Bioorganic & Medicinal Chemistry 21 (2013) 13051311

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Selective inhibition of glycosyltransferases by bivalent imidazolium

salts
Yin Gao a,b, Jason Z. Vlahakis c, Walter A. Szarek c,, Inka Brockhausen a,b,
a

Department of Medicine, Division of Rheumatology, Queens University, Kingston, Ontario, Canada K7L 3N6
Department of Biomedical and Molecular Sciences, Queens University, Kingston, Ontario, Canada K7L 3N6
c
Department of Chemistry, Queens University, Kingston, Ontario, Canada K7L 3N6
b

a r t i c l e

i n f o

Article history:
Received 31 October 2012
Revised 5 December 2012
Accepted 14 December 2012
Available online 3 January 2013
Keywords:
Galactosyltransferases
GlcNAc-transferases
GalNAc-transferases
b3-GalT5
Inhibitors
Imidazolium salts

a b s t r a c t
Galactosyltransferases (GalTs) extend the glycan chains of mammalian glycoproteins by adding Gal to
terminal GlcNAc residues, and thus build the scaffolds for biologically important glycan structures. We
have shown that positively charged bivalent imidazolium salts in which the two imidazolium groups
are linked by an aliphatic chain of 20 or 22 carbons form potent inhibitors of puried human b3-GalT5,
using GlcNAcb-benzyl as acceptor substrate. The inhibitors are not substrate analogs and also inhibited a
selected number of other glycosyltransferases. These bis-imidazolium compounds represent a new class
of glycosyltransferase inhibitors with potential as anti-cancer and anti-inammatory drugs.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
In mammals, the extension of glycoconjugate oligosaccharide
chains by galactosyltransferases (GalTs) is important for a variety
of biological recognition processes, such as cell adhesion, microbial
adhesion, apoptosis, inammation, and cancer.1 Two types of carbohydrate chain extensions (type 1 and type 2 chains) are synthesized by b3-GalTs and b4-GalTs, respectively.2 These chains can be
further modied to produce various epitopes, such as Lewis antigens. Specic Lewis antigens can serve as biomarkers for certain
cancers35 and function in cellcell adhesion in the immune system, during inammation and in the metastatic process of cancer
cells.1 In humans, the serum or tissue levels of GalT activities appear to be effectively regulated, and are often found abnormal in
cancer.610 Therefore, the design of biologically applicable GalT
inhibitors is important for the development of both anti-cancer
and anti-inammatory drugs.1,11,12 Previously we have described
acceptor substrate-analog inhibitors13 that inhibited b4-GalT but
not b3-GalT.14 UDP-Gal donor substrate-based inhibitors have also
been developed and shown to inhibit a number of mammalian and
bacterial GalTs.15 In addition, based on the structure of the
Corresponding authors. Tel.: +1 613 533 2643 (W.A.S.); tel.: +1 613 533 2927
(I.B.).
E-mail addresses: szarekw@chem.queensu.ca (W.A. Szarek), brockhau@queensu.ca (I. Brockhausen).
0968-0896/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.034

UDP-Gal binding pocket of blood group B GalT, nonionic mimics

of UDP-Gal have been developed as GalT inhibitors.16
Both monovalent and bivalent imidazolium salts have been
shown to have anti-Plasmodium activity, probably by binding to
specic cell-surface receptors of Plasmodium falciparum merezoites
and thus blocking cell replication.17 Here we report that bivalent
imidazolium chloride and mesylate salts, in which the two imidazolium groups are linked by an aliphatic chain, strongly inhibit
human b3-GalT5. The compounds also inhibited a selected number
of other glycosyltransferases. This information is useful for studying the functional role of glycans and is a basis for the development
of biologically applicable glycosylation modiers.
A series of bis-imidazolium salt compounds was prepared that
contained two terminal positively charged imidazolium groups
and an aliphatic chain linker of different lengths (see Table 1). These
compounds were screened as inhibitors of puried human recombinant soluble b3-GalT5 expressed in insect cells. This enzyme is
abundantly expressed in mammalian tissues and extends the glycan chains of glycoproteins, with a preference for O-glycans.2,14
2. Results and discussion
2.1. Synthesis
The bis-imidazolium compounds in the dichloride form (2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, and 24) were synthesized from the

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Y. Gao et al. / Bioorg. Med. Chem. 21 (2013) 13051311

Table 1
Inhibition of mammalian b4-GalT1 and b3-GalT5 using bis-imidazoles and bis-imidazolium saltsa

Bis-Imidazolium Salts
H3C
Bis-Imidazoles
N

CH2

1
3
5
7
9
11
13
15
17
19
21
23

2HCl

n=4
n=6
n=8
n = 10
n = 11
n = 12
n = 13
n = 14
n = 15
n = 16
n = 18
n = 20

CH2

2
4
6
8
10
12
14
16
18
20
22
24
25
26

CH3

2Cl

n=4
n=6
n=8
n = 10
n = 11
n = 12
n = 13
n = 14
n = 15
n = 16
n = 18
n = 20
n = 20, dimesylate salt
n = 22, dimesylate salt

Compound

% Inhibition of b4-GalT1

% Inhibition of b3-GalT5

120
21
22
23
24
25b
26b

4, 6, 8, 10, 11, 12, 13, 14, 15, 16

18
18
20
20
20
22

<1
<1
<1
<1
54 3
40.7 0.5
78 4

<1
<1
<5
<1
84 1
79.2 0.7
95 2

a
Human recombinant soluble b3-GalT5 was expressed in insect cells and puried. Human recombinant soluble, His-tagged b4-GalT1 protein was expressed in BL21 cells
and puried. The GalT assays contained 0.5 mM GlcNAcb-Bn as the acceptor substrate, 0.5 mM UDP-Gal donor substrate, 0.37 lg b3-GalT5 or 0.34 lg b4-GalT1, and 0.5 mM
bis-imidazole or bis-imidazolium compound. Negative controls had no acceptor and positive (100% activity) controls had no inhibitor.
b
Compound synthesized as the dimesylate (2CH3SO3 ) salt instead of the dichloride salt. Data represent mean values standard deviation of replicate experiments.

corresponding diiodide salts18 by treatment with an excess of

Amberlite IRA-410 (anion-exchange resin in the chloride form) using
1:1 (v/v) acetonitrile/methanol as solvent. The bis-imidazolium compounds in the dimesylate form (25 and 26) were synthesized in three
steps as follows. Treatment of ecosanedioic acid or docosanedioic
acid with lithium aluminium hydride in THF gave the diols 1,20-eicosanediol and 1,22-docosanediol, respectively. Treatment of the
respective diol with methanesulfonyl chloridetriethylamineTHF
gave the alkylene dimesylates 1,20-bis-(methanesulfonyloxy)eicosane and 1,22-bis-(methanesulfonyloxy)docosane. The nal bis-imidazolium dimesylate salts 25 and 26 were synthesized by a
nucleophilic displacement reaction on the corresponding alkylene
dimesylate using 1-methylimidazole.
2.2. Biological evaluation
Of the large series of positively charged imidazolium compounds examined, only compounds having an aliphatic chain
length of 20 or 22 carbons, namely, compounds 24, 25, and 26,
effectively inhibited b3-GalT5 with IC50 values of 0.44 mM,
0.42 mM, and 0.40 mM, respectively (Fig. 1, Panel C). In spite
of structural similarities, positively charged bis-imidazolium
compounds having shorter aliphatic chains did not inhibit b3GalT5. The structurally similar neutral bis-imidazole compounds
showed no inhibition (Table 1), indicating that a specic structure was necessary for the inhibitory activity. To conrm that
the imidazolium moiety of 24 and not the chloride ion was
responsible for the inhibition, we prepared the dimesylate salt
analog 25. Both compounds, 24 and 25, inhibited b3-GalT similarly, conrming that the aliphatic bis-imidazolium cationic
structure was critical for inhibition. The results indicated that
it was the positively charged imidazolium groups, spaced apart
by an aliphatic chain of 20 carbons in length, that had inhibitory
properties.

Inhibition kinetics (Fig. 1) of b3-GalT5 indicated that the inhibition by 24 was not competitive with the acceptor substrate GlcNAcb-Bn. This result was expected since the structure of 24 does
not bear resemblance with the acceptor. However, the inhibition
appeared to have a mixed uncompetitive/noncompetitive nature.
Similar results were obtained with the dimesylate analog 25.
The addition of increasing concentrations of the non-ionic
detergent Triton X-100 completely blocked the inhibitory activity
of 24 on b3-GalT5 at 0.5% (Fig. 2). Similarly, the addition of phosphatidylcholine vesicles at 7.5 mg/mL resulted in complete restoration of b3-GalT5 activity in the presence of inhibitor 24 (Fig. 3).
Since bis-imidazolium salts of the type studied have detergent-like
hydrophilic as well as hydrophobic moieties, it is possible that Triton X-100, as well as phosphatidylcholine, support an unfavourable micelle formation that no longer allows the interaction
between 24 and the enzyme protein. Surface tension experiments
were conducted using compound 20 and 24 in 10% EtOH in water
(results not shown) in an effort to obtain critical micelle concentrations. There was no indication that micelle formation occurred
even at concentrations (1000 lM) double that involved in the reported studies. Interestingly, DNA from calf thymus also blocked
the inhibitory activity of 24 on b3-GalT5 in a dose-related fashion.
This suggested that the positively charged imidazolium moiety
was the inhibiting factor, possibly by binding to negatively charged
groups on the enzyme protein.
To determine the specicity of inhibition by 24, a number of
glycosyltransferases were investigated. Among the b4-GalT family,
human recombinant soluble b4-GalT1, as well as bovine milk b4GalT, were also inhibited by 24 and 25. The IC50 values for 24 were
1.96 mM for b4-GalT1 and 0.73 mM for bovine milk b4-GalT. However, another ubiquitous GalT, human core 1 b3-GalT5, showed no
inhibition. These results suggested that the mechanism of inhibition did not involve the binding and elimination of UDP-Gal as a
donor substrate.

Y. Gao et al. / Bioorg. Med. Chem. 21 (2013) 13051311

1307

Figure 1. Inhibition of human b3-GalT5 by different concentrations of inhibitors. (A) Assay mixtures for GalT contained different concentrations of GlcNAcb-Bn as the
acceptor substrate and inhibitor 24. No 24 (rectangles), apparent KM = 9.2 mM, apparent Vmax = 224.3 lmol/h/mg; 0.5 mM 24 (circles), KM = 16.3 mM, Vmax = 169.2 lmol/h/
mg; 0.75 mM 24 (diamonds), KM = 12.5 mM, Vmax = 62.2 lmol/h/mg; 1.0 mM 24 (triangles), KM = 10.0 mM, Vmax = 38.4 lmol/h/mg. (B) Reciprocal plot. With increasing
inhibitor concentrations, the Vmax values were reduced. (C) Inhibition curves of human b3-GalT5 with different concentrations of compound 24, 25, or 26. GlcNAcb-Bn
(0.5 mM) was used as the acceptor substrate in the assay mixture. For 24 (closed rectangles/dark solid line) IC50 = 0.44 mM; for 25 (opened rectangles/dashed line)
IC50 = 0.42 mM; for 26 (closed circles/light solid line) IC50 = 0.40 mM. The IC50 values were calculated using the Origin 8.0 program.

Figure 2. Inhibition of human b3-GalT5 and b4-GalT1 in the presence of non-ionic

detergent. Human b3-GalT5 and b4-GalT1 were assayed in the presence of
compound 24 (at 0.5 mM) which strongly inhibits the enzymes. The presence of
Triton X-100 in the assay blocked the inhibition in a dose-dependent manner and
restored activity at 0.5%. Closed circles, human b3-GalT5; open circles, human b4GalT1.

Figure 3. Inhibition of human b3-GalT5 and b4-GalT1 in the presence of phosphatidylcholine. Human b3-GalT5 and b4-GalT1 were assayed in the presence of
phosphatidylcholine vesicles, and in the presence of compound 24 (at 0.5 mM)
which strongly inhibits the enzymes. The presence of phosphatidylcholine vesicles
in the assay blocked the inhibition in a dose-dependent manner. Closed diamonds,
human b3-GalT5; open diamonds, human b4-GalT1.

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Y. Gao et al. / Bioorg. Med. Chem. 21 (2013) 13051311

Table 2
Inhibition of selected glycosyltransferases by bis-imidazolium saltsa
Enzyme
Gal-transferases
Human b3-GalT5
Human b4-GalT1
Bovine milk b4GalT1
Human C1GalT
GlcNAc-transferases
Human C2GnT1
Human B3GnT6
Human C2GnT2
Human C2GnT2
Human GnT-I
Human GnT-II
GalNAc-transferase
Bovine
ppGalNAcT1

Acceptor

Control activity (without inhibitor)

(lmol h 1 mg 1)

% Inhibition by
compound 24

% Inhibition by
compound 25

% Inhibition by
compound 26

GlcNAcb-Bn
GlcNAcb-Bn
GlcNAcb-Bn

6.7 0.3
1.3 0.3
9.1 0.9

84 1
41 2
54 3

79.2 0.7
35 5
40.7 0.5

95 2
53 2
78 4

GalNAca-Bn

0.042 0.003

<1

<1

<1

Galb3GalNAca-Bn
GalNAca-Bn
Galb3GalNAca-Bn
GlcNAcb3GalNAcapnp
0.1 mM Man3octyl
0.1 mM GnMan3octyl

0.034 0.003
0.190 0.004
0.077 0.008
0.074 0.001

100 1
94.4 0.6
<1
<1

100 1
80 2
<1
<1

100 1
78 4
<1
<1

0.012 0.001

<1

<1

<1

0.015 0.002

<1

<1

<1

AQPTPPP

2.6 0.4

91 5

96.8 0.6

100 1

a
Mammalian glycosyltransferases were assayed in the absence and presence of 0.5 mM bis-imidazolium compound 24, 25, or 26. Human b4-GalT1 was expressed in
bacteria and puried, bovine milk b4-GalT1 was from Sigma, and bovine ppGalNAcT was highly puried. All other glycosyltransferases were produced as soluble enzymes in
insect cells. All assays contained 10% ethanol. Control assays lacked the acceptor substrate or inhibitor. Man3-octyl, Mana1-6(Mana1-3)Manb-octyl; GnMan3-octyl, Mana16(GlcNAcb1-2Mana1-3)Manb-octyl; pnp, p-nitrophenyl; ppGalNAcT, polypeptide GalNAc-transferase. Data represent mean values standard deviation of replicate
experiments.

Polypeptide GalNAc-transferases initiate the synthesis of Oglycan structures of glycoproteins and an inhibition could be very
useful to study the role of O-glycans. Polypeptide GalNAc-transferase 1 is an abundant member of a large family of these enzymes.
The activity of puried bovine polypeptide GalNAc-transferase 1,
using AQPTPPP as the acceptor peptide,19 was strongly inhibited
by 24 (Table 2). A previously described inhibitor of this activity
was nucleotide sugar-based and also contained an aliphatic
chain.20 In comparison, another O-glycan synthesis inhibitor,
GalNAca-Bn, is a competitive substrate and only blocks the extension of O-glycans after GalNAc has been added to Ser/Thr. Several
recombinant soluble GlcNAc-transferases expressed in Sf9 insect
cells were also tested with 24. GlcNAc-transferases involved in
the synthesis of O-glycans with core 2 and core 3 structures19,21
(C2GnT1 and puried C3GnT6, respectively) showed almost complete inhibition by 24, and similarly by the dimesylate analog 25
(Table 2). In contrast, the GlcNAc-transferase involved in the synthesis of core 2 and core 4 (C2GnT2) which is related to C2GnT1,
as well as two unrelated GlcNAc-transferases involved in N-glycan
synthesis21 (GnT-I and GnT-II) were not inhibited. C2GnT2 activity
and inhibition was examined with the same acceptor as for
C2GnT1, in addition to its specic GlcNAcb1-3GalNAca-p-nitrophenyl acceptor, but no inhibition was observed (Table 2). These
results indicated that the inhibition by 24 was enzyme-selective,
and appeared to be independent of the structure of the nucleotide
sugar or acceptor substrate.
3. Conclusions
In summary, we have shown that a number of glycosyltransferases that utilize various nucleotide sugars and different acceptor
substrates were found to be inhibited by bis-imidazolium salts
24 and 25, while other enzymes that use the same donor substrates
and the same or different acceptor substrates were not inhibited.
We conclude that it is not the interaction of 24 with the nucleotide
sugar donors or with specic acceptor substrates that caused the
inhibition but rather an effect on selected enzyme proteins. Bisimidazolium salts may bind to negatively charged as well as hydrophobic amino acids that are critical for protein structure, substrate

binding, or catalysis. This effect appears to be based on a combination of a specic spacing between positively charged imidazolium
groups mediated by a hydrophobic chain of dened length, and
thus depends on the physical properties of the inhibitor. These
bis-imidazolium salts, due to their detergent-like properties, may
be incorporated into cell membranes, but it remains to be shown
if these novel glycosyltransferase inhibitors cross the membrane,
are delivered to the Golgi apparatus, and inhibit glycosyltransferases in vivo.
4. Experimental
4.1. General
Preparatory-scale thin-layer chromatography was performed
on SiliaPlate TLC glass-backed silica gel plates (Silicycle, 2000 lm
thickness), and the compounds were visualized by UV illumination
(254 nm). Melting points were measured on a Mel-Temp II apparatus and are uncorrected. 1H and 13C NMR spectra were recorded on
a Bruker Avance 400 spectrometer in DMSO-d6, CD3OD, or CDCl3.
The chemical shifts are reported in d (ppm) relative to tetramethylsilane.22 The compounds synthesized were deemed >95% pure
by 1H NMR analysis. High-resolution ESI mass spectra were recorded on an Applied Biosystems/MDS Sciex QSTAR XL mass spectrometer with an Agilent HP1100 Cap-LC system. Samples were
run in 50% aqueous MeOH at a ow rate of 6 lL/min. High-resolution EI mass spectra were recorded on a Waters/Micromass GC-TOF
instrument.
4.2. Materials
The bis-imidazoles in the dihydrochloride form (1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, and 23) were prepared according to
the published procedure.18 1,4-Bis-(3-methyl-1H-imidazolium1-yl)butane diiodide, 1,6-bis-(3-methyl-1H-imidazolium-1-yl)
hexane diiodide, 1,8-bis-(3-methyl-1H-imidazolium-1-yl)octane
diiodide, 1,10-bis-(3-methyl-1H-imidazolium-1-yl)decane diiodide, 1,11-bis-(3-methyl-1H-imidazolium-1-yl)undecane diiodide,
1,12-bis-(3-methyl-1H-imidazolium-1-yl)dodecane diiodide, 1,

Y. Gao et al. / Bioorg. Med. Chem. 21 (2013) 13051311

13-bis-(3-methyl-1H-imidazolium-1-yl)tridecane
diiodide,
1,
14-bis-(3-methyl-1H-imidazolium-1-yl)tetradecane diiodide, 1,15bis-(3-methyl-1H-imidazolium-1-yl)pentadecane diiodide, 1,
16-bis-(3-methyl-1H-imidazolium-1-yl)hexadecane diiodide, 1,
18-bis-(3-methyl-1H-imidazolium-1-yl)octadecane diiodide, and
1,20-bis-(3-methyl-1H-imidazolium-1-yl)eicosane diiodide were
prepared according to the published procedure.18 Eicosanedioic
acid was obtained from Alfa Aesar. All of the other chemicals were
obtained from SigmaAldrich.
4.3. Representative procedure for the formation of bisimidazolium compounds (dichloride salt form)
4.3.1. 1,4-Bis-(3-methyl-1H-imidazolium-1-yl)butane dichloride
(2)
A sample of 1,4-bis-(3-methyl-1H-imidazolium-1-yl)butane
diiodide (318 mg, 0.67 mmol, 1 equiv) was dissolved in methanol
(0.5 mL) and loaded onto a preparatory-scale silica gel TLC plate
(20 cm  10 cm  2 mm). The plate was dried with warm air, then
eluted with acetonitrile. The section corresponding to Rf = 0.32
0.06 was excised and ground into a ne powder. To the powder
was added 1:1 acetonitrile/methanol (40 mL) and Amberlite IRA410 anion-exchange resin in the chloride form (2 g) and the mixture stirred for 30 min. The solids were removed by ltration and
washed with 1:1 acetonitrile/methanol (2  40 mL). Treatment of
the ltrate with Amberlite IRA-410 followed by stirring and ltration as before was repeated two more times; the nal ltrate was
concentrated, the residue treated with diethyl ether (10 mL), and
then the mixture was concentrated again. High-vacuum drying
gave the product (102 mg, 0.35 mmol, 52%) as a white solid; mp
164165 C; 1H NMR (400 MHz, DMSO-d6): d 1.731.86 (m, 4H),
3.86 (s, 6H), 4.204.31 (m, 4H), 7.73 (s, 2H), 7.82 (s, 2H), 9.35 (s,
2H); 13C NMR (100 MHz, DMSO-d6): d 25.9, 35.7, 47.7, 122.3,
123.6, 136.9; HRMS (ESI) [M Cl]+ Calcd for C12H20ClN4:
255.1376. Found: 255.1372.
4.4. Characterization of the bis-imidazolium compounds
(dichloride salt form) synthesized following the representative
procedure above for compound 2
4.4.1. 1,6-Bis-(3-methyl-1H-imidazolium-1-yl)hexane
dichloride (4)
This compound was prepared in 48% yield from 1,6-bis-(3methyl-1H-imidazolium-1-yl)hexane diiodide and was obtained
as a white solid; mp 105106 C; 1H NMR (400 MHz, DMSO-d6):
d 1.201.32 (m, 4H), 1.721.84 (m, 4H), 3.87 (s, 6H), 4.19 (t,
J = 7.0 Hz, 4H), 7.76 (s, 2H), 7.88 (s, 2H), 9.49 (s, 2H); 13C NMR
(100 MHz, DMSO-d6): d 24.7, 29.1, 35.7, 48.5, 122.3, 123.6, 136.7;
HRMS (ESI) [M Cl]+ Calcd for C14H24ClN4: 283.1689. Found:
283.1686.
4.4.2. 1,8-Bis-(3-methyl-1H-imidazolium-1-yl)octane dichloride
(6)
This compound was prepared in 58% yield from 1,8-bis-(3methyl-1H-imidazolium-1-yl)octane diiodide and was obtained
as a hygroscopic white sticky solid; mp 80 C; 1H NMR
(400 MHz, DMSO-d6): d 1.171.33 (m, 8H), 1.711.83 (m, 4H),
3.87 (s, 6H), 4.17 (t, J = 7.2 Hz, 4H), 7.74 (s, 2H), 7.82 (s, 2H), 9.38
(s, 2H); 13C NMR (100 MHz, DMSO-d6): d 25.3, 28.1, 29.3, 35.7,
48.6, 122.2, 123.5, 136.6; HRMS (ESI) [M Cl]+ Calcd for
C16H28ClN4: 311.2002. Found: 311.2005.
4.4.3. 1,10-Bis-(3-methyl-1H-imidazolium-1-yl)decane
dichloride (8)
This compound was prepared in 37% yield from 1,10-bis-(3methyl-1H-imidazolium-1-yl)decane diiodide and was obtained

1309

as a clear oil; 1H NMR (400 MHz, DMSO-d6): d 1.151.35 (m,

12H), 1.751.85 (m, 4H), 3.87 (s, 6H), 4.17 (t, J = 7.2 Hz, 4H), 7.76
(s, 2H), 7.84 (s, 2H), 9.43 (s, 2H); 13C NMR (100 MHz, DMSO-d6):
d 25.5, 28.3, 28.7, 29.4, 35.7, 48.7, 122.2, 123.5, 136.7; HRMS
(ESI) [M Cl]+ Calcd for C18H32ClN4: 339.2315. Found: 339.2327.
4.4.4. 1,11-Bis-(3-methyl-1H-imidazolium-1-yl)undecane
dichloride (10)
This compound was prepared in 64% yield from 1,11-bis-(3methyl-1H-imidazolium-1-yl)undecane diiodide and was obtained
as a clear oil; 1H NMR (400 MHz, DMSO-d6): d 1.181.32 (m, 14H),
1.711.83 (m, 4H), 3.86 (s, 6H), 4.17 (t, J = 7.2 Hz, 4H), 7.75 (s, 2H),
7.83 (s, 2H), 9.38 (s, 2H); 13C NMR (100 MHz, DMSO-d6): d 25.5,
28.3, 28.7, 28.8, 29.4, 35.7, 48.7, 122.2, 123.5, 136.6; HRMS (ESI)
[M Cl]+ Calcd for C19H34ClN4: 353.2472. Found: 353.2461.
4.4.5. 1,12-Bis-(3-methyl-1H-imidazolium-1-yl)dodecane
dichloride (12)
This compound was prepared in 57% yield from 1,12-bis-(3methyl-1H-imidazolium-1-yl)dodecane diiodide and was obtained
as a clear oil; 1H NMR (400 MHz, DMSO-d6): d 1.161.33 (m, 16H),
1.711.82 (m, 4H), 3.86 (s, 6H), 4.17 (t, J = 7.0 Hz, 4H), 7.75 (s, 2H),
7.82 (s, 2H), 9.36 (s, 2H); 13C NMR (100 MHz, DMSO-d6): d 25.5,
28.4, 28.8, 28.9, 29.4, 35.7, 48.7, 122.3, 123.6, 136.6; HRMS (ESI)
[M Cl]+ Calcd for C20H36ClN4: 367.2628. Found: 367.2633.
4.4.6. 1,13-Bis-(3-methyl-1H-imidazolium-1-yl)tridecane
dichloride (14)
This compound was prepared in 50% yield from 1,13-bis-(3methyl-1H-imidazolium-1-yl)tridecane diiodide and was obtained
as a clear oil; 1H NMR (400 MHz, DMSO-d6): d 1.121.34 (m, 18H),
1.711.83 (m, 4H), 3.86 (s, 6H), 4.17 (t, J = 7.0 Hz, 4H), 7.75 (s, 2H),
7.82 (s, 2H), 9.37 (s, 2H); 13C NMR (100 MHz, DMSO-d6): d 25.5,
28.4, 28.8, 28.9 (2C), 29.4, 35.7, 48.7, 122.2, 123.5, 136.6; HRMS
(ESI) [M Cl]+ Calcd for C21H38ClN4: 381.2785. Found: 381.2781.
4.4.7. 1,14-Bis-(3-methyl-1H-imidazolium-1-yl)tetradecane
dichloride (16)
This compound was prepared in 39% yield from 1,14-bis-(3methyl-1H-imidazolium-1-yl)tetradecane diiodide and was obtained as a hygroscopic white sticky solid; 1H NMR (400 MHz,
DMSO-d6): d 1.151.32 (m, 20H), 1.721.82 (m, 4H), 3.86 (s, 6H),
4.16 (t, J = 7.2 Hz, 4H), 7.74 (s, 2H), 7.81 (s, 2H), 9.35 (s, 2H); 13C
NMR (100 MHz, DMSO-d6): d 25.5, 28.4, 28.8, 28.9, 29.0, 29.4,
35.7, 48.7, 122.2, 123.5, 136.6; HRMS (ESI) [M Cl]+ Calcd for
C22H40ClN4: 395.2941. Found: 395.2951.
4.4.8. 1,15-Bis-(3-methyl-1H-imidazolium-1-yl)pentadecane
dichloride (18)
This compound was prepared in 35% yield from 1,15-bis-(3methyl-1H-imidazolium-1-yl)pentadecane diiodide and was obtained as a hygroscopic white sticky solid; 1H NMR (400 MHz,
DMSO-d6): d 1.151.32 (m, 22H), 1.711.83 (m, 4H), 3.86 (s, 6H),
4.16 (t, J = 7.0 Hz, 4H), 7.74 (s, 2H), 7.81 (s, 2H), 9.32 (s, 2H); 13C
NMR (100 MHz, DMSO-d6): d 25.5, 28.4, 28.8, 28.9, 29.0 (2C),
29.4, 35.7, 48.7, 122.2, 123.6, 136.6; HRMS (ESI) [M Cl]+ Calcd
for C23H42ClN4: 409.3098. Found: 409.3095.
4.4.9. 1,16-Bis-(3-methyl-1H-imidazolium-1-yl)hexadecane
dichloride (20)
This compound was prepared in 51% yield from 1,16-bis-(3methyl-1H-imidazolium-1-yl)hexadecane diiodide and was obtained as a white sticky solid; 1H NMR (400 MHz, DMSO-d6): d
1.121.33 (m, 24H), 1.701.83 (m, 4H), 3.85 (s, 6H), 4.15 (t,
J = 7.0 Hz, 4H), 7.72 (s, 2H), 7.79 (s, 2H), 9.22 (s, 2H); 13C NMR
(100 MHz, DMSO-d6): d 25.5, 28.4, 28.9, 29.0, 29.05, 29.08, 29.4,

1310

Y. Gao et al. / Bioorg. Med. Chem. 21 (2013) 13051311

35.7, 48.7, 122.3, 123.6, 136.7; HRMS (ESI) [M Cl]+ Calcd for
C24H44ClN4: 423.3254. Found: 423.3248.
4.4.10. 1,18-Bis-(3-methyl-1H-imidazolium-1-yl)octadecane
dichloride (22)
This compound was prepared in 51% yield from 1,18-bis(3-methyl-1H-imidazolium-1-yl)octadecane diiodide and was
obtained as a white solid; mp 9295 C; 1H NMR (400 MHz,
DMSO-d6): d 1.051.35 (m, 28H), 1.681.84 (m, 4H), 3.86 (s, 6H),
4.084.23 (m, 4H), 7.75 (s, 2H), 7.82 (s, 2H), 9.34 (s, 2H); 13C
NMR (100 MHz, DMSO-d6): d 25.5, 28.3, 28.8, 28.91, 28.97, 29.0
(2C), 29.4, 35.7, 48.7, 122.2, 123.5, 136.6; HRMS (ESI) [M Cl]+
Calcd for C26H48ClN4: 451.3568. Found: 451.3554.
4.4.11. 1,20-Bis-(3-methyl-1H-imidazolium-1-yl)eicosane
dichloride (24)
This compound was prepared in 52% yield from 1,20-bis-(3methyl-1H-imidazolium-1-yl)eicosane diiodide and was obtained
as a white solid; mp 103108 C; 1H NMR (400 MHz, DMSO-d6):
d 1.181.32 (m, 32H), 1.721.82 (m, 4H), 3.85 (s, 6H), 4.15 (t,
J = 7.2 Hz, 4H), 7.71 (s, 2H), 7.78 (s, 2H), 9.19 (s, 2H); 13C NMR
(100 MHz, DMSO-d6): d 25.5, 28.4, 28.8, 28.9, 29.0, 29.1 (3C),
29.3, 35.7, 48.7, 122.2, 123.6, 136.5; HRMS (ESI) [M Cl]+ Calcd
for C28H52ClN4: 479.3875. Found: 479.3876.
4.5. Other synthetic procedures
4.5.1. 1,20-Eicosanediol
Under an atmosphere of nitrogen, eicosanedioic acid
(890 mg, 2.60 mmol, 1 equiv) was dissolved in freshly distilled
THF (60 mL). The solution was cooled to 0 C and to this was
added in small portions lithium aluminum hydride (370 mg,
9.75 mmol, 3.75 equiv). The mixture was warmed to rt and then
heated at reux temperature for 12 h. The mixture was cooled
to 0 C, quenched with wet THF and then water (5 mL), stirred
at rt for 1 h, then concentrated and dried under high vacuum.
The remaining residue was extracted with boiling methanol,
then boiling ethyl acetate, and the combined organic extracts
were ltered, and the ltrate concentrated. High-vacuum drying
gave a white solid that was washed with a solution of 1% w/w
sodium carbonate in water (100 mL). The solid was collected by
ltration and washed with water (100 mL). High-vacuum drying
gave the product (416 mg, 1.32 mmol, 51%) as a white solid;
mp >260 C; 1H NMR (400 MHz, CD3OD): d 1.181.38 (m,
32H), 1.461.57 (m, 4H), 3.54 (t, J = 6.6 Hz, 4H); 13C NMR
(100 MHz, DMSO-d6 + CDCl3): d 25.4, 28.8, 28.9 (5C), 29.0,
32.4, 60.9.
4.5.2. 1,20-Bis-(methanesulfonyloxy)eicosane
Under an atmosphere of nitrogen, 1,20-eicosanediol (403 mg,
1.28 mmol, 1 equiv) was dissolved in warm THF (10 mL) and
the solution was then cooled to rt. To this solution was added triethylamine (0.44 mL, 320 mg, 3.16 mmol, 2.5 equiv), the mixture
cooled to 0 C, and methanesulfonyl chloride (0.22 mL, 326 mg,
2.85 mmol, 2.2 equiv) was added. The mixture was stirred at rt
for 2 h, methylene chloride (5 mL) added, and the mixture heated
at reux temperature for 1 min. The mixture was then stirred at
rt overnight, concentrated and dried under high vacuum. The
resulting white solid was washed with water (250 mL) and the
solid collected by ltration. High-vacuum drying gave the product
(533 mg, 1.13 mmol, 88%) as a white solid; mp 9495 C; 1H NMR
(400 MHz, DMSO-d6): d 1.101.40 (m, 32H), 1.571.71 (m, 4H),
3.15 (s, 6H), 4.17 (t, J = 6.2 Hz, 4H); 13C NMR (100 MHz, DMSOd6): d 24.6, 28.2, 28.3, 28.5, 28.6, 28.7 (4C), 36.5, 70.1; HRMS
(ESI) [M+Na]+ Calcd for C22H46O6S2Na: 493.2634. Found:
493.2641.

4.5.3. 1,20-Bis-(3-methyl-1H-imidazolium-1-yl)eicosane
dimesylate (25)
Under an atmosphere of nitrogen, 1,20-bis-(methanesulfonyloxy)eicosane (100 mg, 0.21 mmol, 1 equiv) was mixed with 1methylimidazole (0.17 mL, 175 mg, 2.13 mmol, 10 equiv) and the
mixture was heated at 8090 C for 1 h; acetone (20 mL) was then
added and the mixture was stirred at reux temperature for 24 h,
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. High-vacuum drying gave the product (126 mg,
0.20 mmol, 95%) as a white waxy oily solid; mp 55 C; 1H NMR
(300 MHz, DMSO-d6): d 1.151.40 (m, 32H), 1.651.85 (m, 4H),
2.31 (s, 6H), 3.85 (s, 6H), 4.15 (t, J = 9.2 Hz, 4H), 7.71 (s, 2H), 7.78
(s, 2H), 9.14 (s, 2H); 13C NMR (100 MHz, DMSO-d6): d 25.5, 28.4,
28.8, 28.9, 29.00, 29.04 (3C), 29.4, 35.7, peak under solvent, 48.7,
122.2, 123.6, 136.6; HRMS (ESI) [M-O3SCH3]+ Calcd for
C29H55N4O3S: 539.3994. Found: 539.3991.
4.5.4. 1,22-Docosanediol
Under an atmosphere of nitrogen, docosanedioic acid (1.04 g,
2.81 mmol, 1 equiv) was dissolved in freshly distilled THF
(55 mL). The solution was cooled to 0 C and to this solution was
added in small portions lithium aluminum hydride (650 mg,
17.13 mmol, 24.4 equiv). The mixture was warmed to rt and then
heated at reux temperature for 18 h. The mixture was cooled to
0 C, quenched with wet THF and then water (10 mL), stirred at
rt for 1 h, then diluted with water (50 mL). The mixture was cooled
to 0 C and the resulting white solid precipitate was collected by
ltration and washed with water (3  50 mL). The solid was dried
under high vacuum and extracted with boiling ethyl acetate
(300 mL), then boiling methanol (150 mL). The combined organic
extracts were ltered, and the ltrate concentrated. High-vacuum
drying gave the product (350 mg, 1.02 mmol, 36%) as a white solid;
mp 9698 C; 1H NMR (400 MHz, 1:1 CD3OD/CDCl3): d 1.181.37
(m, 36H), 1.461.56 (m, 4H), 3.53 (t, J = 6.8 Hz, 4H); 13C NMR
(100 MHz, 1:1 CD3ODCDCl3): d 26.3, 30.0, 30.1, 30.2 (6C), 33.1,
62.8; HRMS (EI) [M]+ Calcd for C22H46O2: 342.3498. Found:
342.3483.
4.5.5. 1,22-Bis-(methanesulfonyloxy)docosane
Under an atmosphere of nitrogen, 1,22-docosanediol (340 mg,
0.99 mmol, 1 equiv) was dissolved in warm THF (20 mL) and the
solution was then cooled to rt. To this solution was added triethylamine (0.34 mL, 251 mg, 2.48 mmol, 2.5 equiv) and then methanesulfonyl chloride (0.17 mL, 250 mg, 2.18 mmol, 2.2 equiv). The
mixture was stirred at rt for 19 h, methylene chloride (10 mL)
added, and the mixture heated at reux temperature for 1 min.
The mixture was then stirred at rt for 2 h, concentrated and dried
under high vacuum. The resulting white solid was washed with
water (250 mL) and the solid collected by ltration. High-vacuum
drying gave the product (397 mg, 0.80 mmol, 81%) as a white solid;
mp 9596 C; 1H NMR (400 MHz, 1:1 DMSO-d6CDCl3): d 1.13
1.40 (m, 36H), 1.611.71 (m, 4H), 3.02 (s, 6H), 4.14 (t, J = 6.4 Hz,
4H); 13C NMR (100 MHz, 1:1 DMSO-d6CDCl3): d 24.7, 28.3, 28.4,
28.7, 28.8, 28.9 (5C), 36.5, 69.9; HRMS (ESI) [M+Na]+ Calcd for
C24H50O6S2Na: 521.2947. Found: 521.2941.
4.5.6. 1,22-Bis-(3-methyl-1H-imidazolium-1-yl)docosane
dimesylate (26)
Under an atmosphere of nitrogen, 1,22-bis-(methanesulfonyloxy)docosane (100 mg, 0.20 mmol, 1 equiv) was mixed with 1methylimidazole (0.16 mL, 165 mg, 2.01 mmol, 10 equiv) and the
mixture was heated at 8090 C for 1 h; acetone (15 mL) was then
added and the mixture was stirred at reux temperature for 12 h,

Y. Gao et al. / Bioorg. Med. Chem. 21 (2013) 13051311

then 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
(40 mL). This dissolution/concentration/washing procedure was
repeated two more times. High-vacuum drying gave the product
(112 mg, 0.17 mmol, 85%) as a grey oily solid; mp 70 C; 1H
NMR (400 MHz, DMSO-d6): d 1.121.42 (m, 36H), 1.681.85 (m,
4H), 2.29 (s, 6H), 3.84 (s, 6H), 4.14 (t, J = 7.2 Hz, 4H), 7.70 (s, 2H),
7.76 (s, 2H), 9.11 (s, 2H); 13C NMR (100 MHz, DMSO-d6): d 25.5,
28.3, 28.8, 28.9, 29.0, 29.1 (4C), 29.3, 35.7, peak under solvent,
48.7, 122.2, 123.6, 136.5; HRMS (ESI) [M O3SCH3]+ Calcd for
C31H59N4O3S: 567.4307. Found: 567.4305.
4.6. Assay conditions
Insect cell supernatants containing soluble human b3-GalT5
were used for the purication as described previously.14 Enzyme
solutions were stored at 80 C in the presence of 20% glycerol.
BL21 bacteria expressing human recombinant b4-GalT1 (mutant
M340H) were obtained from Pradman K. Qasba, U.S. National Institutes of Health. Bacteria were grown and protein production induced as described.23 Enzyme was puried from inclusion bodies
by the denaturing and renaturing process. Bovine milk b4-GalT
was from Sigma. Insect cell supernatant containing human recombinant soluble b3-GalT5 was kindly donated by Henrik Clausen,
University of Copenhagen, Denmark. b3-GalT5 was puried by QSepharose anion-exchange chromatography and further puried
by S-Sepharose cation-exchange chromatography. The following
glycosyltransferases were produced in insect cells as soluble enzymes: Human C1GalT was donated by Zongchong Ju, Atlanta,
GA, USA. Human core 3-synthase (B3GnT6) was donated by Don
Jarvis, Laramie, WY, USA, and puried by Nickel-NTA Sepharose
afnity chromatography. Human C2GnT that acts on Galb1-3GalNAca-Bn was obtained as described.24 Human C2GnT2 that acts
on both Galb1-3GalNAca-Bn and GlcNAcb1-3GalNAca-p-nitrophenyl (Toronto Research Chemicals) was obtained from Don Jarvis, University of Wyoming, USA and was puried by Nickel-NTA
Sepharose afnity chromatography. Puried bovine polypeptideGalNAc-transferase14 was obtained from A. Elhammer, Kalamazoo,
MI, USA. Human GnT-I and GnT-II were obtained from Harry
Schachter, Toronto, Ontario, Canada.
Glycosyltransferases
were
assayed
as
previously
described.1214,19,24 Briey, mammalian b3/4-GalTs were assayed
in a total volume of 40 lL containing different concentrations of
acceptor substrate, 0.251 lg recombinant enzyme, 0.81.6 mM
UDP-[3H]Gal (2266 cpm/nmol), 0.125 M MES buffer (N-morpholino-ethanesulfonic acid) (pH 7), 12.5 mM MnCl2, 10 mM AMP,
and 5 mM c-galactonolactone. The standard GalT assays contained
0.5 mM GlcNAcb-Bn as the acceptor substrate. Inhibition assays
contained different concentrations of bis-imidazolium salt compound and 10% ethanol. C1GalT was assayed with 0.5 mM
GalNAca-Bn as the acceptor substrate, 0.8 mM UDP-[3H]Gal as donor substrate, and 0.5 mM bis-imidazolium salt compound.
C2GnT1 and C2GnT2 assay mixtures contained 0.125 M MES buffer
(pH 7), 10 mM AMP, 0.125 M GlcNAc, 0.5 mM Galb1-3GalNAca-Bn
or GlcNAcb1-3GalNAca-p-nitrophenyl, and 1 mM UDP-[3H]GlcNAc
(5800 cpm/nmol). C3GnT6 was assayed similarly, using 0.5 mM
GalNAca-Bn acceptor in the presence of 12.5 mM MnCl2. The assay
mixtures for GnT I and II contained 1 mM UDP-[3H] GlcNAc
(5800 cpm/nmol), 0.1 mM Mana6(Mana3)Manb-octyl (Toronto

1311

Research
Chemicals)
for
GnT-I,
or
Mana6(GlcNAcb2
Mana3)Manb-octyl (for GnT-II),14 0.125 M MES (pH 7), 0.125 M
GlcNAc, 10 mM AMP, and 12.5 mM MnCl2. Polypeptide GalNAcT
activities were determined in a total volume of 40 lL containing
0.125 M MES (pH 7), 0.125% Triton X-100, 10 mM AMP, 12.5 mM
MnCl2, 1.2 mM UDP-[3H]GalNAc (1700 cpm/nmol), and 0.5 mM
AQPTPPP acceptor substrate.19 Control assays lacked the acceptor
substrate or inhibitors. Inhibition assays were performed with different concentrations of bis-imidazolium salt compound and 10%
ethanol. Reaction mixtures were incubated for 1 h at 37 C, and
products isolated using 0.4 mL AG1  8 (100200 mesh) anionexchange resin (chloride form); elution was performed using water
(see Ref. 14 for more details). Radioactivity of the eluate was
determined by scintillation counting. KM and Vmax values were
calculated with the Origin 8.0 program. Phosphatidylcholine vesicles were freshly prepared by sonication.
Acknowledgments
We thank John Schutzbach, Queens University, for valuable discussions and advice. I.B. acknowledges the nancial support of the
Natural Sciences and Engineering Council of Canada and the Prostate Cancer Fight Foundation (Motorcycle Ride for Dad). I.B. and
W.A.S are supported by a grant from the Canadian Institutes of
Health Research. Y.G. is supported by an Ontario graduate student
fellowship award.
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18.
19.
20.
21.
22.
23.
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