Synthesis of 1,2,3-triazole linked galactopyranosides and evaluation of cholera toxin inhibition

Article abstract of DOI:10.1039/c1ob06317k

We report the synthesis of a series of bivalent 1,2,3-triazole linked galactopyranosides as potential inhibitors of cholera toxin (CT). The inhibitory activity of the bivalent series was examined (ELISA) and the series showed low inhibitory activity (millimolar IC₅₀s). Conversely, the monomeric galactotriazole analogues were strong inhibitors of cholera toxin (IC ₅₀ = 71-75 μM). The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1ob06317k

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PAPER
Synthesis of 1,2,3-triazole linked galactopyranosides and evaluation of
cholera toxin inhibition†
David J. Leaver,a Raymond M. Dawson,c Jonathan M. White,d Anastasios Polyzos*b and Andrew B. Hughes*a
Received 3rd August 2011, Accepted 22nd September 2011
DOI: 10.1039/c1ob06317k
We report the synthesis of a series of bivalent 1,2,3-triazole linked galactopyranosides as potential
inhibitors of cholera toxin (CT). The inhibitory activity of the bivalent series was examined (ELISA)
and the series showed low inhibitory activity (millimolar IC50s). Conversely, the monomeric
galactotriazole analogues were strong inhibitors of cholera toxin (IC50 = 71–75 mM).
This is largely attributed to the mild conditions under which the
Introduction
cycloaddition usually proceeds, strong functional group tolerance,
Glycoconjugates are important as molecular probes for the in-
stereospecificity, high yield, ease of isolation and purification, and
biocompatibility.4,9 The versatility of the transformation has been
demonstrated in the synthesis of glycoconjugates, where anomeric
glycotriazoles have been readily prepared from azido sugars in high
yields and excellent anomeric purity.10–14 The choice of ligation
ameliorates often difficult conditions associated with glycosidic
bond-forming reactions15 and the cycloaddition proceeds readily
with O-protected sugars as well as with those containing free
hydroxyl groups.15–23
It was reasoned that the use of galactotriazoles facilitates the
rapid and efficient synthesis of galactoconjugates in a regiospecific
and stereoselective fashion. We sought to devise the synthesis
of C- and N-linked galactotriazoles to enable access to suitable
galactoconjugates that inhibit cholera toxin (CT). CT is the
enterotoxin secreted by Vibrio cholerae and is the agent responsible
for the symptoms presented by cholera-infected patients.
terrogation of biological processes, biomolecular communication
events and related processes. Glycoconjugates are rich in struc-
tural and functional diversity and involve protein–carbohydrate
interactions that play important roles in biology (i.e. immune
function and fertilization) and various disease states.1–3 There are
early events in the infectious cycles of many bacteria, viruses,
mycoplasma and parasites that involve carbohydrate-mediated
recognition of the host by the pathogen.1 The ability to control
or prevent these events from occurring via the use of small or
multivalent ligands is highly desirable.
In recent years, the CuI-catalysed 1,3-dipolar cycloaddition
of terminal azides and alkynes (the Huisgen reaction), which is
commonly abbreviated as CuAAC (CuI-catalysed azide-terminal
alkyne cycloaddition), has emerged in glycoconjugation chemistry
for linking carbohydrate moieties to desired scaffolds.4,5 CuAAC
yields 1,4-disubstituted-1,2,3-triazoles in a regiospecific and usu-
ally efficient fashion and was independently discovered by the
laboratories of Sharpless and Meldal.6–8 Since the designation
of the CuAAC transformation by Sharpless as a ‘click’ chemical
reaction, the 1,4-disubstituted-1,2,3-triazole ring has emerged as
an important heterocycle in medicinal and material chemistry.7
Cholera is a severe diarrheal disease associated with infection by
the gram-negative bacterium V. cholerae and is often correlated to
high infective and mortality rates. Since 1961 there have been ap-
proximately 5 million recorded cases of cholera infection, with over
250 000 deaths.24 CT is of the AB5 class of toxins,25 comprised of a
catalytic A-subunit and multiple B-subunits that bind specifically
to the ganglioside GM1; a complex glycolipid found in the epithelial
aDepartment of Chemistry, La Trobe University, Victoria, 3086, Australia.
E-mail: tahughes@optusnet.com.au; Fax: +613 9479 1226; Tel: +613 9479
1353
cytoplasmic membrane.24,26 A large association constant (ka
=
1.05 ¥ 106 M-1) for the oligosaccharide-GM1:B pentamer complex26
permits the receptor mediated endocytosis of the toxic A subunit.
Cellular internalization of the A-subunit activates adenyl cyclase,
leading to the disruption of the normal sodium influx, thereby
effecting an uncontrolled loss of intracellular electrolytes into the
intestinal lumen. This action leads to the establishment of severe
diarrheal symptoms and onset of dehydration.24 Crystallographic
studies have led to the extensive characterisation of the recognition
of GM1 by the pentameric CTB-subunits.27,28 These studies reveal
that the terminal galactose of the GM1 oligosaccharide binds to
a shallow binding epitope on each B subunit.26,29 The intrinsic
dissociation constant for this terminal galactose in the CTB:GM1
bCSIRO Materials Science and Engineering, Clayton South, Victoria, 3169,
Australia. E-mail: tash.polyzos@csiro.au; Fax: +613 9545 2446; Tel: +613
9545 8172
cHuman Protection and Performance Division, DSTO Melbourne, 506
Lorimer Street, Fishermans Bend, Victoria, 3207, Australia
dBio21 Institute, School of Chemistry, University of Melbourne, Parkville,
Victoria, 3010, Australia
† Electronic supplementary information (ESI) available: Experimental
procedures for compounds 3, 4, 6, 8, 10, 12–18, 20, 21, 24, 27–30, 36, 37,
39–42 and bioassay procedures. 1H and 13C NMR spectra for compounds
7–12, 19–23, 25, 26, 31–37, 41 and 42. CCDC reference numbers 833575
and 833576. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c1ob06317k
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complex is around 50 mM.30 Thus the recognition of CT by GM1
represents a target for therapeutic intervention and an opportunity
to design antagonists as prophylactic and therapeutic drugs
against the symptoms of infection by V. cholerae.
Classical approaches to CT anti-infective therapies have relied
on small molecule inhibitors that reversibly bind to the B
subunit (forming an association complex), which disrupts host
cell surface recognition. Such molecules are generally deriva-
tives of the entire GM1 oligosaccharide unit31 or mimics of the
GM2, or GM1 derivatised headgroups that exhibited substantial
gains in affinity in comparison to the corresponding monovalent
ligands.43–47 Some of the most potent bivalent CT inhibitors possess
improved affinity for CT over that of GM1, which illustrates the
importance of increasing the valence of the inhibitors.
The incorporation of triazoles adjacent to galactose and sialic
acid scaffolds, via CuAAC reactions, has been utilized in devel-
oping small molecules, polymer-based heterobifunctional ligands,
glycopeptides, and glycopolypeptides against CT that have shown
improvement in activity in comparison to galactose and small
molecule derivatives.22,23,30 This body of work suggests that the
triazole is a suitable isostere for the m-nitrophenyl group found in
MNPG derivatives. This approach is particularly attractive owing
to the synthetic accessibility of triazoles under mild conditions
relative to those required for the installation of the m-nitrophenyl
group in glycoside forming reactions. The mild conditions may
provide greater stereochemical control enabling the isolation
of either pure a- or b-configured galactosides and perhaps a
reduced requirement for OH-protecting groups, thereby reducing
the number of synthetic steps. We believe these considerations will
allow rapid access to a library of derivatives, enabling optimization
of bivalent compounds following biological assay.
We now report the synthesis and biological evaluation of
bivalent 1,2,3-triazole linked galactopyranosides. The general
structure of the bivalent CT inhibitors reported herein is shown
in Fig. 2. They comprise the following design elements: 1.
piperazine core, 2. polyethylene glycol (PEG) linker, 3. pendant
galactotriazole units. The piperazine scaffold was chosen on the
basis that MNPG derivatised propylpiperazine analogues have
been shown to have improved activity against CT.27,48 The PEG
linker groups were considered to be essential for water solubility.
The length of the PEG oligomers was varied within the series to
enable access to spanning and non-spanning derivatives.
terminal binding galactose and/or sialic acid residues of GM1
,
and include conformationally restricted GM1 mimics.32–37 For the
small molecule antagonists, galactose itself has been shown to
have a low but specific affinity for CT (IC50 = 45 mM).38,39
These design strategies, however, suffer from difficulties relating to
complex oligosaccharide synthesis and consequently unfavourable
economic considerations (in the case of GM1 mimics), in addition
to the relatively low binding affinities of galactose derivatives. In
the latter case, the poor affinities are largely due to the inherent
weak interaction of protein–carbohydrate binding.
In contrast, multivalent antagonists have resulted in signifi-
cant improvement in inhibitory activity relative to monovalent
analogues.40,41 Multivalent antagonists employ multiple copies of
an inhibitor to take advantage of the symmetric distribution of
binding epitopes on the pentameric B subunit, thereby effecting the
simultaneous occupation of the binding sites on the CTB subunit.
The simplest multivalent inhibitors are bivalent derivatives and
their general structure is shown in Fig. 1. Bivalent inhibitors
consist of two terminal galactose groups, a core and an appropriate
linker. Work by Hol and Fan27 established that bivalent inhibitors
can be classified as “spanning”, where the galactose binding can
reach the required binding epitopes on CT, or as “non-spanning”,
where the galactose can not reach adjacent galactose binding sites
(in the latter case, the bivalent linkers can bind in an intermolecular
fashion, in which the second unbound galactose binds to a second
CT molecule). The length of the chosen linker will determine
whether or not the bivalent inhibitor will be spanning or non-
spanning. Generally, a small molecule lead compound, such as
m-nitrophenyl-a-D-galactopyranoside (MNPG), is attached to a
bivalent scaffold that enables the affinity for CT to be enhanced
significantly. A series of non-spanning bivalent ligands based on
MNPG were observed to have a gain in activity of two orders
of magnitude in comparison to monovalent MNPG.27 This gain
in activity was greater than that expected for the presence of an
additional copy of a monovalent ligand, which could be a result of
steric blocking playing a role in the competitive surface receptor
binding inhibition process.27 Some pseudo-bivalent linkers have
shown non-specific binding interactions between the linkers and
CT.42 It was observed that these non-specific interactions were
enhanced as the linker lengths were increased. Other bivalent
inhibitors have been synthesized with galactose, lactose, GM1
,
Fig. 2 Target bivalent galactotriazole CT ligands.
It is well established that N- and C-glycosides are stable and do
not possess the enzymatic lability of traditional O-glycosides.5,49–51
A survey of multivalent galactotriazole-linked CT inhibitors
Fig. 1 General structure of bivalent CT inhibitors.
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previously reported,22,23,30 reveals that they contain metabolically
unstable amide bonds and enzymatically labile O-glycosidic
linkages. As we envisage the bivalent inhibitors to comprise an
adjunct therapy, in which oral administration would serve to
reduce the symptoms of the toxin following V. cholerae infection,
these derivatives are required to have prolonged stability in the
gastric environment. It was therefore of interest to prepare both
N- and C-galactotriazoles. It was of further interest to develop
syntheses which preserved the stereochemistry at the anomeric
position. This would be achievable by constructing galactose
derivatives with either a terminal alkyne or an azide group at
the anomeric position. These could then undergo cycloaddition
with the appropriately functionalised bi-functional cross-linker.
C-linked galactotriazoles, in which the anomeric alkynyl-glycoside
replaced the respective azido-glycoside. The synthesis of the target
compounds is shown in Schemes 2 and 3, respectively. For the
synthesis of the target compounds [b-C-GTPEG3 (23) and b-
C-GTPEG4 (26)], the preparation of the piperazinyl-azides (19
and 20) was required. Accordingly, triethylene and tetraethylene
glycol were both mono-tosylated to afford tosylates 13 and 14.55–59
These were then treated with sodium azide to furnish the mono-
azides 15 and 16.56,60 Subsequent tosylation of the remaining
alcohol61 to give 17 and 18 provided sufficient activation to permit
the double nucleophilic addition of piperazine that resulted in
the production of 19 and 20 in good yields. The cycloaddi-
tion of 3,7-anhydro-4,5,6,8-tetra-O-benzyl-1,1,2,2-tetradehydro-
1,2-D-glycero-L-mannooctitol62,63 (21) to PEG3-azide (19), pro-
ceeded smoothly giving adduct 22. This was followed by hy-
drogenolysis to afford the target compound b-C-GTPEG3 (23).
The complete removal of the benzyl protecting groups for the
analogous tetraethylene glycol compound of 22 proved difficult by
hydrogenolysis. This problem was overcome through conversion
of the O-benzyl groups in 21 (Scheme 3) to the corresponding O-
acetates via the use of acetic anhydride and trimethylsilyl triflate,
which yielded compound 24.62 The crystal structure for 24 was
solved (Fig. 3) and comparison with other galactoacetylenes is
discussed (vide infra). The click reaction was carried out under
the same conditions as described for b-C-GTPEG3 (23) and the
Results and discussion
1. Synthesis of bivalent inhibitors.
b-N-Galactotriazoles [b-N-GTPEG3 (11), b-N-GTPEG4 (12)].
Scheme 1 shows the synthesis for target bivalent compounds
b-N-GTPEG3 (11) and b-N-GTPEG4 (12). The synthesis was
initiated by the installation of the propargyl moiety on the
appropriate ethylene glycol unit via the use of propargyl bromide
(Scheme 1), affording the intermediates 3 and 4 in moderate
yields (58% and 41%, respectively).52,53 Mono-tosylation gave the
activated alkynyl-ethyleneglycols52 (5 and 6), which enabled double
alkylation of piperazine to yield the bis-adducts 7 and 8. The
1,3-dipolar cycloaddition of 1-b-azido-2,3,4,6-tetra-O-acetyl-D-
galactose54 with 7 furnished the bis-galactotriazole (9), although in
a low yield. Subsequent deprotection of the methyl ethers afforded
the target dimeric compound 11 (b-N-GTPEG3). Compound 12
(b-N-GTPEG4) was prepared in a similar fashion, however with
improved yields in the cycloaddition step, which may be attributed
to the increased water solubility derived from the additional
polyethylene glycol unit.
Scheme 2 Reagents and conditions: a Et3N, TsCl, CH2Cl2; b EtOH, NaN3,
85 ◦C; c Et3N, TsCl, CH2Cl2; d piperazine, NaH, THF, -78 ◦C/rt/75 ◦C;
e 21, 19, Cu(0), TBTA, 2 : 1 t-BuOH : H2O; f 5% Pd/C, H2, EtOH.
Scheme 1 Reagents and conditions: a NaH, propargyl bromide, THF;
b DMAP, Et3N, TsCl, THF; c piperazine, NaH, 75 ◦C, THF; d
Cu(0), 1-b-azido-2,3,4,6-tetra-O-acetyl-D-galactose, 2 : 1 t-BuOH : H2O; e
MeOH, K2CO3.
b-C-Galactotriazoles [b-C-GTPEG3 (23), b-C-GTPEG4 (26)].
Attention was then turned to the preparation of the corresponding
Scheme 3 Reagents and conditions: a Ac2O, TMSOTf; b 20, 24, Cu(0),
TBTA, 1 : 1 : 1 DMSO : H2O : CH2Cl2; c MeOH, K2CO3.
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Scheme 4 Reagents and conditions: a TMSOTf, CH2Cl2; b 1 M NaOH,
CH3OH/CH2Cl2; c 20, Cu(0), TBTA, 2 : 1 : 1 t-BuOH : H2O : CH2Cl2; d
10% Pd/C, H2, EtOH : AcOH.
Cu(I) [generated from Cu(0)] and tris(benzyltriazolylmethyl)amine
(TBTA), followed by debenzylation to give the target compound
a-C-GTPEG4 (32) with a 36% yield for the final step (Scheme
4). Application of this synthesis to the second target in this
series, a-C-GTPEG3 (35) was unsuccessful owing to incomplete
benzyl deprotection in the final step of the sequence. However,
this problem was ameliorated by conversion of the 2,3,4,6-tetra-
O-benzyl-protected alkynyl galactose (30) to the corresponding
2,3,4,6-tetra-O-acetate (33) (Scheme 5). The crystal structure
of 33 was solved and is shown in Fig. 3. It should be noted
that the Ac2O/TMSOTf mediated conversion of the a-alkynyl
2,3,4,6-tetra-O-benzyl-protected galactose 30 to 33 has not been
previously reported and 33 was obtained in a yield of 86%. The
acetate deprotection proceeded smoothly to give a-C-GTPEG3
(35) with an overall yield of 12% starting from D-galactose.
Fig. 3 Thermal ellipsoid plot for a- and b-alkynes 33 and 24, respectively.
Ellipsoids are at the 20% probability level.
acetate removal proceeded smoothly with methanolic potassium
carbonate affording b-N-GTPEG4 (26) in a quantitative conver-
sion. Thus, b-N-GTPEG4 (26) was obtained and critical to its
success was the method of transferring the O-benzyl protecting
groups of 21 to O-acetate in a single step procedure.
Scheme 5 Reagents and conditions: a Ac2O, TMSOTf; b 19, Cu(0), TBTA,
1 : 1 : 1 DMSO : H2O : CH2Cl2; c MeOH, K2CO3.
a-C-Galactotriazoles [a-C-GTPEG3 (35), a-C-GTPEG4 (32)].
Following the preparation of the corresponding b-C-linked
galactotriazoles, the synthesis of the a-anomeric derivatives [a-
C-GTPEG3 (35) and a-C-GTPEG4 (32)] was performed. The
syntheses of the target compounds are shown in Schemes 4 and 5.
In order to obtain these a-C linked bivalent inhibitors (35 and 32)
the synthesis of the a-alkynyl galactose 30 was required, which was
accomplished by coupling TMS-protected acetylenylbutylstan-
nane (27) with acetyl 2,3,4,6-tetra-O-benzyl-D-galactopyranoside
(28), followed by basic trimethylsilyl deprotection of 29.64,65 The
cycloaddition of the tetraethylene glycoyl azide (20) with the
a-alkynyl galactose 30 was straightforward in the presence of
The thermal ellipsoid plot for 33 establishes this derivative
to have the a-configuration, while 24 has the b-configuration.
˚
The C1–C7 bond distance in the a-alkyne 33 (1.481(2) A) is
significantly longer than the corresponding distance in the b-
63
˚
alkyne 24 (1.459(3) A) and the previously reported b-alkyne 21
for which the corresponding bond distance is 1.462(4) (Fig. 4). This
bond lengthening is consistent with the presence of (nO-s*CC)
orbital interactions between the ring oxygen lone pair and the
antibonding orbital of the C1–C7 bond and is a clear example of
the anomeric effect.66,67 The differences between the C1–C7 bond
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ring, and that the most active compounds are the ones originating
from the alkyne group on the sugar and not the azide group. This
lack of activity could be due to the beta anomeric configuration
of the products, which may thus adopt an incorrect conformation
in solution, preventing access to the active site in CT. The triazole
ring may also be forcing the galactose to be in an incompatible
conformation that results in no observable activity.
Fig. 4 Comparison of the C1–C7 bond distances for the a- and b-alkynes
(33, 24, 2163).
We next considered inhibitory activity based on the anomeric
configuration. As a result the alpha analogues of b-C-GTPEG3
(23) and b-C-GTPEG4 (26) were synthesized [a-C-GTPEG3 (35)
and a-C-GTPEG4 (32)] and biologically tested. These compounds
did not bind strongly to CT, and as a result IC50 values could
not be obtained, i.e. it seemed that the two bivalent alpha
analogues a-C-GTPEG3 (35) and a-C-GTPEG4 (32) were less
active than their beta counterparts. This result was surprising
since the majority of active compounds against CT are alpha
configured. Disappointingly, inhibitory activity based on the
anomeric configuration was not observed for any of the analogues.
The lack of activity led to the consideration that the triazole-
sugar head group is not a suitable isostere for the MNPG func-
tionality. In order to test this last hypothesis, two small molecules
were synthesized [b-C-GT (36) and a-C-GT (37)] (Fig. 5) which
comprise the triazole-sugar head group with a single PEG unit.
The synthesis for b-C-GT (36) and a-C-GT (37) is detailed in the
supporting information. The C-linked galactotriazoles were cho-
sen for comparison owing to the improved activity relative to the
N-linked galactotriazole derivatives, which is evidenced in Table 1.
distances for 33 and 24 are not as pronounced as those observed
for 2,3,4,6-tetraacetate-b-D-glucopyranosyl azide [1.460 A (C1–
˚
N distance)] and 2,3,4,6-tetraacetate-a-D-glucopyranosyl azide
˚
[1.510 A (C1–N distance)], which indicates that the anomeric effect
is much stronger for the more electronegative azides compared to
the acetylenes.68
2. Cholera toxin inhibition assay
With the synthesis of the bivalent galactotriazoles complete,
the inhibitory activity of the derivatives against the binding
of 10 ng mL-1 cholera toxin (CT) to its cell surface receptor
(GM1) was determined as described previously.69 The results
from the biological analysis are shown in Table 1. The b-N-
linked galactotriazoles, b-N-GTPEG3 (11) and b-N-GTPEG4
(12) showed minimal inhibitory potency (0–11% at concentrations
of 11–12 mM), suggesting that they would be no more potent
than D-galactose (IC50 = 45 mM).38 We next examined the C-
linked galactosides, compounds b-C-GTPEG3 (23) and b-C-
GTPEG4 (26), the regioisomers for b-N-GTPEG3 (11) and b-N-
GTPEG4 (12). These possess a 1,4-disubstituted triazole, however
the glycosidic linkage is at the 4-position of the triazole ring (cf.
the 1-position for b-N-GTPEG3 (11) and b-N-GTPEG4 (12)).
The IC50 values were 0.51 mM and ꢀ 2.5 mM for b-C-GTPEG3
(23) and b-C-GTPEG4 (26), respectively. It should be noted that
b-C-GTPEG3 (23) is more potent than galactose by almost two
orders of magnitude (Table 1). This significant improvement in
activity of b-C-GTPEG3 (23) in comparison to b-N-GTPEG3
(11) and b-N-GTPEG4 (12) could be attributed to b-C-GTPEG3
(23) possessing a C-glycoside bond of the sugar, which results
in the atoms of the triazole group being optimally arrayed. It
is therefore surprising that b-N-GTPEG4 (12), with a similar
structure, does not mirror the potency of b-C-GTPEG3 (23). By
considering the overall improvement in biological activity of b-
C-GTPEG4 (26) and b-C-GTPEG3 (23) in comparison to b-N-
GTPEG3 (11), and b-N-GTPEG4 (12), it can be deduced that
one of the key components of these CT inhibitors is the triazole
Fig. 5 Monovalent inhibitors.
Table 1 Data for inhibition of binding of cholera toxin to its receptora
Both compounds were found to be potent inhibitors of cholera
toxin binding, with IC50 values in the low micromolar range. Both
of these analogues possessed better activity in comparison to
MNPG (IC50 = 0.72 mM)38 by one order of magnitude and are
of a comparable potency with respect to each other. In addition
b-C-GT (36) and a-C-GT (37) were respectively 600 and 634 times
more potent than galactose (see Table 1). This suggests that the
triazole is indeed a suitable isosteric substitute for MNPG, whether
as the beta or alpha glycoside, although from these results it
suggests that CT does have a slight preference for the galactose to
be an alpha anomer. The significant improvement in the biological
activity of the monovalent compounds b-C-GT (36) and a-C-
GT (37) compared with the bivalent series is counter-intuitive.
Entry
Triazole
% Inhib.
0
IC50 (mM)
Gain over galactoseb
1
2
3
4
5
6
7
8
11
12
23
26
35
32
36
37
—
—
89
11
3
506 42
19 12
8
16
4
3
75
71
5
4
600
634
a Data are quoted as mean (arithmetic for % inhibition, geometric for IC50)
standard error of the mean from 2–5 experiments (n = 1 for compound
11). b Based on galactose IC50 = 45 mM.38
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It is plausible that the PEG chains could be coiling around the
sugar groups in solution and thus preventing the desired extended
conformation in the assay. This suggests that the next generation of
bivalent inhibitors should include rigid spacer groups between the
triazole ring and the PEG chain to inhibit intramolecular hydrogen
bonding of the PEG chain oxygen atoms to the sugar hydroxyls.
We anticipate that the highly modular synthetic approach adopted
will enable the rapid synthesis of such derivatives.
infrared spectrometer. Samples were analysed using either sodium
chloride plates (NaCl) or attenuated total reflection (ATR), and
absorptions are given in wavenumbers (cm-1). Low resolution mass
spectra were obtained on a Bruker Daltronics Esquire 6000 Ion-
Trap mass spectrometer employing electrospray ionisation (ESI)
with 40 eV cone voltage; samples were run in 0.1% formic acid.
High resolution mass spectra were obtained on a Waters Q-
TOF II, employing electrospray ionisation (ESI) with 35 eV cone
voltage, using lock spray and sodium iodide as a reference sample.
High performance liquid chromatography (HPLC) was carried
out using a GBC instrument with a photodiode array detector.
Analytical HPLC was carried out with an Alltech Apollo C18 5 mm
150 ¥ 4.6 mm column. Semi-preparative HPLC was carried out
with a Grace Apollo C18 5 mm 250 ¥ 10 mm column. Optical
rotation ([a]) values were determined using an Atago Polax-2L
polarimeter with a 1 dm path length glass cell using the sodium D
line (589 nm) at the temperature specified.
Conclusions
A series of potential bivalent CT inhibitors was successfully
synthesized; the key to the synthesis was the ‘click’ reaction that
yielded the corresponding 1,2,3-triazole linked galactopyrano-
sides. It was observed that these triazole galactopyranosides could
be enantioselectively prepared in both monovalent and bivalent
systems. On the whole there was not much difference between the
alpha and beta analogues for inhibition of the binding of cholera
toxin to its receptor, despite a-C-GT (37) (alpha configured)
being the most potent molecule described herein, with an IC50
of 71 mM. These 1,2,3-triazole linked galactopyranosides can
effectively act as an isostere for the MNPG group due to b-C-
GT (36) and a-C-GT (37) being more potent than MNPG by
an order of magnitude. There would need to be some general
improvements in the efficiency of the synthesis before a viable
cholera prophylactic/therapeutic drug could be obtained from
such a method. Compound a-C-GT (37) is a potent small
molecule CT inhibitor that can serve as an improved lead for
the development of a multivalent CT inhibitor.
2-[2-(2-Prop-2-ynoxyethoxy)ethoxy]ethyl-4-methylbenzene sul-
fonate (5). Compound 3 (3.08 g, 16.4 mmol) was dissolved in
170 mL dry THF (170 mL) to which was added TsCl (3.43 g,
18.0 mmol), Et3N (6.80 mL, 49.1 mmol), and DMAP (80 mg,
0.65 mmol). The reaction mixture was allowed to stir at room
temperature for 24 h and then the solvent was removed in vacuo.
The residue was dissolved in EtOAc and washed with water. The
combined organic layers were dried over MgSO4, concentrated in
vacuo, and then purified by column chromatography on silica gel
eluting with 33–60% EtOAc : hexane to yield 5 as a colourless oil
{3.52 g, 63% (lit.[52] 87%)}. Data for compound 5 were identical to
literature values.52
1,4-Bis-{2-[2-(2-prop-2-ynyloxyethoxy)ethoxy]ethyl}piperazine
(7). Piperazine (124 mg, 1.4 mmol) was dissolved in dry THF
(15 mL), to which was added sodium hydride (60% dispersion
in oil, 234 mg) and the reaction mixture was stirred at room
temperature for 2.5 h. A solution of toluene-4-sulfonic acid 2-[2-(2-
prop-2-ynyloxy-ethoxy)-ethoxy]-ethyl ester 5 (980 mg, 2.86 mmol)
in dry THF (3◦mL + 10 mL rinse) was added. This mixture was then
heated at 75 C (reflux) for 24.5 h, cooled, diluted with CH2Cl2
and the organic layers were washed with water. The combined
organic extracts were dried over MgSO4 and concentrated under
vacuum to yield a crude oil. The crude oil was dry loaded onto
silica and purified by column chromatography eluting initially with
EtOAc, which was the followed with a 50% CH2Cl2 : MeOH that
contained 1% Et3N. Compound 7 was obtained as a brown oil
(298 mg, 49%). IR (ATR, cm-1) 3246, 2916, 2868, 2818, 2112,
Experimental section
General methods
All non-aqueous reactions were performed under an atmosphere
of dry nitrogen or argon, unless otherwise specified. CH2Cl2 and
Et2O were distilled from CaH2 in a recycling still. THF was
dried with LiAlH4 and then distilled from potassium metal in
a recycling still. Et3N was freshly distilled from CaH2 under N2.
Reaction progress was monitored via thin-layer chromatography
(TLC) using kieselgel 60 F254 plates (aluminium backed) and
ethyl acetate/hexane or CH2Cl2/MeOH. Aluminium oxide 60 F254
neutral (Type E) plates were also used with ethyl acetate/hexane.
TLC plates were visualised using a 254 nM UV lamp and/or a 10%
w/v molybdophosphoric acid/EtOH stain or permanganate stain
consisting of KMnO4 (3.0 g), K2CO3 (20 g) and 5% w/v aqueous
NaOH (5 mL) in H2O (300 mL). Flash column chromatography
was performed using silica gel (silica gel 60, 230–400 mesh ASTM)
or aluminium oxide GF254 (Type 60/E) as the stationary phase
and ethyl acetate/hexane or CH2Cl2/MeOH mixtures as the
mobile phase. 1H, 13C, DEPT, 1H/13C-HSQC, 1H/13C-HMBC
1
1456, 1327, 1304, 1244, 1092, 1032, 1011, 943, 841, H NMR d
(CDCl3, 300 MHz), 4.09 (4H, d, J = 2.4 Hz), 3.58–3.52 (20H, m),
2.57–2.53 (12H, m), 2.36 (2H, t, J = 2.3 Hz); 13C NMR d (CDCl3,
75 MHz), 79.4, 74.4, 70.3, 70.09, 70.14, 68.8, 68.5, 58.1, 57.3 and
52.9. MS-ESI: m/z [M + H]+ for C22H39N2O6 calculated 427.3;
+
+
observed 427.3. HRMS-EI: m/z [M]+ for C22H38N2O6 , calculated
1
and H/1H-COSY nuclear magnetic resonance (NMR) spectra
426.2730; observed 426.2718.
were obtained on a Bruker AV-300 spectrometer (1H at 300.13
MHz and 13C at 75.47 MHz, respectively) and a Bruker AV-
500 spectrometer (1H at 500.19 MHz and 13C at 125.78 MHz,
respectively). Proton and carbon chemical shifts are reported as
d values in parts per million (ppm) and are relative to residual
solvent; coupling constants (J) are in Hz. Infrared (IR) spectra
were recorded on a Shimadzu IRPrestige-21 Fourier-Transform
[(2R,3S,4S,5R,6R)-3,5-Diacetoxy-2-(acetoxymethyl)-6-[(1R)-
4-[2-[2-[2-[4-[2-[2-[2-[[1-[(2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6-
(acetoxymethyl)tetrahydropyran-2-yl]triazol-4-yl]methoxy]etho-
xy]ethoxy]ethyl]piperazin-1-yl]ethoxy]ethoxy]ethoxymethyl]triazol-
1-yl]tetrahydropyran-4-yl] acetate (9). Compound 7 (298 mg,
0.7 mmol) was suspended in a 2 : 1 t-BuOH : H2O mixture (3 mL)
8470 | Org. Biomol. Chem., 2011, 9, 8465–8474
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to which was added b-D-galactopyranosyl azide 2,3,4,6-tetra-O-
acetate (575 mg) and Cu(0) mesh (444 mg). The reaction was
allowed to stir vigorously for 23 h at room temperature. Then it
was diluted with CH2Cl2 and filtered through a bed of celite. The
filtrate was washed with water and the organic layer was dried
over Na2SO4 and concentrated to yield a crude oil. The crude
oil was dry loaded onto a silica column and purified by column
chromatography eluting with MeOH. Compound 9 was obtained
as a light brown oil (181 mg, 22%). [a]2D0 -4.0 (c 1.7, DCM); IR
(NaCl, cm-1) 2923, 2874, 2831, 1749, 1454, 1370, 1221, 1092,
(CDCl3, 300 MHz), 3.66–3.58 (16H, m), 3.36 (4H, t, J = 5.1 Hz),
2.63–2.60 (12H, m);13C NMR d (CDCl3, 75 MHz), 70.6, 70.4,
70.0, 68.6, 57.5, 53.1 and 50.7. MS-ESI: m/z [M – N6H + Na]+
for C16H31N2O4Na+ calculated 338.2; observed 338.3. HRMS-ESI:
m/z [M + H]+ for C16H33N8O4 , calculated 401.2625; observed
+
401.2607.
1-(2-(2-(2-(4-((2S,3S,4R,5S,6R)-3,4,5-tris(benzyloxy)-6-(benz-
yloxymethyl)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-1-yl)-
ethoxy)ethoxy)ethyl)-4-(2-(2-(3-(4-((2S,3S,4R,5S,6R)-3,4,5-tris-
(benzyloxy)-6-(benzyloxymethyl)tetrahydro-2H-pyran-2-yl)-1H-
1
1063, H NMR d (CDCl3, 300 MHz), 7.78 (2H, s), 5.80 (2H, d,
J = 9.0 Hz), 5.51–5.45 (4H, m), 5.20 (2H, dd, J = 3.3, 10.5 Hz),
4.62 (4H, s), 4.18–4.07 (6 H, m), 3.60–3.51 (20 H, m), 2.53–2.49
(12H, m), 2.15 (6H, s), 1.97 (6H, s), 1.93 (6H, s), 1.81 (6H, s);
13C NMR d (CDCl3, 75 MHz), 170.2, 169.9, 169.7, 168.9, 145.8,
121.0, 86.1, 73.9, 70.7, 70.5, 70.4, 70.3, 69.7, 68.9, 67.8, 66.8, 64.4,
61.1, 57.7, 53.4, 20.6, 20.4 and 20.2. MS-ESI: m/z [M + H]+ for
1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)piperazine
(22). Com-
pound 20 (116 mg, 0.29 mmol) was suspended in a 2 : 1 : 1
mixture of t-BuOH : H2O : DCM (6 mL), to which was added 21
(325 mg, 0.61 mmol), Cu(0) (184 mg, 2.90 mmol), and TBTA
(3.1 mg, 0.006 mmol). The reaction mixture was allowed to
stir vigorously at room temperature for 23 h after which it was
diluted with DCM, and then filtered through a bed of celite to
remove the excess copper. The filtrate was then washed with
H2O, dried over Na2SO4, and concentrated in vacuo to yield
crude material. The crude material was dry loaded onto alumina
and purified by column chromatography eluting initially with
14% EtOAc : hexane that was followed by 9–17% MeOH : EtOAc
to yield compound 22 as a colourless oil (340 mg, 78%). [a]D20
+17 (c 0.72, DCM); IR (ATR, cm-1) 3088, 3063, 2869, 2243,
+
C50H77N8O24 calculated 1173.5; observed 1173.4. HRMS-ESI:
m/z [M + H]+ for C50H77N8O24+, calculated 1173.5051; observed
1173.5088.
(2R,2¢R,3R,3¢R,4S,4¢S,5R,5¢R,6R,6¢R)-6,6¢-(4,4¢-(2,2¢-(2,2¢-(2,
2¢-(Piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-
diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(methylene)bis(1H-1,
2,3-triazole-4,1-diyl))bis(2-(hydroxymethyl)tetrahydro-2H-pyran-
3,4,5-triol) (11). Compound 9 (113 mg, 0.1 mmol) was dissolved
in distilled methanol (4 mL), to which was added potassium
carbonate (1.2 mg). The reaction was stirred at room temperature
for 24 h after which the solvent was removed under vacuum
to yield a crude oil. Purification was carried out by column
chromatography eluting with 0–1% Et3N : MeOH to give 11 as
a colourless oil (26 mg, 32%). [a]2D0 +9.0 (c 0.79, MeOH); IR
(ATR, cm-1) 3372, 2914, 2877, 2833, 2833, 1656, 1643, 1633, 1454,
1
1733, 1652, 1607, 1496, 1454, 1363, 1308, 1208, 1105, 911, H
NMR d (CDCl3, 300 MHz), 7.61 (2H, s), 7.34–7.17 (36H, m),
7.02–7.00 (4H, m), 4.96 (2H, d, J = 11.7 Hz), 4.74–4.62 (8H, m),
4.51–4.24 (14H, m), 4.05 (2H, d, J = 2.4 Hz), 3.80–3.69 (8H, m),
3.58–3.43 (16H, m), 2.65–2.53 (12H, m);13C NMR d (CDCl3, 75
MHz), 145.8, 138.7, 138.33, 138.26, 137.8, 128.3, 128.1, 128.0,
127.94, 127.90, 127.7, 127.6, 127.5, 127.4, 123.6, 84.4, 78.4, 74.9,
74.6, 73.9, 73.4, 72.4, 70.4, 70.1, 69.4, 68.7, 57.4, 53.0 and 50.1.
1
1444, 1350, 1308, 1234, 1079, 1056, 1019, H NMR d (CD3OD,
MS-ESI: m/z [M + H]+ for C88H105N8O14 calculated 1497.8;
+
300 MHz), 8.23 (2H, s), 5.59 (2H, d, J = 9.0 Hz), 4.66 (4H,
s), 4.16 (2H, t, J = 9.3 Hz), 3.99 (2H, d, J = 3.0 Hz), 3.85–
3.58 (28H, m), 2.57 (12H, t, J = 5.7 Hz); 13C NMR d (CD3OD,
75 MHz), 146.2, 123.9, 90.2, 80.0, 75.3, 71.5, 71.4, 71.3, 70.8,
70.4, 69.6, 65.0, 62.5, 58.6 and 54.0. MS-ESI: m/z [M + H]+
observed 1497.6. HRMS-ESI: m/z [M + H]+ for C88H105N8O14
,
+
calculated 1497.7750; observed 1497.7703.
(2R,3R,4R,5R,6S)-2-(Hydroxymethyl)-6-[1-[2-[2-[2-[4-[2-[2-[2-
[4-[(2S,3R,4R,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetra-
hydropyran-2-yl]triazol-1-yl]ethoxy]ethoxy]ethyl]piperazin-1-yl]-
ethoxy]ethoxy]ethyl]triazol-4-yl]tetrahydropyran-3,4,5-triol (23).
Compound 22 (159 mg, 0.11 mmol) was dissolved in EtOH (5 mL,
required heating) and acetic acid (1 mL). To this mixture was added
5% Pd/C catalyst (610 mg). The reaction flask was evacuated
and the atmosphere was replenished with hydrogen gas. This
was repeated three times. The reaction was allowed to proceed
at room temperature under an atmosphere of hydrogen for 3 d.
The palladium catalyst was then removed by passing the reaction
mixture through a bed of celite. The filtrate was evaporated to
yield 22 (43.6 mg, 53%) as a yellow/brown oil. 1H NMR d (D2O,
300 MHz), 8.19 (2H, s), 4.69 (4H, d, J = 4.5 Hz), 4.56 (2H d, J =
9.9 Hz), 4.10 (2H, d, J = 3.0 Hz), 4.03–3.98 (8H, m), 3.89–3.87
(4H, m), 3.83 (2H, d, J = 3.3 Hz), 3.79–3.67 (16H, m), 3.15–
3.07 (8H, m); 13C NMR d (D2O, 75 MHz), 144.8, 124.6, 78.9,
73.7, 73.5, 69.9, 69.3, 69.0, 68.8, 68.3, 64.9, 60.9, 55.4, 49.9 and
+
for C34H61N8O16 calculated 837.4; observed 837.4. HRMS-ESI:
m/z [M + H]+ for C34H61N8O16+, calculated 837.4206; observed
837.4241.
1,4-Bis[2-[2-(2-azidoethoxy)ethoxy]ethyl]piperazine (19). To a
solution of piperazine (41 mg, 0.47 mmol) in dry THF (4 mL) was
added sodium hydride (60% dispersion in oil, 77 mg). The reaction
mixture was cooled to -78 ◦C and stirred for 1 h before a solution
of 2-[2-(2-azidoethoxy)ethoxy]ethyl 4-methylbenzenesulfonate 17
(318 mg, 0.96 mmol) in THF (3 mL + 3 mL rinse) was added
dropwise. The resulting mixture was allowed to stir at -78 ◦C
for 3 h. The reaction mixture was then allowed to warm up
to room temperature and refluxed at 75 ◦C for 15.5 h. The
reaction mixture was diluted with DCM and washed with water.
The aqueous extracts were then backwashed with DCM and
the combined organic extracts were dried over MgSO4, and
concentrated in vacuo to yield crude material that was purified
by column chromatography on silica gel eluting initially with 33–
100% EtOAc : hexane, which was followed with 1% Et3N : MeOH
to yield compound 19 as a yellow oil (116 mg, 62%). IR (NaCl,
cm-1) 2933, 2871, 2813, 2106, 1456, 1348, 1303, 1119, 1H NMR d
49.7. MS-ESI: m/z [M + H]+ for C32H57N8O14 calculated 777.4;
+
observed 777.3. HRMS-ESI: m/z [M + Na]+ for C32H56N8NaO14
,
+
calculated 799.3814; observed 799.3799.
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Hz), 3.64–3.53 (24H, m), 3.39–3.33 (2H, m), 2.60 (12H, br s); 13
[(2R,3S,4R,5S,6S)-4,5-Diacetoxy-2-(acetoxymethyl)-6-[1-[2-[2-
[2-[2-[4-[2-[2-[2-[2-[4-[(2S,3S,4R,5S,6R)-3,4,5-triacetoxy-6-(acet-
oxymethyl)tetrahydropyran-2-yl]triazol-1-yl]ethoxy]ethoxy]etho-
xy]ethyl]piperazin-1-yl]ethoxy]ethoxy]ethoxy]ethyl]triazol-4-yl]-
tetrahydropyran-3-yl] acetate (25). Compounds 24 (26 mg,
0.07 mmol) and 20 (16 mg, 0.03 mmol) were combined and
dissolved in 1 : 1 : 1 DCM : DMSO : H2O (3 mL). To this reaction
mixture TBTA (0.2 mg, 0.1 eq) was added, followed by Cu(0)
(0.2 mg, 0.1 eq). The reaction mixture was stirred at room tem-
perature for 16 h and worked up as for compound 22. The crude
material was dry loaded and purified by column chromatography
on silica gel eluting initially with 20–100% EtOAc : hexane that
was followed with 6–100% MeOH : EtOAc to yield compound 25
as a colourless oil (24 mg, 62%). [a]2D0 +23 (c 1.2, DCM); IR (NaCl,
cm-1) 2876, 2112, 1748, 1636, 1456, 1435, 1371, 1231, 1092, 1053,
1H NMR d (CDCl3, 300 MHz), 7.75 (2H, s), 5.49–5.39 (4H, m),
5.13 (2H, dd, J = 10.2, 3.3 Hz), 4.73 (2H, d, J = 10.2Hz), 4.51(4H, t,
J = 5.1 Hz), 4.12–4.06 (6H, m), 3.86–3.81 (4H, m), 3.61–3.58 (20H,
m), 2.75–2.62 (12H, m), 2.15 (6H, s), 2.00 (6H, s), 1.96 (6H, s), 1.83
(6H, s); 13C NMR d (CDCl3, 75 MHz), 170.4, 170.2, 170.1, 144.2,
123.3, 74.7, 73.8, 72.0, 70.6, 70.4, 70.3, 69.3, 68.5, 67.6, 61.5, 57.4,
C
NMR d (CDCl3, 75 MHz), 144.7, 138.19, 138.16, 137.94, 137.89,
127.9, 127.7, 127.6, 127.5, 127.4, 127.3, 127.2, 127.1, 124.2, 73.8,
73.2, 73.1, 72.8, 72.6, 70.33, 70.25, 70.19, 70.15, 70.09, 69.9, 69.1,
68.1, 67.1, 66.9, 57.1, 52.7 and 49.8. MS-ESI: m/z [M + H]+ for
+
C92H113N8O16 calculated 1585.8; observed 1585.3. HRMS-ESI:
m/z [M + H]+ for C92H113N8O16+, calculated 1585.8275; observed
1585.8262.
(2R,3R,4R,5R,6R)-2-(Hydroxymethyl)-6-[1-[2-[2-[2-[2-[4-[2-[2-
[2-[2-[4-[(2R,3R,4R,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-
tetrahydropyran-2-yl]triazol-1-yl]ethoxy]ethoxy]ethoxy]ethyl]pipe-
razin-1-yl]ethoxy]ethoxy]ethoxy]ethyl]triazol-4-yl]tetrahydropy-
ran-3,4,5-triol (32). Compound 32 was obtained by the same pro-
cedure as used for compound 23 with the following modifications:
compound 31 (143 mg, 0.09 mmol), 10% Pd/C catalyst (71 mg),
glacial acetic acid (8 mL) were used. The reaction mixture was
stirred for a total of 7 d, and toluene was used to azeotropically
remove the glacial acetic acid (¥3). Compound 32 was obtained as
a light brown oil (28 mg, 36%). 1H NMR d (D2O, 300 MHz), 8.19
(2H, s), 5.36 (2H, d, J = 6.0 Hz), 4.68 (4H, t, J = 4.5 Hz), 4.30–
4.25 (2H, m), 4.17–4.00 (6H, m), 3.80–3.65 (28H, m), 2.73 (12H,
br s); 13C NMR d (D2O, 75 MHz), 142.6, 125.9, 73.3, 71.3, 69.9,
69.8, 69.2, 69.0, 68.5, 68.4, 67.3, 66.5, 60.6, 60.0, 55.9, 51.1 and
+
52.9, 50.4, 20.7 and 20.6. MS-ESI: m/z [M + H]+ for C52H81N8O24
calculated 1201.5; observed 1201.4. HRMS-ESI: m/z [M + H]+ for
C52H81N8O24+, calculated 1201.5364; observed 1201.5344.
49.6. MS-ESI: m/z [M + H]+ for C36H65N8O16 calculated 865.5;
+
observed 865.3. HRMS-ESI: m/z [M + H]+ for C36H65N8O16
,
+
(2R,3R,4R,5R,6S)-2-(Hydroxymethyl)-6-[1-[2-[2-[2-[2-[4-[2-[2-
[2-[2-[4-[(2S,3R,4R,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-
tetrahydropyran-2-yl]triazol-1-yl]ethoxy]ethoxy]ethoxy]ethyl]pipe-
razin-1-yl]ethoxy]ethoxy]ethoxy]ethyl]triazol-4-yl]tetrahydro-
pyran-3,4,5-triol (26). Compound 26 was obtained by the same
procedure as used for compound 11 with the following modifica-
tions. Compound 25 (22 mg, 0.02 mmol) was dissolved in distilled
MeOH (3 mL) to which was added a catalytic amount of potassium
carbonate. The reaction was stirred at room temperature for 19 h.
Compound 26 was obtained as a pale brown oil (15.8 mg, 100%).
1H NMR d (D2O, 300 MHz), 8.18 (2H, s), 4.70 (4H, t, J = 5.1
Hz), 4.51 (2H, d, J = 9.9 Hz), 4.09 (2H, d, J = 3.0 Hz), 4.04–3.98
(6H, m), 3.89 (2H, t, J = 6.3 Hz), 3.83–3.77 (6H, m), 3.70–3.65
(20H, m), 2.62 (12H, t, J = 5.7 Hz); 13C NMR d (D2O, 75 MHz),
144.8, 124.7, 78.8, 73.8, 73.5, 69.9, 69.2, 69.1, 69.0, 68.7, 68.4, 67.1,
calculated 865.4519; observed 865.4540.
[(2R,3S,4R,5S,6R)-3,4,5-Triacetoxy-6-ethynyl-tetrahydropy-
ran-2-yl]methyl acetate (33). Compound 30 (851 mg, 1.55 mmol)
was dissolved in acetic anhydride (46 mL) and the solution was
cooled to 0 ◦C before trimethylsilyl triflate (1.88 mL, 10.4 mmol)
was added dropwise. The reaction mixture was then allowed to
warm to room temperature and stirred for 7 d. The solution was
◦
then cooled to 0 C and quenched by the cautious addition of a
saturated solution of NaHCO3. Dilution of the reaction mixture
with EtOAc was followed by washing the organic layer with a
solution of NaHCO3, then water, and then brine. The organic layer
was dried over Na2SO4 and concentrated in vacuo with silica gel.
The crude material was added to the top of a silica gel column and
eluted with 66–90% EtOAc : hexane. Compound 33 was obtained
as a light yellow solid (473 mg, 86%). [a]2D0 +93 (c 0.94, DCM);
IR (NaCl, cm-1) 2965, 2114, 1748, 1371, 1219, 1087, 1H NMR d
(CDCl3, 300 MHz), 5.43 (1H, dd, J = 3.3, 1.2 Hz), 5.33 (1H, dd,
J = 10.5, 3.3 Hz), 5.18–5.12 (1H, m), 5.04 (1H, dd, J = 5.7, 2.1 Hz),
4.38 (1H, td, J = 6.6, 0.9 Hz), 4.13–4.01 (2H, m), 2.57 (1H, d, J =
2.4 Hz), 2.11 (3H, s), 2.06 (3H, s), 2.02 (3H, s), 1.97 (3H, s); 13C
NMR d (CDCl3, 75 MHz), 170.3, 170.1, 170.0, 169.9, 78.0, 76.5,
69.6, 68.5, 67.7, 66.7, 65.8, 61.5, 20.7, 20.62, 20.57 and 20.5. MS-
60.8, 56.0, 51.6 and 49.7. MS-ESI: m/z [M + H]+ for C36H65N8O16
+
calculated 865.5; observed 865.3. HRMS-ESI: m/z [M + Na]+ for
C36H64N8NaO16+, calculated 887.4338; observed 887.4357.
1,4-Bis[2-[2-[2-[2-[4-[(2R,3S,4R,5S,6R)-3,4,5-tribenzyloxy-6-
(benzyloxymethyl)tetrahydropyran-2-yl]triazol-1-yl]ethoxy]etho-
xy]ethoxy]ethyl]piperazine (31). Compound 31 was obtained via
the same procedure as that used for compound 25 with the
following modifications: compound 30 (434 mg, 0.79 mmol),
compound 20 (187 mg, 0.38 mmol), TBTA (9 mg, 0.4 eq.), Cu(0)
(1.1 mg, 0.04 eq.) and a 1 : 1 : 1 mixture of DCM : DMSO : H2O
(9 mL) were used. The reaction was allowed to proceed at room
temperature for 17.5 h and the crude material was dry loaded
onto silica and purified via column chromatography eluting with
0–17% MeOH : EtOAc containing 1% Et3N to yield compound
31 as a pale yellow oil (512 mg, 84%). [a]2D0 +28 (c 1.41, DCM);
IR (NaCl, cm-1) 3028, 2870, 2106, 1722, 1495, 1452, 1275, 1096,
1H NMR d (CDCl3, 300 MHz), 7.70 (2H, s), 7.28–7.11 (40H, m),
5.26 (2H, d, J = 4.2 Hz), 4.74–4.40 (18H, m), 4.30–4.26 (2H, m),
4.18–4.15 (2H, m), 4.08 (4H, q, J = 3.6 Hz), 3.82 (4H, t, J = 5.1
ESI: m/z [M + Na]+ for C16H20NaO9 calculated 379.1; observed
+
379.1. MS-EI: m/z [M]+ for C16H20O9+ calculated 356.1; observed
+
356.1. HRMS-EI: m/z [M]+ for C16H20O9 , calculated 356.1107;
observed 356.1095.
[(2R,3S,4R,5S,6R)-4,5-Diacetoxy-2-(acetoxymethyl)-6-[1-[2-
[2-[2-[4-[2-[2-[2-[4-[(2R,3S,4R,5S,6R)-3,4,5-triacetoxy-6-(aceto-
xymethyl)tetrahydropyran-2-yl]triazol-1-yl]ethoxy]ethoxy]ethyl]-
piperazin-1-yl]ethoxy]ethoxy]ethyl]triazol-4-yl]tetrahydropyran-3-
yl] acetate (34). Compound 34 was obtained via the same
procedure as that used for compound 25 with the following
8472 | Org. Biomol. Chem., 2011, 9, 8465–8474
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modifications: compound 19 (53.8 mg, 0.13 mmol), compound
33 (101 mg, 0.28 mmol), TBTA (7.1 mg, 0.1 eq.), Cu(0) (0.9 mg,
0.1 eq), and a 1 : 1 : 1 mixture of DCM : DMSO : H2O (3 mL) was
used. The reaction was allowed to proceed at room temperature for
21 h and the crude material was dry loaded onto silica and purified
via column chromatography eluting with 10–100% MeOH : EtOAc
to yield compound 34 as a colourless oil (120 mg, 80%). [a]2D0 +65
(c 2.9, DCM); IR (NaCl, cm-1) 2961, 2874, 2816, 2104, 1748, 1639,
1549, 1456, 1369, 1227, 1049, 1H NMR d (CDCl3, 300 MHz), 7.72
(2H, s), 5.93 (2H, dd, J = 9.3, 3.3 Hz), 5.53 (2H, dd, J = 3.3, 1.5
Hz), 5.49–5.43 (4H, m), 4.54–4.46 (6H, m), 4.09–4.03 (4H, m),
3.84 (4H, t, J = 5.1 Hz), 3.55–3.53 (12H, m), 2.57–2.51 (12H, m),
2.13 (6H, s), 2.03 (12H, s), 2.00 (6H, s);13C NMR d (CDCl3, 75
MHz), 170.4, 170.2, 170.0, 169.7, 142.4, 125.2, 70.5, 70.1, 69.3,
68.8, 68.2, 68.1, 68.0. 67.6, 61.4, 57.6, 53.4, 50.2 and 20.6. MS-
on a rocking platform mixer (Ratek Instruments, Melbourne,
Australia) during the incubation of the assay plate with 360 mL
blocking buffer. A 100 mL aliquot from each test tube was then
added to a well of the 96-well plate, after which the assay proceeded
as normal. The amount of unbound CT was determined by
reference to a control plot of rate of absorbance change vs. CT
concentration that was performed on the same plate. The amount
of unbound CT was plotted against the concentration of inhibitor.
If the values of percent inhibition of binding approached or
spanned 50%, the IC50 for inhibition of binding was calculated
from a second-order polynomial fit to the data. Examples of a
control plot and an inhibition plot are shown in the supplementary
information.
Crystallography
ESI: m/z [M + K]+ for C48H72KO22 calculated 1151.4; observed
+
Crystals of 33 and 24 were mounted in low temperature oil then
flash cooled to 130 K using an Oxford low temperature device.
Intensity data were collected at 130 K with an Oxford SuperNova
X-ray diffractometer with CCD detector using Cu-Ka radiation
1151.7. HRMS-ESI: m/z [M + H]+ for C48H73N8O22+, calculated
1113.4839; observed 1113.4825.
(2R,3R,4R,5R,6R)-2-(Hydroxymethyl)-6-[1-[2-[2-[2-[4-[2-[2-[2-
[4-[(2R,3R,4R,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetra-
hydropyran-2-yl]triazol-1-yl]ethoxy]ethoxy]ethyl]piperazin-1-yl]-
ethoxy]ethoxy]ethyl]triazol-4-yl]tetrahydropyran-3,4,5-triol (35).
Compound 35 was obtained by the same procedure used for
compound 11 with the following modifications. Compound 34
(45 mg, 0.04 mmol) was dissolved in distilled MeOH (3 mL) to
which was added potassium carbonate (0.2 mg). The reaction was
stirred at room temperature for 17 h. Compound 35 was obtained
as a thick colourless oil (28 mg, 89%). 1H NMR d (D2O, 300 MHz),
8.18 (2H, s), 5.39 (2H, d, J = 6.3 Hz), 4.67 (4H, t, J = 4.5 Hz), 4.29
(2H, dd, J = 10.2, 6.3 Hz), 4.12 (2H, dd, J = 9.9, 3.3 Hz), 4.07 (2H,
d, J = 3.3 Hz), 4.02 (4H, t, J = 4.8 Hz), 3.82–3.62 (18H, m), 2.62
(12H, t, J = 5.7 Hz); 13C NMR d (D2O, 75 MHz), 142.6, 125.9, 73.4,
69.9, 69.8, 69.3, 68.9, 68.5, 68.4, 67.4, 67.1, 60.6, 56.0, 51.6 and
˚
˚
for 33 (l = 1.54184 A) and Mo-Ka radiation (l = 0.71073 A) for 24.
Data were reduced and corrected for absorption.70 The structures
were solved by direct methods and difference Fourier synthesis
using the SHELX suite of programs71 as implemented within the
WINGX72 software. Thermal ellipsoid plots were generated using
the program ORTEP-3.
Crystal data for 33
The crystals obtained for 33 were recrystallized from hexane–
dichloromethane, mp 137–138 ◦C. C16H20O9, M = 356.32, T =
130.0(2) K, l = 0.71069, Orthorhombic, space group P212121
3
˚
˚
a = 7.1296(2) b = 9.0495(4), c = 27.544(1) A, V = 1777.1(1) A ,
Z = 4, Dc = 1.332 mg M-3 m(Cu-Ka) 0.943 mm-1, F(000) = 752,
crystal size 0.52 ¥ 0.27 ¥ 0.18 mm. 7003 reflections measured, 3448
independent reflections (Rint = 0.0123), the final R was 0.0360 [I >
2s(I)] and wR(F2) (all data) was 0.0961.
+
49.6. MS-ESI: m/z [M + H]+ for C32H57N8O14 calculated 777.4;
observed 777.3. HRMS-ESI: m/z [M + H]+ for C32H57N8O14
,
+
calculated 777.3994; observed 777.3975.
Crystal data for 21
Biological studies
The crystals obtained for 24 were recrystallized from hexane–
dichloromethane, mp 154–155 ◦C. C16H20O9, M = 356.32, T =
The binding of cholera toxin (CT) to its cell surface receptor (GM1
)
was determined as described previously.69 In brief, a solution of
GM1 in phosphate-buffered saline (PBS) was used to coat wells of a
Nunc-Immuno Maxisorp 96-well plate (100 mL per well) overnight
at room temperature. After this and subsequent incubations, the
plate was washed 3 times with PBS. The wells of the plate were
initially incubated for one hour with 360 mL blocking buffer (0.25%
bovine serum albumin in PBS–0.05% Tween 20) to block non-
specific binding sites, and then with 100 mL CT (1–100 ng mL-1
in blocking buffer), 100 mL goat anti-CT antibody in blocking
buffer, and 100 mL rabbit anti-goat alkaline phosphatase conjugate
in blocking buffer. These incubations were at room temperature
for one hour, except for CT (1.5 h). Finally, the plate received an
extra wash with distilled water, and 100 mL alkaline phosphatase
substrate (para-nitro phenol) in Tris buffer was added. The rate
of change of absorbance at 405 nm was measured over 10 min
using a Bio-Tek Synergy HT plate reader (Bio-Tek, Winooski,
VT, USA). Inhibition of the binding of CT to immobilised GM1
was determined by incubating the inhibitor with CT (total volume
130 mL) for 1 h in a disposable glass tube with gentle rocking
130.0(2) K, l = 0.71069, Monoclinic, space group C2, a =
3
˚
˚
18.1428(8) b = 8.3337(5), c = 11.7816(9) A, V = 1776.1(2) A ,
Z = 4, Dc = 1.333 mg M-3 m(Mo-Ka) 0.110 mm-1, F(000) = 752,
crystal size 0.6 ¥ 0.3 ¥ 0.2 mm. 4376 reflections measured, 2620
independent reflections (Rint = 0.0260), the final R was 0.0374 [I >
2s(I)] and wR(F2) (all data) was 0.0906.
Acknowledgements
DJL acknowledges the Australian Government for the provision
of an Australian Postgraduate Award (APA) and La Trobe
University/CSIRO for the La Trobe University/CSIRO Aurora
PhD top-up Scholarship.
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