for 15 sec and then centrifuged. The supernatant was an excellent source of active
and stable enzyme and was used to determine the IC50 values. The expressed WbwC protein was
7
also purified using a Ni-NTA column as described. Gal-transferase activities were assayed in
standard assays in a total volume of 40 μL in the presence of 5 mM MnCl
2
, 0.125M Tris-HCl buffer
pH 7.5, with donor substrate 0.8 mM UDP-[ H]Gal and 0.1 mM acceptor substrate GalNAc-PO
PO -(CH 11-O-phenyl. 10% MeOH was present in control assays. Mixtures were incubated for 1 h
3
3
-
3
2
)
o
at 37 C. Reaction products were isolated using a C18 Sep-Pak cartridge (short), washed first with 4
mL water and then eluted with 3 mL MeOH, and evaluated by scintillation counting. Candidate
compounds (Table 1) were added from 5 mM stock solutions in MeOH, and were present at 0.5
mM in the assay mixtures, and from 0.01 to 0.5 mM for inhibition assays. A 10 min pre-incubation
of the enzymes did not increase the inhibition. The inhibitor of bovine β1,4-Gal-transferase, 1-thio-
5
N-butyrylGlcNβ-(2-naphthyl) was tested with WbwC under the same conditions. Bovine milk
3
β1,4-galactosyltransferase (Sigma) was assayed similarly, but with UDP-[ H]Gal, acceptor
substrate GlcNAcβ-Bn and candidate compounds at 1 mM and 0.05% bovine serum albumin in the
4
assay, as described before.
4
.7. Growth of bacteria
Antimicrobial assays were performed using the colony-forming unit (CFU) assay. Gram-negative
bacteria Escherichia coli (BL21) or Pseudomonas aeruginosa PAO1, and Gram-positive bacteria
Bacillus subtilis and Staphylococcus epidermidis were grown in LB medium from a single colony
with agitation at 37 °C. The bacterial number was then evaluated by measuring the optical density
8
(
OD) at 550 nm corresponding to approximately 10 CFU/mL). The bacteria were pelleted, washed
8
with phosphate-buffered saline (PBS) and then concentrated to 5×10 CFU/mL in PBS. The
bacterial suspension (100 µL) was incubated with candidate compound and a final concentration of
1
% MeOH in PBS for 4 h and poured onto solid LB agar. Bacterial colonies were counted after
incubation overnight at 37 °C.
4
.8. Mammalian cell cultures
Human embryonal kidney cells (AD293), lung cancer (A549) and prostate cancer (PC-3,
1
2
LNCaP) cells were obtained from ATCC and grown as recommended. Human ovarian cancer cells
OC-3-VGH) were donated by G. Lee, University of British Columbia. Candidate compounds (0.5
mM final concentration in the growth medium) were dissolved in DMSO (final concentration 0.1
) and added to the cell medium; cells were then grown on 96-well plates and incubated for 24 h at
7°C followed by analyses of cell proliferation by MTT assays in at least three identical
(
%
3
experiments. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, Thermo-Fisher)
was used to assess cell proliferation accroding to the manufacturer's instructions.
Acknowledgments

1
4
This research was supported in part by the Natural Sciences and Engineering Research Council of
Canada, the Canadian Institutes of Health Research, and GlycoNet.
References
1
2
3
4
5
.
.
.
.
.
Cui X, Vlahakis JZ, Crandall IE, Szarek WA. Anti-Plasmodium activity of tetrazolium salts.
Bioorg Med Chem. 2008;16:1927–1947.
Vlahakis JZ, Lazar C, Crandall IE, Szarek WA. Anti-Plasmodium activity of imidazolium and
triazolium salts. Bioorg Med Chem. 2010;18:6184–6196.
Vlahakis JZ, Mitu S, Roman G, Rodriguez EP, Crandall IE, Szarek WA. The anti-malarial
activity of bivalent imidazolium salts. Bioorg Med Chem. 2011;19:6525–6542.
Gao Y, Vlahakis JZ, Szarek WA, Brockhausen I. Selective inhibition of glycosyltransferases
by bivalent imidazolium salts. Bioorg Med Chem. 2013;21:1305–1311.
Wang S, Hao, Y, Lam JS, Vlahakis JZ, Szarek WA, Vinnikova A, Veselovsky VV, Brockhausen I.
Biosynthesis of the common polysaccharide antigen of Pseudomonas aeruginosa PAO1:
Characterization and role of WbpZ – a GDP-D-rhamnose: GlcNAc/GalNAc-diphosphate-
D-rhamnosyltransferase WbpZ. J Bacteriol. 2015;197:2012–2019.
-
6
7
.
.
Czuchry D, Desormeaux P, Stuart M, Jarvis D, Matta KL, Szarek WA, Brockhausen I.
Identification and biochemical characterization of the novel α2,3-sialyltransferase WbwA
from pathogenic Escherichia coli serotype O104. J Bacteriol. 2015;197:3760–3768.
Wang S, Czuchry D, Liu B, Vinnikova AN, Gao Y, Vlahakis JZ, Szarek WA, Feng L, Wang
L, Brockhausen I. Characterization of two UDP-Gal: GalNAc-diphosphate-lipid
β1,3-Galactosyltransferases WbwC from Escherichia coli Serotypes O104 and O5.
J Bacteriol. 2014;196:3122–3133.
8
.
.
Czuchry D, Szarek WA, Brockhausen I. Identification and biochemical characterization of
WbwB, a novel UDP-Gal: Neu5Ac-R 1,4-galactosyltransferase from the intestinal
pathogen Escherichia coli serotype O104. Glycoconj J. 2017;35:65–76.
Asano K, Matsubara S. Effects of a flexible alkyl chain on a ligand for CuAAC reaction. Org
Lett. 2010;12:4988–4991, Supporting information, page S4.
9
1
0. Brockhausen I, Benn M, Bhat S, Marone S, Riley, JG, Montoya-Peleaz P, Vlahakis JZ,
Paulsen H, Schutzbach JS, Szarek WA. UDP-Gal: GlcNAc-R β1,4-galactosyltransferase—a
target enzyme for drug design. Acceptor specificity and inhibition of the enzyme. Glycoconj J.
2
006;23:525-541.
1. Xu C, Liu B, Hu B, Han Y, Feng L, Allingham JS, Szarek WA, Wang L, Brockhausen I.
Biochemical characterization of UDP-Gal:GlcNAc-pyrophosphate-
1
-
galactosyltransferase WfeD, a new enzyme from Shigella boydii type 14 that catalyzes the
second step in O-antigen repeating-unit synthesis. J Bacteriol. 2011;193:449–459.
2. Gao Y, Chachadi VB, Chen P-W, Brockhausen I. Glycosylation potential of human prostate cancer
cell lines. Glycoconj J. 2012;29:525–537.
3. Gottlieb HE, Kotlyar V, Nudelman A. NMR chemical shifts of common laboratory solvents as
trace impurities. J Org Chem. 1997;62:7512–7515.
1
1

1
5
Declaration of interests
X The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

1
6