Synthesis of soluble multivalent glycoconjugates that target the Hc region of botulinum neurotoxin A

Article abstract of DOI:10.1016/j.bmcl.2007.02.028

The design, synthesis, and initial inhibitory studies of di- and tetravalent glycoconjugates that target the heavy chain of botulinum neurotoxin A are reported.

Full text of DOI:10.1016/j.bmcl.2007.02.028

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2459–2464
Synthesis of soluble multivalent glycoconjugates that target
the Hc region of botulinum neurotoxin A
Ramesh R. Kale,a Colin M. Clancy,b Rebecca M. Vermillion,a
Eric A. Johnsonb and Suri S. Iyera,*
a301 Clifton Court, Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA
b1925 Willow Drive, Department of Food Microbiology and Toxicology, Food Research Institute,
University of Wisconsin–Madison, Madison, WI 53706, USA
Received 26 December 2006; revised 6 February 2007; accepted 9 February 2007
Available online 14 February 2007
Abstract—The design, synthesis, and initial inhibitory studies of di- and tetravalent glycoconjugates that target the heavy chain of
botulinum neurotoxin A are reported.
Ó 2007 Elsevier Ltd. All rights reserved.
Botulinum toxin, produced by the gram-positive, spore
forming Clostridium botulinum, is one of the most poi-
sonous proteins found in Nature.1 It has been estimated
that a gram of aerosolized botulinum neurotoxin A
(BoNT/A) could potentially cause over a million deaths
in a densely populated area.2 The clinical manifestations
of botulism are based on observation alone and are sub-
ject to misdiagnosis, particularly in the early stages of
intoxication. Current treatment is limited to administra-
tion of neutralizing antibodies and life support to pre-
vent respiratory failure. Furthermore, effective small
molecule inhibitors are not available as therapeutics or
prophylactics. The ease of transmission and the paralyz-
ing nature of the toxin have led the CDC and NIAID to
classify botulinum as a Category A Select Agent. The
high potency of the toxin coupled with recent bioterror-
ist events and lack of small molecule drugs has led to a
renewed interest in developing therapeutics and diagnos-
tics for these toxins.
would also be valuable.4 The 100 kDa fragment consists
of HN and HC fragments, of which the HC is responsible
for binding to complex glycolipids, GD1a/b and GT1a/
b. Indeed, polysialylated GT1b has been shown to inhi-
bit synaptosomes and to quench BoNT/A fluores-
cence.5,6 Recent crystal structures of clostridial
neurotoxins provide the first direct identification and
characterization of the binding sites to their natural gan-
glioside receptors.7 It is obvious that multivalent bind-
ing is prevalent, as X-ray structures reveal that the
binding sites for individual units (monosaccharide) of
glycolipid are widely separated. The terminal sialic acid
of the two branches of the gangliosides is crucial in
binding and it induces cross-linking by binding to two
distinct sites on the Hc fragment. The structure of the
GT1b is shown in Figure 1 (left). Our interest in this
area is twofold: (1) Using the structure of the ganglio-
side as a starting point combined with a modular
synthetic approach, we were interested in developing a
panel of glycoconjugates that could potentially block
the toxin from cell entry. (2) The tailor-made soluble
GT1a/b analogues could be used for Bot epitope
mapping to define the intricate mechanism of infection.
Tailor-made soluble glycoconjugates are important for
structural biology studies; recent studies have shown
that the structural differences are present in the different
serotypes which can be exploited to differentiate between
the serotypes, and develop better inhibitors.7a,17a–c Thus,
this report describes our efforts to develop soluble glyco-
conjugates that could be used for structural biology
All seven serotypes of botulinum toxin consist of two
fragments, a 50 kDa catalytic domain and a 100 kDa
binding and translocation domain. Several recent re-
ports have targeted the 50 kDa light chain to develop
small molecule inhibitors of the zinc protease,3 however,
suitable drug candidates specific to the heavy chain
Keywords: Glycoconjugates; Multivalent; Botulinum A.
*
Corresponding author. Tel.: +1 513 556 9273; fax: +1 513 556
9239; e-mail: suri.iyer@uc.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.028

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R. R. Kale et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2459–2464
Figure 1. Structures of GT1b (left) and designer divalent ligand (right).
studies or as competitive inhibitors to prevent toxin
binding or find use in diagnostics as front end capture
ligands.
with aqueous ammonia and the amine formed was pro-
tected with a CBz group in high yields.9 Next, the acid
groups were activated using CDMT/NMM and reacted
with 2.2 equivalents of propargyl amine to yield 5. The
terminal alkynes of 5 resonated at 3.15 and 66.0 ppm
We reasoned that a tailored bivalent molecule with car-
bohydrate residues at the termini of variable spacers
would mimic the two arms of the natural ganglioside.
The designer molecule is shown in Figure 1 (right).
The three components of the molecule are the carbohy-
drate residues, the oligoethylene oxide spacers, and the
dimeric scaffold bearing a protected amine at the meta
position. The first critical component is the variability
of the oligoethylene spacers. We have chosen tetraethyl-
ene glycol spacers as our starting material; however, we
can vary the length of the spacers to potentially control
the binding stoichiometry. Shorter spacers should result
in aggregation as the terminal sugars will be prevented
from wrapping around the molecule for a perfect fit.
The bifunctional oligoethylene glycols are designed to
terminate in an azide, which allows for facile 1,3 dipolar
addition to an alkyne bearing molecule.8 The second
component is the dimeric scaffold. The dimeric scaffold
bears two alkyne functionalities and a protected amine.
5-amino-isophthalic acid was chosen as the rigid core
because the two acid functionalities are in the meta po-
sition, reducing the possible formation of a lactone by-
product. The amine functionality is critical as it can be
used to tether the molecule to a surface/magnetic bead/
fluorophore or extended to a higher order dendron.
The third and most important piece of the molecule is
the recognition element. While we have concentrated
on the development of high affinity ligands for botu-
linum neurotoxins, any azide bearing sugar/antibody/
aptamer can be used. This modular design strategy
was utilized to synthesize five compounds (Fig. 2).
1
in the H and 13C NMR spectra in DMSO-d6, respec-
tively (HRMS M+H+ = 447.2182). We were also inter-
ested in studying the binding affinity of multiple sialic
acid residues with the toxin and therefore, we synthe-
sized a tetrameric scaffold as shown in Scheme 2. The
amine group of 1 was protected using Boc anhydride
and the acid groups were activated using CDMT/
NMM and reacted with 2.2 equivalents of propargyl
amine to give 7. Deprotection of the Boc group, fol-
lowed by sequential treatment with bromoacetyl bro-
mide and methanolic ammonia, furnished amine 9.
Two equivalents of 9 was reacted with the carboxylic
acid residues of the amino protected isophthalic acid
derivative 4 in the presence of CDMT/NMM to yield
a dendron, 10, bearing four terminal alkynes, in good
yields. The terminal alkynes resonated at 3.16 and
1
66.0 ppm in the H and 13C NMR spectra in DMSO-
d6, respectively (HRMS M+H+ = 961.3275).
Next, we synthesized two carbohydrate–oligoethylene
conjugates bearing an azide functionality at the termi-
nus of an oligoethylene oxide spacer. Briefly, the ano-
meric acetate of lactose octaacetate 11 was specifically
cleaved in the presence of hydrazine acetate and acti-
vated to trichloroacetimidate 13 using K2CO3. Reaction
with a tetraethylene glycol spacer in the presence of
TMSOTf yielded the desired b-lactoside 14 in reason-
able yields as shown in Scheme 3. The anomeric protons
resonated at 4.53 ppm (J1,2 = 8.0 Hz) and 4.44 ppm
1
(J1,2 = 8.0 Hz) in the H NMR spectrum, respectively,
which is in good agreement with expected values. While
a similar methodology could be used to attach an oligo-
ethylene oxide spacer to the sialic acid residue, we chose
to synthesize the thiosialoside for two reasons. One, the
The synthesis of the dimeric scaffold is shown in
Scheme 1. First, 5-amino isophthalic acid was reacted
with chloroacetyl chloride and subsequently reacted

R. R. Kale et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2459–2464
2461
Figure 2. Structures of homodimers (A and C), mixed heterodimer (B), and tetramers (D and E).
18. The proton of the triazole ring resonated at
8.23 ppm and integrated very well with the acetate pro-
tons in the 1H NMR. 18 was treated with 1.1 equivalent
of azide 14 to give mixed heterodimer which was sub-
jected to acetate deprotection and deesterification using
NaOMe and aqueous NaOH, respectively. The final
product was purified using a Biogel P-2 column and
lyophilized to yield a white foam. The presence of
characteristic signals in the 13C NMR spectrum at
174.9 (–COOH) along with re0sonance signals at 102.0
and 102.9 ppm (C-1 and C-1 from the lactose unit)
and 85.2 ppm (C-2 from the sialic acid unit) further cor-
roborated the formation of compound 19 (HRMS =
1505.4847 for C57H87N12Na3O29S). We are currently
furthering the synthetic methodology to mix and match
sugars on different multivalent scaffolds. All other li-
gands were synthesized and purified using a similar
strategy.
Scheme 1. Reagents and conditions: (a) chloroacetyl chloride, 4 M
NaOH 82%; (b) aq NH3, 12 h, rt, 91%; (c) CBzCl, NaHCO3, rt, 12 h,
79%; (d) propargyl amine, CDMT, NMM, THF/DMF, 0 °C ! rt,
32 h, 81%.
The five compounds (Fig. 2) were examined for cleavage
of SNAP-25 by BoNT/A. SNAP-25 (SyNaptosomal-
Associated Protein, 25 kDa) is the intracellular protein
target selectively cleaved by BoNT/A1,2 and cleavage of
SNAP-25 by BoNT/A provides a convenient in vitro high
throughput assay to test the efficacy of potential inhibi-
tors.12 Briefly, 250 lM solutions of the compounds were
dissolved in PBS buffer and added to 500 pg of BoNT/A.
The solutions were incubated at 37 °C for 1 h and added
to single wells of a cell culture dish containing spinal cord
cells harvested from rats. After incubation for 24 h at
37 °C, LDS buffer was added and subjected to Western
blot analysis using polyclonal antibodies to SNAP-25.
The results are shown in Figure 3. Lanes A, B, C, D,
and E represent compounds A, B, C, D, and E of Figure
2, respectively. Lane F represents the mixture of ligands.
Lane G contains no sample. Lanes H, I and J are controls;
H has anti-toxin, I represents only toxin, and J has no
toxin. The Western blot analysis indicates that significant
inhibition of BoNT/A was achieved with ligands A and D,
and when a mixture of ligands (Lane F) was used. Com-
pounds A and D, and the mixture of compounds caused
significant inhibition of BoNT/A mediated cleavage of
SNAP-25.
formation of the undesirable b-glycoside is limited and
second, the thioglycoside is more stable to cleavage reac-
tions.10 Thio-N-acetyl neuraminic acid 1511 was subse-
quently reacted with
a bifunctional oligoethylene
linker 16 as shown in Scheme 4 to yield a sialic acid -
azide terminated oligoethylene oxide conjugate 17. The
1
reaction was monitored by H and 13C NMR spectros-
copy; loss of the peak at 2.28 ppm in the 1H NMR
and observation of a concomitant peak in the 13C
NMR spectrum at 32.6 ppm confirmed the formation
of the product (HRMS M+H+ = 766.2814). The cou-
pling of the carbohydrate bearing azides to the dimeric
or the tetrameric scaffold was carried out in a mixture
of THF/H2O in the presence of Cu(I) as catalyst.8
Briefly, reaction of 2.2 or 4.2 equivalents of the carbohy-
drate–oligoethylene conjugates with the dimeric (or tet-
rameric) scaffold yielded the desired products. A
representative example is shown in Scheme 5. The di-
meric scaffold 5 was treated with 1.1 equivalent of azide
17 which furnished 57% of the monomeric compound

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R. R. Kale et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2459–2464
Scheme 2. Reagents and conditions: (a) NaOH, di-tert-butyl-dicarbonate, DMF/H2O, rt, 30 h, 67%; (b) propargyl amine, CDMT, NMM, THF/
DMF, 0 °C ! rt, 32 h, 85%; (c) TFA, triisopropylsilane, rt, 8 h, 80%; (d) bromoacetyl bromide, Na2CO3, CH3CN, rt, 96%; (e) NH3 in MeOH, rt,
81%; (f) 4, CDMT, NMM, THF/DMF, 0 °C! rt, 32 h, 78%.
Scheme 3. Reagents and condition: (a) NH2NH2ÆAcOH, THF, 85%; (b) Cl3CCN, K2CO3, DCM, 82%; (c) H(OCH2CH2)3N3, TMSOTf, DCM, 0 °C,
74%.
Scheme 4. Reagents: (a) Et2NH, DMF, 92%.
This study indicates that the penultimate sugars are crit-
ical for tighter binding and the binding pockets are dee-
per. In general, carbohydrate–protein interactions can
be characterized as weak and multivalent binding is nec-
essary to increase the binding affinity.13 However, in this
situation, increase in the valency of the sialic acids does
not lead to an increase in complete inhibition, which is
in good agreement with a recent report that character-
izes the binding of botulinum neurotoxin A with gan-
gliosides as unimolecular.14 Thus, the penultimate
sugars are more critical for tighter binding as opposed
to multivalency, which is in contrast to pathogens such
as Influenza virus, where multivalency is critical for cap-
turing the pathogen.15
In summary, we have used a rational approach to syn-
thesize robust, carbohydrate based ligands for botu-
linum toxin. In this report, we have outlined our

R. R. Kale et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2459–2464
2463
Scheme 5. Reagents: (a) CuSO4, sodium ascorbate, THF/H2O, 57%; (b) 14, CuSO4, sodium ascorbate, THF/H2O, 71%; (c) NaOMe, MeOH ; (d) aq
NaOH, 86% (over two steps).
ever, for complete inhibition, a chimeric ligand incorpo-
rating a peptide and a ganglioside mimic is essential.
This is based on recent crystal structures of botulinum
neurotoxin B with cell surface receptors, which suggests
a double receptor model, where synaptotagmin and the
ganglioside synergistically bind the neurotoxin.17 Our
modular synthesis, presented here, allows us to intro-
duce or delete sugars/peptides and develop high affinity
inhibitors. We are currently exploring these options in
addition to obtaining quantitative binding affinity data
to develop future generation ligands.
Acknowledgments
We thank the Chemical and Biological Sensors Group
at the University of Cincinnati for funding. We also
thank Dr. Steven Macha for obtaining the mass spectral
Figure 3. Western blot analysis depicting the inhibitory capacity of the
ligands. Lanes A, B, C, D, and E represent compounds A, B, C, D, and
data and Dr. Alison Weiss for valuable suggestions.
E of Figure 2, respectively. Lane F represents the mixture of ligands.
Lane G contains no sample. Lanes H, I, and J are controls; H has anti-
toxin, I represents only toxin, and J has no toxin (Please see text for
Supplementary data
details) MM is the molecular marker.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.02.028.
modular synthetic strategy and successfully synthesized
dimeric and tetrameric glycoconjugates. Select first gen-
eration compounds show significant inhibition of
SNAP-25 of BoNT/A in a cell culture based assay.
The compounds could potentially be used to reduce
the toxicity when Bot NT/A is used to treat disorders
associated with the central nervous system.1a,16 How-
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