Multifunctional multivalency: A focused library of polymeric cholera toxin antagonists

Article abstract of DOI:10.1039/c0ob01089h

Structural pre-organization of the multivalent ligands is important for successful interaction with multimeric proteins. Polymer-based heterobifunctional ligands that contain pendant groups prearranged into heterodimers can be used to probe the active site and surrounding area of the receptor. Here we describe the synthesis and activities of a series of galactose conjugates on polyacrylamide and dextran. Conjugation of a second fragment resulted in nanomolar inhibitors of cholera toxin, while the galactose-only progenitors showed no detectable activity.

Full text of DOI:10.1039/c0ob01089h

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PAPER
Multifunctional multivalency: a focused library of polymeric cholera toxin
antagonists†
Huu-Anh Tran,a,b Pavel I. Kitov,a Eugenia Paszkiewicz,a Joanna M. Sadowskaa and David R. Bundle*a
Received 26th November 2010, Accepted 9th February 2011
DOI: 10.1039/c0ob01089h
Structural pre-organization of the multivalent ligands is important for successful interaction with
multimeric proteins. Polymer-based heterobifunctional ligands that contain pendant groups
prearranged into heterodimers can be used to probe the active site and surrounding area of the
receptor. Here we describe the synthesis and activities of a series of galactose conjugates on
polyacrylamide and dextran. Conjugation of a second fragment resulted in nanomolar inhibitors of
cholera toxin, while the galactose-only progenitors showed no detectable activity.
antibiotic resistance6–8 encourages the investigation of alternative
Introduction
therapies that exert less evolutionary pressure, for example, by
Discovery of novel ligands that can disrupt the binding of
targeting bacterial adhesion and colonization or deactivation of
proteins to their target receptors is an important branch of
bacterial toxins. Cholera toxin (CT), an AB5 toxin, is the most
medicinal chemistry. Rational design is based on structural
important virulence factor responsible for the disease. The A
information previously obtained for known complexes, whereas,
subunit is an enzyme that activates adenylate cyclase and elevates
activity screening of available compound libraries or ligands
the level of cyclic AMP, which in turn affects ion transport
obtained by combinatorial chemistry relies on chance and high
across the cell membrane and upsets osmotic balance within the
intestine leading to diarrhea.9,10 The B subunit (CTB) binds to the
surface of the apical side of epithelial cells in the host intestines
and facilitates endocytosis and intracellular transport of the A
subunit. The principle receptor of CT is the complex glycol-
ipid GM1 (Gal-b(1-3)-GalNAc-b(1-4)-[NeuNAc-a(2-3)]-Gal-b(1-
4)-Glc-b(1-3)-ceramide) but other gangliosides (GM2, GD1b) have
also been shown to bind CT.11
A variety of approaches have been pursued in the search for
potent inhibitors of CT. The highest activities were attained
via multivalent presentation of various carbohydrate ligands. A
decameric Starfish-type inhibitor12 applied to cholera toxin was
reported by Zhang et al.13 Octavalent GM1 dendrimer derivatives
exhibited exceptionally high activity14 but more surprisingly a
simple b-galactopyranoside dendrimer constructed by similar
chemistry showed activity comparable to GM1.15 Several reviews
have described progress in this area.16–20
Our research is aimed at development of effective ligands that
would bind CT and could be orally administered to patients to re-
duce toxin burden during Vibrio cholerae infection. Here we report
a novel approach for the design and synthesis of a small library
of heterobifunctional ligands conjugated to a polymer carrier. A
polymer carrier provides a framework that facilitates synthesis,
purification and biological assay of the library members. This
polymer scaffold is also necessary for multivalent presentation
of the ligands to achieve high avidity for the homopentameric
protein, CTB. In the case of gastrointestinal infections when
a drug does not need to cross biological barriers in order to
engage its target, the high molecular weight of the polymeric
ligand might be beneficial to ensure noninvasiveness of anti-toxin
throughput techniques. A fragment-based approach attempts to
capitalize on serendipity and design by incremental improvement
of the lead inhibitor via attachment of moieties that, individually,
bind only weakly to adjacent areas on the protein surface but,
together, substantially enhance the potency of composite ligands.1
The fragment-based approach seems particularly appropriate for
carbohydrate-binding proteins,2–4 where low-molecular-weight in-
hibitors have traditionally met with limited success. Weak intrinsic
binding constants for native ligands often necessitates aggregation
of lectins into multimeric assemblies in order to perform their
function. Functional multivalency is usually expected from the
carbohydrate ligands to achieve biologically relevant strength of
interaction. New methods that allow facile identification of non-
competitive weak binding moieties are required. We propose here
a fragment-based ligand discovery approach that takes advantage
of multivalency in the early phase of the drug development process.
Cholera is a severe diarrheal disease that is caused by con-
sumption of food and water contaminated with the bacterium
Vibrio cholerae. Treatment for Vibrio cholerae consists of fluid
replacement therapy to restore electrolyte balance5 and, in se-
vere cases, administration of antibiotics. Concern with growing
aAlberta Ingenuity Centre for Carbohydrate Science, Department of Chem-
istry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2. E-mail:
dave.bundle@ualberta.ca; Fax: (+1)780-492-7705; Tel: (+1)780-492-8808
bTheraCarb Inc., 243-3553 31st Street N.W., Calgary, Alberta, Canada, T2L
2K7
† Electronic supplementary information (ESI) available: NMR and IR
spectra, solid-phase assay data for inhibition of heat-labile enterotoxin
(LT). See DOI: 10.1039/c0ob01089h
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Fig. 1 General design of a focused library of heterobifunctional polymers.
therapy. In this study we report the generation of a polymer-based
diversity-oriented compound library, in which the pendant ligands
contain an invariable anchor fragment, galactose, and variable
non-galactose fragments, which probe the second part of the GM1
binding site that normally accommodates neuraminic acid (Fig. 1).
to cross-linking and gel formation. Degree of incorporation was
deduced from integration of appropriate signals in NMR spectra.
For functionalization of dextran we followed a two-step pro-
cedure described by Lukyanov et al22 involving first activation of
the polysaccharide with carbonyl diimidazole then introduction
of the linker in the form of a diamine derivative.
Results
Synthesis of heterobifunctional polymers containing a-Gal and
b-Gal moieties
Synthesis of aminated polymers
Two polymers were used in this study: polyacrylamide was
employed for facile synthesis and screening for activity of the
library compounds and dextran was used in combination with
select derivatives as a prototype of a clinically acceptable polymer
for in vivo trials and future drug development (Scheme 1).
Polyacrylamide was rendered amenable for conjugation by par-
tial trans-amination of amide side chains at 80 ◦C, the procedure
is analogous to that previously described for trans-amination of
cross-linked polyacrylamide.21 Increased incubation times led to
increasing content of amino groups in the polymer. Up to 10%
NH2 per repeat unit can be achieved without significant loss due
Two series of galactopyranose derivatives were synthesized in
uniform fashion starting from the corresponding peracetylated
allyl glycosides (Scheme 2). Treatment of 1a and 1b with m-CPBA
gave the corresponding epoxides 2a and 2b, the oxirane ring of
which was opened with azide to provide 3-azido-3-deoxy-glycerol
derivatives 3a and 3b. Activation of the hydroxyl group as a 4-
nitrophenyl carbonate gave 4a and 4b, which were conjugated to a
polymer scaffold to afford key library precursors 5a, 5b, 6a, and 6b.
Introduction of the second component was achieved by con-
jugation of various propargyl derivatives under conditions of
the copper-catalyzed cycloaddition “click chemistry” reaction23
Scheme 1 Preparation of aminated polymeric scaffolds, polyacrylamide and dextran.
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Scheme 2 Synthesis of galactoside containing polymeric precursors of heterobifunctional ligands. Identical procedures were implemented separately for
a- and b-galactoside series.
Scheme 3 Conjugation of the second moiety by “click chemistry”
reaction.
(Scheme 3). Completion of the reaction was confirmed by NMR
spectra and the disappearance in the IR spectra of the charac-
teristic band at 2100 cm-1 corresponding to the azide stretching
frequency.
Scheme 4 Preparation of coating reagent 9 for solid-phase assay.
Evaluation of inhibitory activity
Enzyme-linked immunoassay (ELISA) was performed in 96-
well plates coated with a GM1 N-acylated glycoside 9, which
was synthesized from GM1-oligosaccharide (GM1OS) 824 via
amination with liquid ammonia followed by acylation with the
NHS ester of 10-undecenoic acid (Scheme 4). We found that
during amination of a reducing sugar in liquid ammonia addition
of ~1% water improved the yield and accelerated the reaction
approximately 10 fold.
CTB (Sigma) conjugated to horseradish peroxidase (HRP) was
incubated in the wells in the presence of a serially diluted inhibitor.
After washing with buffer the presence of bound CTB was detected
by color reaction with 3,3¢,5,5¢-tetramethylbenzidine (TMB). As
positive control, serially diluted 8 was included on every plate to
ensure consistent assay conditions. For routine screening, one well
per data point was used while assays for select compounds were
performed in triplicates. The results are presented in Table 1.
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Table 1 Inhibitory activities of heterobifunctional polymers against CT
Alkyne derivative 7.n
a-Gal polymer
IC50, mMa
b-Gal polymer
IC50, mMa
None
None
5a
Inactive
5b
Inactive
6a
Inactive
6b
Inactive
5a1
828 (hb = 1.0)
5b1
584 (hb = 0.9)
5a2
5a3
832 (h = 1.3)
623 (h = 1.1)
5b2
5b3
1246 (h = 1.0)
630 (h = 0.8)
5a4
5a5
5a6
5a7
5a8
1286 (h = 0.85)
Inactive
5b4
5b5
5b6
5b7
5b8
629 (h = 0.9)
1934
153 (h = 4.3)
339 (h = 1.5)
389 (h = 2.6)
122 (h = 3.3)
325 (h = 1.6)
230 (h = 2.1)
5a9
200 (h = 3.8)
243 (h = 3.0)
5b9
156 (h = 2.6)
225 (h = 3.0)
5a10
5b10
5a11
80 (h = 1.8)
5b11
104 (h = 3.0)
5a12
5a13
108 (h = 1.9)
100 (h = 2.7)
5b12
5b12
124 (h = 3.1)
58 (h = 2.5)
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Table 1 (Contd.)
Alkyne derivative 7.n
a-Gal polymer
5a14
IC50, mMa
b-Gal polymer
5b14
IC50, mMa
0.24 (h = 0.4)
1.8 (h = 0.6)
5a15
5.9 (h = 0.6)
5b15
5.2 (h = 0.5)
5a16
5a17
45 (h = 1.4)
61 (h = 3.0)
5b16
5b17
23 (h = 1.3)
53 (h = 2.2)
5a18
103 (h = 3.5)
5b18
109 (h = 3.3)
5a19
5a20
5a21
0.9 (h = 0.2)
0.3 (h = 0.3)
Inactive
5b19
5b20
5b21
0.023 (h = 0.4)
0.19 (h = 0.3)
1934
5a22
5a23
5a24
209 (h = 2.5)
0.17 (h = 0.3)
31 (h = 2.3)
5b22
5b23
5b24
169 (h = 3.5)
0.02 (h = 0.3)
39 (h = 2.6)
5a25
3.2 (h = 0.8)
5b25
9.0 (h = 0.45)
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Table 1 (Contd.)
Alkyne derivative 7.n
a-Gal polymer
5a26
IC50, mMa
b-Gal polymer
5b26
IC50, mMa
8.2 (h = 0.7)
0.06 (h = 0.25)
5a27 (3%)
0.0055 (h = 0.3)
5b27 (3%)
0.014 (h = 0.4)
5a27 (5%)
5a27 (9%)
6a27 (6.6%)
0.0012 (h = 0.4)
0.0014 (h = 0.7)
0.005 (h = 0.3)
5b27 (5%)
5b27 (9%)
6b27 (6.6%)
0.013 (h = 0.2)
0.056 (h = 0.3)
0.018 (h = 0.2)
5a28 (3%)
5a28 (9%)
6a28 (6.6%)
0.07 (h = 0.3)
0.008 (h = 0.3)
0.004 (h = 0.2)
5b28 (3%)
5b28 (9%)
6b28 (6.6%)
0.02 (h = 0.3)
0.01 (h = 0.3)
0.024 (h = 0.3)
a activities per pendant ligand. b Hill coefficient.
of side chains. Polymer-supported synthesis is often used for
generation of compound libraries.29–32 We propose to employ
a polymeric support both to facilitate synthesis and biological
assays of novel ligands. Since the basic anchoring ligand was only
a monosaccharide a multivalent scaffold was also necessary to
permit detection of weak activities.
High solubility in water and ease of modifications established
polyacrylamide as a standard polymeric carrier used in studies on
multivalency.33–35 However, limited solubility of polyacrylamide
in organic solvents imposes substantial limitations to the scope
of chemical modifications. Since dipolar cycloaddition of alkynes
to azides known as “click chemistry” is one of a few reactions
suitable for efficient conjugation in aqueous solutions, we utilized
it to generate a library of pendant ligands. Both a-Gal and b-Gal
glycosides 3a and 3b were used as an invariant pendant ligand
in the corresponding series of heterobifunctional inhibitors. The
mixture of water and DMSO was found to be an appropriate media
for performing conjugation of a large variety of alkyne derivatives
under homogeneous conditions.
The original polymers 5a and 5b that are devoid of the variable
fragment failed to inhibit cholera toxin at 10 mg mL-1 and were
considered inactive under the assay conditions. Surprisingly, both
hetero-bifunctional ligands 5a1 and 5b1 containing neuraminic
acid as a complementary ligand have shown only partial inhibition
at the same high concentration. The length of the linker is sufficient
for both Gal and Neu to reach their respective positions in the GM1
binding site on the surface of cholera toxin; however, entropy loss
due to linker flexibility offsets the contribution from this additional
interaction.
The galactose moiety occupies the deepest indentation in
the GM1 binding pocket on the surface of cholera toxin.36
Thermodynamic analysis of the CTB-GM1 interaction37 identified
galactose as the keystone for affinity and selectivity albeit with
intrinsic dissociation constant of ~50 mM. The other important
fragment, neuraminic acid, which binds with Kd 210 mM, would
Discussion
Due to the synthetic complexity of the native ligand, GM1
oligosaccharide, several attempts to simplify the structure have
been reported. Bernardi and co-workers have designed a number
of conformationally restricted GM1 mimetics, in which the core
lactose moiety is replaced by a conformationally constrained cis-
cyclohexanediol-1,2, thus, reducing synthetic complexity some-
what while relying on both Gal and Neu for the interaction.25
Recently, they evaluated a large set of non-hydrolysable Gal-Neu
dimers.26 Hol and Fan have discovered m-nitrophenyl galactoside
(MNPG) to be about 50–100 fold more potent than galactose.27
However, since none of the unimeric mimics approached the
activity level of GM1OS (100–200 nM) most researchers resort
to multivalency for the construction of viable inhibitors.13–20 The
crystal structure of the CTB-GM1OS complex shows that the
˚
nearest bound ligands are separated by ~31 A as measured between
the anomeric oxygen atoms of the terminal galactose. Therefore,
spanning this distance requires long linker-arms making multi-
valent display rather challenging though rewarding considering
the substantial activity enhancements that could be attained. In
recent years, a variety of dendrimers and polymers bearing ligands
as simple as galactose monosaccharide15 to ligands as complex
as the GM1 pentasaccharide14,28 were employed to achieve up
to subnanomolar inhibitors; albeit very high activities were only
observed with pendant GM1 pentasaccharide ligands. Tailored
multivalent ligands possessing the same 5-fold symmetry as CTB
were shown to attain activities that approach that of GM1OS
even though just a simple monosaccharide, meta-nitrophenyl a-
galactopyranoside (MNPG), was used as specific ligand.13
In our study, we needed a multivalent platform for generation
and screening of focused compound libraries. Polymers provide
convenient albeit less structurally defined scaffolds for multimeric
display of a ligand. The separation distance between pendant
ligands is tunable by controlling the degree of incorporation
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have never been selected as a lead ligand in fragment-based design
of inhibitors. A large negative enthalpy of neuraminic acid binding
to CTB is largely offset by an almost equal and unfavourable
entropy term (negative entropy). Nevertheless, essentially only
these two monosaccharides, galactose and neuraminic acid,
contribute substantially to formation of the GM1-CTB complex,
a lectin-carbohydrate complex with one of the highest known
intrinsic affinity constants. The rest of the GM1 pentasaccharide
forms a scaffold that severely restricts the conformational space
sampled by Gal and Neu in solution37 and, thus, greatly reduces
the entropic penalties that GM1 has to pay upon binding to
the protein. Recently reported CTB inhibitors containing Gal
and Neu connected via flexible linkers show modest activities in
mM range,26 again underscoring the importance of optimal rigid
scaffold.
Most of the sulfonamide derivatives (5a2–5a10 and 5b2–
5b10) that were generated from arbitrarily chosen, commercially
available sulfonyl chlorides had a modest contribution to binding,
while some carbamate analogs (i.e. 5b12) exceeded the activity of
neuraminic acid derivative by 10 fold. Further activity improve-
ments were observed when substituted benzamides 5a14–5a28 and
5b14–5b28 were tested. For several compounds, IC50 values in the
low nanomolar region were attained.
While activities of most compounds in two parallel series
were not affected by the anomeric form of the galactopyranose
residue, some analogs showed a substantial difference. Of these,
the b-analogs had substantially higher activity. An important
exception, the a-Gal analog of p-fluorobenzamide derivative,
had superior inhibitory power not only with respect to the b-
counterpart but generally to all tested compounds. This, in some
cases dramatic, difference between a- and b-series is indicative
of specific interactions of the variable ligand with the protein
surface.
In an attempt to further increase activities of the multivalent
ligands presenting mono- and difluorobenzamide moieties we
explored the influence of pendant ligand density. Generally,
no significant avidity gains were observed for polymers with
a higher payload of heterobifunctional head groups (Table
1, entries 5.27 and 5.28). This modification caused the sol-
ubility of the polymers to be substantially decreased com-
pared to the standard 3% payload on polyacrylamide. Ac-
tivities of dextran-based polymers were generally similar to
polyacrylamide analogs (Table 1, entries 6.27 and 6.28). The
greater water solubility of dextran over polyacrylamide and
its biodegradability renders dextran-based heterobifunctional
ligands promising drug candidates in future biological evalua-
tion.
Fig. 2 Bell-shape relationship between avidities and Hill coefficients.
activities (Fig. 3, 5b12), while high and low avidity ligands show
weak concentration dependence. Moreover, the highest avidity
ligands present even more complex shape of binding curves: part
of the curve corresponding to low concentration of inhibitor
can be fitted to near perfect log function with a Hill coefficient
close to unity and higher. However, when approximately 80–90%
inhibition is attained the second, extremely shallow part of the
curve begins and complete inhibition is finally attained at high
mM concentrations. Several derivatives of 7.27 and 7.28 have also
been tested for inhibitory activity against heat labile toxin (LT),
a toxin homologous to CT and produced by enteropathogenic
E. coli (ESI,† Table 1S). Activities in the picomolar range were
observed for some compounds with Hill coefficients close to
unity. However, although less pronounced, an inhibitory profile
(resistance to complete inhibition after reaching a certain limit)
similar to that with CT was observed.
The multivalent format for measuring activities permits higher
discrimination between pendant ligands with similar affinity due
to amplification of small differences in binding energies. What is
rarely discussed though is that the multistage binding of multi-
valent ligands may result in a staggered shape of the inhibition
curve and apparent decrease in Hill coefficients. Curiously, when
data from the Table 1 are plotted as a scatter graph, a bell shaped
correlation between Hill coefficients and avidity of the polymeric
inhibitors could be observed (Fig. 2). It appears that the shape
of the binding curve depends not on the nature of the polymeric
scaffold or the pendant ligand but solely on the range of IC50
value. Very sharp inhibition curves are observed for midrange
Fig. 3 Representative examples of solid-phase inhibition data.
Increase in Hill coefficients with increase in avidity followed
by some reduction of their value upon further avidity increase
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was previously observed for dendrimer-based ligands for Cholera
toxin on a limited set of inhibitors,14 the present work may permit
generalization of this phenomenon. As a tentative explanation for
the observed tendency we propose to rationalize reduction of Hill’s
coefficients in terms of a multistep binding of CTB to the plate due
to incomplete saturation of multiple binding sites by the flexible
polymeric ligand. At low concentrations of the polymeric ligand,
only one polymer chain binds to the pentameric toxin. Since the
intrinsic binding constant for the pendant ligands is low, the intra-
molecular binding results in only partial saturation of the binding
sites. Therefore, the initial part of the inhibition curve appears as a
normal log curve but reaches a limit corresponding to 1 : 1 complex
formation. At this point no free toxin is left. The second phase
requires inhibition of the binding of 1 : 1 complex between toxin
and the single chain of a multivalent inhibitor. The binding of the
second chain of the inhibitor to this complex is less efficient since
fewer binding sites are available for this interaction. As a result,
the complete inhibition of toxin occurs at substantially higher
concentration of the inhibitor. The partially inhibited species of
CTB are able to bind to the plate via binding sites that remain free
and, after washing off the ligand, a fraction of these weakly bound
toxin species rebinds via the liberated binding sites and remains on
the plate. This process may be facilitated by essentially irreversible
deposition of the toxin to a GM1 coated plate as revealed by
detailed investigation of ELISA conditions.38
instead of measuring low intrinsic binding constants of unimeric
ligands.
Two pendant hetero-bifunctional ligands, which possessed the
higher inhibitory power when presented on polyacrylamide, were
evaluated as univalent analogs 10 and 11. They show approx-
imately 100-fold higher activities than galactose measured in a
similar solid phase assay and compare well with a benchmark uni-
valent ligand, MNPG, which also contains only a monosaccharide
binding fragment (Fig. 5). The apparent 10-fold discrepancy in the
activities of MNPG as reported in literature39 and obtained in this
work could be explained by different solid-phase assay conditions,
particularly, the different ganglioside, GD1b, previously employed
as the solid phase capture reagent for cholera toxin. This ligand
is known to have substantially weaker affinity for the toxin than
GM1.11
The idealized mechanism, a process involving only two steps, is
illustrated in Fig. 4; the higher heterogeneity of the real polymeric
ligand would result in more staggered binding phases and less
distinct transitions than shown in Fig. 3. Two step inhibition
due to significant interaction of toxin species with partially
saturated binding sites on a GM1-coated plate results in the
staggered shape of the inhibition curve. With these caveats, the
ELISA assay is clearly suitable for ranking multivalent inhibitors
according to their relative potencies. The uniform preparation
enables direct comparison of affinities in a multivalent format
Fig. 5 Inhibitory activities of unimeric analogs.
Conclusions
A polymer-supported focused library of hetero-bifunctional lig-
ands assisted in identifying weak ligands that have the potential to
complement the affinity of galactose for cholera toxin by binding
to a complementary binding site on the protein surface. The
choice of the linker separating Gal from the second fragment
was dictated by synthetic expediency and was not intended to
provide a comparable level of structural pre-organization to that
seen in GM1. The relatively high activities were achieved here
via multivalent presentation rather than as the result of high
intrinsic binding constants, which explains the shallow shape of
the inhibition curves and difficulty to attain complete inhibition
of CTB binding to GM1-coated plates. Optimization of the
heterobivalent scaffold should be the next step in search for potent
cholera toxin antagonists.
Fig. 4 Proposed mechanism of sequential inhibition of multivalent
proteins by polymeric inhibitors.
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mixture of d, 1 H total, OH), 2.17 (2 s, 3 H total, OAc), 2.08 (m, 6
H, OAc), 2.00 (s, 3 H total, OAc); 13C NMR (125 MHz, CDCl3):
d 170.46, 170.43, 170.16, 170.07, 170.06, 169.61, 169.57, 101.97,
101.82, 72.83, 72.20, 71.04, 71.02, 70.71, 70.70, 69.75, 69.60, 68.80,
68.77, 66.98, 66.95, 61.49, 61.43, 53.10, 53.0, 20.77, 20.644, 20.640,
20.63, 20.62, 20.55; ES HRMS: calcd for C17H25N3O11Na ([M +
Na]+) 470.13813, found 470.13824.
Experimental
General methods
Commercially available reagents were used as supplied without
further purification. Evaporation and concentration in vacuo
was conducted under water-aspirator pressure. All solution-phase
reactions were carried out under a nitrogen atmosphere. Reactions
were monitored by analytical thin-layer chromatography (TLC)
with pre-coated silica gel 60 F254 glass plate (Merck). Plates
were visualized under UV light or stained by treatment with
either cerium ammonium molybdate solution or 5% sulfuric
acid in ethanol followed by heating at 180 ◦C. Purification of
products was conducted by column chromatography using silica
(R,S)-3-azido-2-(4-nitrophenyloxycarbonyl)oxypropyl 2,3,4,6-
tetra-O-acetyl-a-D-galactopyranoside (4a). To a stirred solution
of alcohol 3a (2.53 g, 5.66 mmol) and p-nitrophenyl chloroformate
(1.37 g, 6.79 mmol) in dry CH2Cl2 (35 mL) pyridine (0.69 mL,
8.5 mmol) was added dropwise at room temperature. The reaction
mixture was stirred at room temperature for 1.5 h. After the
removal of solvent, the resulting residue was purified by flash
chromatography, eluting with EtOAc–hexane (1 : 1) to yield 4a
(3.28 g, 97%) as a colourless solid: mp 42–44 ◦C; IR (KRS-5
˚
R
gel SiliaFlashF60 (40–63 mm, 60 A) from SiliCycleꢀ Inc. Optical
rotations were measured with a Perkin Elmer 241 polarimeter at
22 ◦C. Melting points (mp) were determined on a Gallenkamp
melting point apparatus and uncorrected. IR data were recorded
on Nic-Plan IR Microscope (solid film); only signals corresponding
to functional groups indicative to the structure are reported. NMR
spectra were recorded at 400, 500 or 600 MHz, at 27 ◦C in CDCl3
or D2O. Coupling constants are first order and chemical shifts
are referenced to residual solvent (CDCl3) at 7.24 ppm for 1H and
77.0 ppm for 13C and relative to 0.1% external acetone at 2.225 ppm
1
disk) n 2109 cm-1; H NMR (300 MHz, CDCl3): d 8.34–8.27
(m, 2 H, arom.), 7.47–7.40 (m, 2 H, arom.), 5.47 (m, 1 H, H-4),
5.37–5.31 (m, 1 H, H-3), 5.22–5.01 (m, 3 H, H-1, H-2, CH),
4.29–4.20 (m, 1 H, H-5), 4.13–3.59 (m, 6 H, H-6a, H-6b, CH2O,
CH2N), 2.15–2.01 (m, 12 H, OAc); 13C NMR (125 MHz, CDCl3):
d 170.40, 170.37, 170.35, 170.31, 170.13, 170.03, 169.99, 155.22,
155.19, 151.87, 151.84, 145.70, 145.64, 125.42, 125.40, 121.88,
121.77, 96.88, 96.65, 76.08, 76.05, 67.99, 67.96, 67.86, 67.84,
67.32, 67.30, 66.84, 66.72, 66.29, 61.62, 61.60, 50.45, 50.19, 20.71,
20.70, 20.67, 20.65, 20.63; ES HRMS: calcd for C24H28N4O15Na
([M + Na]+): 635.14434, found: 635.14460.
1
for H for solution in D2O. Electrospray ionization mass spectra
were recorded on a Micromass Zabspec TOF-mass spectrometer.
(R,S)-3-azido-2-hydroxypropyl 2,3,4,6-tetra-O-acetyl-a-D-gala-
ctopyranoside (3a). A suspension of (R,S)-2,3-epoxypropyl
2,3,4,6-tetra-O-acetyl-a-D-galactopyranoside40 (2a, 3.41 g,
8.43 mmol), NaN3 (1.64 g, 25.3 mmol), NH4Cl (1.35 g,
(R,S)-3-azido-2-(4-nitrophenyloxycarbonyl)oxypropyl 2,3,4,6-
tetra-O-acetyl-b-D-galactopyranoside (4b). The title compound
4b (96%) was obtained from 3b as described for 4a as a colourless
◦
25.3 mmol) in dry DMF (20 mL) was stirred at 55 C for 7.5 h
◦
1
solid: mp 50–52 C; IR (KRS-5 disk) n 2107 cm-1; H NMR
(500 MHz, CDCl3): d 8.34–8.28 (m, 2 H, arom.), 7.49–7.41 (m,
2 H, arom.), 5.42 (d, 1 H, J3,4 3.3 Hz, H-4), 5.27–5.22 (m, 1 H,
H-2), 5.09–5.04 (m, 2 H, H-3, CH), 4.57–4.54 (m, 1 H, H-1),
4.20–4.09 (m, 3 H), 3.97–3.94 (m, 1 H, H-5), 3.86–3.82 (m, 1 H),
3.67–3.58 (m, 2 H), 2.18 (s, 3H, OAc), 2.09–2.04 (m, 6 H, OAc),
2.01 (s, 3H, OAc); 13C NMR (125 MHz, CDCl3): d 170.38, 170.14,
170.07, 169.54, 169.47, 155.29, 155.24, 151.81, 145.62, 125.37,
121.86, 121.79, 101.73, 101.14, 76.25, 76.09, 71.01, 70.99, 70.65,
70.59, 68.55, 68.41, 67.41, 67.08, 66.90, 61.20, 50.47, 50.42, 20.73,
20.65, 20.56; ESI HRMS: calcd for C24H28N4O15Na ([M + Na]+):
635.14434, found: 635.14472.
then cooled and diluted with water (150 mL). The aqueous
solution was extracted with EtOAc (4 ¥ 50 mL). The combined
organic layers were washed with saturated aqueous NH4Cl (2 ¥
100 mL), brine (1 ¥ 100 mL), dried over anhydrous mgSO4 and
filtered. After the removal of solvent, the resulting residue was
purified by flash chromatography, eluting with EtOAc–CH2Cl2
(35 : 65), to yield 3a (2.67 g, 71%) as a colourless, oily mixture of
diastereomers: [a]D +118.05 (c 1.04, CHCl3); IR (KRS-5 disk) n
2098 cm-1, 1H NMR (300 MHz, CDCl3): d 5.46 (dd, 1 H, J3,4 3.3
Hz, J4,5 1.2 Hz, H-4), 5.33 (dd, 1 H, J3,4 3.4 Hz, J2,3 10.6 Hz, H-3),
5.20–5.13 (m, 2 H, H-1, H-2), 4.30–4.24 (m, 1 H, H-5), 4.12–3.34
(m, 7 H, H-6a, H-6b, CH2O, CH2N, CHOH), 2.52 and 2.45 (2d,
1 H total, OH), 2.15 (s, 3 H, OAc), 2.08 (2 s, 3 H total, OAc), 2.06
(2 s, 3 H total, OAc), 2.00 (s, 3 H, OAc), 13C NMR (125 MHz,
CDCl3): d 170.49, 170.48, 170.26, 170.24, 170.18, 170.06, 97.15,
96.92, 70.42, 70.36, 69.46, 69.45, 68.01, 68.00, 67.98, 67.96, 67.47,
67.42, 66.78, 66.69, 61.83, 61.79, 53.36, 53.08, 20.75, 20.74, 20.69,
20.65, 20.63; ES HRMS: calcd for C17H25N3O11Na ([M + Na]+)
470.13813, found 470.13795.
Synthesis of trans-aminated polyacrylamide
To a solution of polyacrylamide (10 g for payload 3% and
5%, 5 g for payload 9%) in water (50 mL) 2,2¢-(ethylene-
dioxy)bis(ethylamine) (50 mL) was added and the reaction mixture
was stirred at 80 ◦C for 20 min (for payload 3%), 40 min (for
payload 5%), or 90 min (for payload 9%) then heat was removed
and ice was added. The mixture was extensively dialyzed against
distilled water then freeze-dried to give colourless powder.
(R,S)-3-azido-2-hydroxypropyl 2,3,4,6-tetra-O-acetyl-b-D-gala-
ctopyranoside (3b). The title compound 3b (58%) was obtained
from 2b40 as described for 3a. [a]D +7.76 (c 1.02, CHCl3); IR (KRS-
Synthesis of galactoside azidopolyacrylamide 5a and 5b
5 disk) n 2103 cm-1, H NMR (300 MHz, CDCl3): d 5.40 (dd, 1
1
H, J3,4 3.2 Hz, J4,5 1.0 Hz, H-4), 5.21 (dd, 1 H, J1,2 7.9 Hz, J2,3 10.5
Hz, H-2), 5.03 (dd, 1 H, H-3), 4.51 (2 : 1 mixture of d, 1 H total,
H-1), 4.16 (m, 2 H, CH2), 3.97 (m, 1 H, H-5), 3.93–3.72 (m, 3 H,
CH2O, CHOH), 3.40–3.30 (m, 2 H, CH2N), 2.98 and 2.81 (2 : 1
To a viscous solution of trans-aminated polyacrylamide (1.00 g) in
water (13 mL) a solution of carbonates 4a or 4b (0.60 g, 1.0 mmol)
in DMSO (8 mL) was added dropwise at room temperature
followed by Et3N (0.15 mL). The reaction mixture was tumbled at
3666 | Org. Biomol. Chem., 2011, 9, 3658–3671
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room temperature for 3 days. Aqueous NH4OH (3 mL) was added
and the reaction mixture was tumbled for 1 day then extensively
dialyzed against water and lyophilized to yield colourless foam.
IR confirmed incorporation of azido groups, and 1H NMR
showed clear peaks of the galactose moiety on the polymer. The
payload was determined by integration of appropriate galactose
and polymer signals.
General synthesis of propargylsulfonamides
To a stirred solution of propargylamine (200 mL, 3.12 mmol)
and Et3N (0.65 mL, 4.7 mmol) in dichloromethane (10 mL) ◦the
corresponding sulfonyl chloride (3.75 mmol) was added at 0 C.
The reaction mixture was stirred at room temperature for 2–16 h.
After removal of the solvent, the resulting residue was purified
by flash chromatography, eluting with EtOAc–hexane to yield
sulfonamides in moderate to high yields.
a-Galactoside azidopolyacrylamide 5a: colourless foam (71%).
IR (KRS-5 disk) n 2106 cm-1.
N-(Prop-2-ynyl)methanesulfonamid (7.2).42
b-Galactoside azidopolyacrylamide 5b: colourless foam (79%).
IR (KRS-5 disk) n 2106 cm-1.
N -(Prop-2-ynyl)-(S)-(+)-10-camphorsulfonyl-methanesulfo-
namide (7.3) as a colourless solid (70%): mp 96–97 ◦C; IR (solid)
n 2115 cm-1; 1H NMR (300 MHz, CDCl3): d 6.09 (s, br, 1 H, NH),
4.1 (ddd, 1 H, J 2.5 Hz, J 8.5 Hz, J 18.2 Hz, propargyl CH2), 3.94
(ddd, 1 H, J 1.2 Hz, J 2.5 Hz, J 18.2 Hz, propargyl CH2), 3.73 (d,
1 H, J 15.2 Hz, CH2S), 3.06 (d, 1 H, J 15.2 Hz, CH2S), 2.43 (ddd, 1
H, J 2.4 Hz, J 4.9 Hz, J 18.6 Hz, CH2), 2.32 (t, 1 H, propargyl CH),
2.16–1.92 (m, 5 H, CH2), 1.51–1.42 (m, 1 H, CH), 1.01 (s, 3 H,
CH3), 0.94 (s, 3 H, CH3). 13C NMR (100 MHz, CDCl3): d 217.85,
80.08, 74.02, 60.38, 52.55, 49.80, 43.79, 43.50, 33.80, 28.35, 27.82,
20.74, 20.09; ES HRMS calcd for C13H19NO3SNa ([M + Na]+)
292.0978, found 292.0977.
Synthesis of aminated dextran
To a stirred, clear solution of dextran (Mn ~100–200 kDa, 2.00 g,
12.3 mmol of glucose) in formamide (50 mL) a solution of 1,1¢-
carbonyldiimidazole (0.50 g, 3.1 mmol) in formamide (15 mL)
was added at room temperature. The reaction mixture was stirred
at room temperature for 6 h then it was added dropwise 2,2¢-
(ethylenedioxy)bis(ethylamine) (135 mL). The reaction mixture
was stirred at room temperature overnight, and extensively
dialyzed using dialysis tube (MWCO 12 000–14 000) for 3 d, and
lyophilized to yield aminated dextran (1.52 g, 72%) as a colourless
foam. 1H NMR is consistent with the conjugate structure.
4-Methyl-N-prop-2-ynylbenzenesulfonamide (7.4).43
4-Isopropyl-N-prop-2-ynylbenzenesulfonamide (7.5) as a colour-
less solid (59%): mp 51–52 ◦C; IR (KRS-5 disk) n 2120 cm-1; 1H
NMR (300 MHz, CDCl3): d 7.81 (m, 2 H), 7.36 (m, 2 H), 4.84 (s,
1 H, NH), 3.83 (dd, 2 H, J 2.5 Hz, J 6.1 Hz, NCH2), 2.98 (m, 1 H,
CHMe2), 2.09 (t, 1 H, J 2.5 Hz, CH), 1.27 (d, 6 H, J 7.0, CH3).13C
NMR (100 MHz, CDCl3): d 154.54, 136.71, 127.51, 127.16, 78.05,
72.87, 34.17, 32.84, 23.65; ES HRMS calcd for C12H15NO2SNa
([M + Na]+) 260.07157, found 260.07125.
Synthesis of galactoside azidodextran 6a and 6b
To a solution of aminated dextran (200 mg, 70 mmol) in water
(2 mL) a solution of carbonate 4a or 4b (85 mg, 140 mmol) in
DMSO (8 mL) was added at room temperature followed by Et3N
(0.15 mL). The reaction mixture was tumbled at room temperature
for 3 d then aqueous NH4OH (3 mL) was added and the mixture
was tumbled for 24 h then dialyzed extensively against water using
dialysis tube (MWCO 12 000–14 000) for 3 d, and lyophilized
to yield colourless foam. IR confirmed incorporation of azido
groups, and 1H NMR showed clear peaks of the galactose moiety
on the polymer.
4-tert-Butyl-N-prop-2-ynylbenzenesulfonamide (7.6) as a colour-
less solid (84%): mp 61–62 ◦C; IR (solid) n 2112 cm-1; 1H NMR
(600 MHz, CDCl3): d 7.82 (m, 2 H, arom.), 7.53 (m, 2 H, arom.),
4.78 (s, 1 H, NH), 3.83 (dd, 2 H, J 2.7 Hz, J 6.0 Hz, CH2), 2.10 (t, 1
H, J 2.5 Hz, CH), 1.35 (s, 9 H, CH3); 13C NMR (100 MHz, CDCl3):
d 156.83, 136.35, 127.22, 126.06, 78.05, 72.86, 35.15, 32.86, 31.07;
ES HRMS calcd for C13H17NO2SNa ([M + Na]+) 274.0872, found
274.0872.
a-Galactoside azidodextran 6a: colourless foam (95%). IR
4-Nitro-N-prop-2-ynylbenzenesulfonamide (7.7).44
(KRS-5 disk) n 2105 cm-1.
4-Nitro-N-(1-ethynylcyclohexyl)benzenesulfonamide (7.8) as a
b-Galactoside azidodextran 6b: colourless foam (100%). IR
(KRS-5 disk) n 2107 cm-1.
◦
colourless solid (35%): mp 144–145 C; IR (KRS-5 disk) n 2117
cm-1; 1H NMR (300 MHz, CDCl3): d 8.33 (m, 2 H, arom.), 8.11 (m,
2 H, arom.), 5.00 (s, 1 H, NH), 2.15 (s, 1 H, CH), 2.1–2.0 (m, 2 H,
CH2), 1.72–1.50 (m, 7 H, CH2), 1.30–1.20 (m, 1 H, CH2); 13C NMR
(100 MHz, CDCl3): d 149.89, 147.62, 128.92, 123.83, 83.01, 74.60,
54.80, 38.93, 24.70, 22.22; ES HRMS calcd for C14H16N2O4SNa
([M + Na]+) 331.07230, found 331.07214.
(Prop-2-ynyl 5-acetamido-3,5-dideoxy-D-glycero-a-D-galacto-2-
nonulopyranosid)onic acid (7.1). To a solution of methyl (prop-
2-ynyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-
a-D-galacto-2-nonulopyranosid)onate41 (31.8 mg, 60.0 mmol) in
MeOH (1 mL) aqueous 1.5 M NaOH (0.3 mL) was added. The
mixture was stirred at room temperature for 24 h, DOWEX (H+)
was added and the mixture was concentrated, taken up in water
and freeze-dried to give the◦title compound (9.8 mg, 95%) as a
colourless foam; mp > 250 C (dec. upon heating); IR (solid) n
2126 cm-1; 1H NMR (400 MHz, D2O): d 4.37 (d, 1 H, Jgem 16 Hz
CH2), 4.23 (d, 1 H, Jgem 16 Hz, CH2), 3.93–3.55 (m, 7 H, H-4, H-5,
H-6, H-7, H-8, H-9a, H-9b), 2.73 (dd, 1 H, J3a,4 4.6 Hz, J3a,3b 12.5
Hz, H-3a), 2.02 (s, 3 H, NHAc), 1.67 (t, 1 H, J3b,4 12.5 Hz, H-3b).
13C NMR (125 MHz, D2O): d 176.05, 173.77, 101.58, 80.07, 73.80,
72.63, 69.17, 69.12, 63.61, 53.49, 52.76, 41.24, 23.00; ES HRMS
calcd for C14H20NO9 ([M-H]-) 346.1144, found 346.1137.
4-Acetyl-N-prop-2-ynylbenzenesulfonamide (7.9) as a colourless
◦
solid (94%): mp 110–111 C; IR (KRS-5 disk) n 2129 cm-1; H
NMR (400 MHz, DMSO-d6): d 8.11 (d, 2 H, J 8.4 Hz, arom.),
7.92 (d, 2 H, J 8.4 Hz, arom.), 4.93 (t, 1 H, J 5.8 Hz, NH), 3.93 (dd,
2 H, J 2.4 Hz, J 6.0 Hz, CH2), 2.68 (s, 3 H, CH3), 2.10 (t, 1 H, J 2.4
Hz, CH); 13C NMR (125 MHz, CDCl3): d 196.83, 143.71, 140.25,
128.92, 128.85, 127.70, 77.60, 73.36, 32.93, 26.91; ES HRMS calcd
for C11H11NO3SNa ([M + Na]+) 260.03519, found 260.03505.
N-Acet◦ylsulfanilylamide (7.10) as a colourless solid (80%): mp
178–179 C (dec.); IR (KRS-5 disk) n 2121 cm-1; 1H NMR
(400 MHz, DMSO-d6): d 10.29 (s, 1 H, NH), 7.96 (t, 1 H, J 6
1
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Hz, NH), 7.72 (m, 4 H, arom.), 3.64 (dd, 2 H, J 2.5 Hz, J 6.0 Hz,
CH2), 3.03 (t, J 2.5 Hz, 1 H, CH), 2.0 (s, 3 H, CH3); 13C NMR
(100 MHz, DMSO-d6): d 168.95, 142.83, 133.98, 127.82, 118.39,
79.40, 74.50, 31.84, 24.09; ES HRMS calcd for C11H12N2O3SNa
([M + Na]+) 275.04609, found 275.04602.
ES HRMS calcd for C10H7N3O5Na ([M + Na]+) 272.02779, found
272.02772.
3,4,5-Trimethoxy-N-(prop-2-ynyl)benzamide (7.18) as a colour-
less solid (87%): mp 56–57 ◦C; IR (solid) n 2116 cm-1; 1H NMR
(400 MHz, CDCl3): d 7.02 (s, 2 H, arom.), 6.57 (t, 1 H, J 4.9
Hz, NH), 4.22 (dd, 2 H, J 2.5 Hz, J 5.3 Hz, CH2), 3.86 (s, 9 H,
OMe), 2.26 (t, 1 H, J 2.5 Hz, CH); 13C NMR (100 MHz, CDCl3): d
166.94, 153.16, 141.11, 129.09, 104.51, 79.52, 71.74, 60.87, 56.28,
29.82. ES HRMS calcd for C13H15NO4Na ([M + Na]+) 272.0893,
found 272.0895.
General synthesis of N-propargyl carbamates
To a stirred solution of propargylamine (100 mL, 1.56 mmol)
and pyridine (189 mL, 2.34 mmol) in dry CH2Cl2 (5 mL) the
corresponding chloroformate (1.87 mmol) was added at 0 ◦C. The
reaction mixture was stirred overnight at room temperature. After
the removal of solvent, the resulting residue was purified by flash
chromatography to yield respective carbamates.
4-Cyano-N-(prop-2-ynyl)benzamide (7.19) as a colourless solid
◦
1
(98%): mp 174–175 C; IR (KRS-5 disk) n 2235, 2120 cm-1; H
NMR (400 MHz, CDCl3): d 7.90 (d, 2 H, J 8.2 Hz, arom.), 7.76 (d,
2 H, J 8.2 Hz, arom.), 6.33 (s, 1 H, NH), 4.28 (dd, 2 H, J 2.5 Hz,
J 5.1 Hz, CH2), 2.32 (t, 1 H, J 2.5 Hz, CH); 13C NMR (100 MHz,
CDCl3): d 164.53, 137.68, 132.45, 128.08, 118.22, 113.81, 80.80,
73.13, 28.65; ES HRMS calcd for C11H8N2ONa ([M + Na]+)
207.05288, found 207.05277.
N-(2,2,2-Trichloroethyloxycarbonyl)propargylamine (7.11) as a
col◦ourless oil (93%) which crystallized upon standing: mp 48–
49 C; IR (KRS-5 disk) n 2128 cm-1; 1H NMR (400 MHz, CDCl3):
d 5.23 (s, 1 H, NH), 4.75 (s, 2 H, CH2), 4.04 (dd, 2 H, J 2.5
Hz, J 5.6 Hz, CH2), 2.29 (t, 1 H, J 2.5 Hz, CH); 13C NMR
(100 MHz, CDCl3): d 154.09, 95.24, 78.87, 74.75, 72.16, 31.08; ES
HRMS calcd for C6H6NO2Cl3Na ([M + Na]+) 251.93563, found
251.93570.
4-(Methylthio)-N-(prop-2-ynyl)benzamide (7.20) as a colourless
solid (87% yield): mp 136–137 ◦C; IR (KRS-5 disk) n 2113 cm-1;
1H NMR (400 MHz, CDCl3): d 7.70 (m, 2 H, arom.), 7.26 (m, 2 H,
arom.), 6.26 (s, 1 H, NH), 4.25 (dd, 2 H, J 2.5 Hz, J 5.2 Hz, CH2),
2.51 (s, 3 H, CH3), 2.28 (t, 1 H, J 2.5 Hz, CH); 13C NMR (100 MHz,
CDCl3): d 166.53, 143.91, 129.77, 127.39, 125.39, 79.51, 71.86,
29.76, 14.99; ES HRMS calcd for C11H11NOSNa ([M + Na]+)
228.04536, found 228.04518.
N-(Benzyloxycarbonyl)propargylamine (7.12).45
N-(4-Nitrobenzyloxycarbonyl)prop◦argylamine (7.13) as
a
colourless solid (70%): mp 125–126 C; IR (KRS-5 disk) n 2116
1
cm-1; H NMR (300 MHz, CDCl3): d 8.21 (d, 2 H, J 8.1 Hz,
4-tert-Butyl-N-(prop-2-ynyl)benzamide (7.21) as a colourless
arom.), 7.51 (d, 2 H, J 8.1 Hz, arom.), 5.22 (s, 2 H, CH2), 5.09 (s, 1
H, NH), 4.01 (dd, 2 H, J 2.3 Hz, J 5.4 Hz, CH2), 2.27 (t, 1 H, J 2.4
Hz, CH); 13C NMR (100 MHz, CDCl3): d 155.35, 147.65, 143.61,
128.17, 123.75, 79.29, 71.88, 65.51, 30.95; ES HRMS calcd for
C11H10N2O4Na ([M + Na]+) 257.05339, found 257.05328.
◦
1
solid (90%): mp 116–117 C; IR (KRS-5 disk) n 2117 cm-1; H
NMR (400 MHz, CDCl3): d 7.73 (m, 2 H, arom.), 7.45 (m, 2 H,
arom.), 6.39 (s, 1 H, NH), 4.25 (dd, 2 H, J 2.6 Hz, J 5.2 Hz,
CH2), 2.27 (t, 1 H, J 2.6 Hz, CH), 1.33 (s, 9 H, CH3); 13C NMR
(100MHz, CDCl3): d 167.06, 155.28, 130.86, 126.88, 125.51, 79.67,
71.70, 34.93, 31.13, 29.69; ES HRMS calcd for C14H17NONa ([M +
Na]+) 238.12024, found 238.11994.
General synthesis of N-propargyl amides
2,4,6-Trichloro-N-(prop-2-ynyl)benzamide (7.22) as a colourless
◦
1
To a stirred solution of proparylamine (150 mL, 2.34 mmol)
and pyridine (284 mL, 3.51 mmol) in dry CH2Cl2 (7.5 mL) the
corresponding acyl chloride (2.81 mmol) was added at 0 ◦C. The
reaction mixture was stirred overnight at room temperature. After
the removal of solvent, the resulting residue was purified by flash
chromatography to yield N-(prop-2-ynyl)amide derivatives.
N-(prop-2-ynyl)benzamide (7.14).46
solid (72%): mp 165–166 C; IR (KRS-5 disk) n 2101 cm-1; H
NMR (300 MHz, CDCl3): d 7.33 (s, 2 H, arom.), 6.08 (broad s,
1 H, NH), 4.26 (dd, 2 H, J 2.6 Hz, J 5.3 Hz, CH2), 2.29 (t, 1 H,
J 2.6 Hz, CH); 13C NMR (100 MHz, CDCl3): d 163.35, 136.03,
133.77, 133.03, 128.11, 78.27, 72.35, 29.73; ES HRMS calcd for
C10H6NOCl3Na ([M + Na]+) 283.94072, found 283.94079.
4-Chloro-N-(prop-2-◦ynyl)benzamide (7.23) as a colourless solid
(92%): mp 145–146 C; IR (solid) n 2120 cm-1; 1H NMR
(300 MHz, CDCl3): d 7.73 (m, 2 H, arom.), 7.42 (m, 2 H, arom.),
6.33 (s, 1 H, NH), 4.25 (q, 2 H, J 2.5 Hz, CH2), 2.29 (t, 1 H, CH);
13C NMR (100 MHz, CDCl3): d 166.04, 138.10, 132.07, 128.89,
128.45, 79.23, 72.05, 29.86; ES HRMS calcd for C10H8NClONa
([M + Na]+) 216.0187, found 216.0187.
3-Cyano-N-(prop-2-ynyl)benzamide (7.15) as a colourless solid
◦
(92%): mp 132–133 C; IR (KRS-5 disk) n 2234 cm-1;1H NMR
(300 MHz, DMSO-d6): d 9.14 (t, 1 H, J 5.4 Hz, NH), 8.26 (t, 1
H, J 1.7 Hz, arom.), 8.15 (dt, 1 H, J 1.3 Hz, J 8.0 Hz, arom.),
8.01 (dt, 1 H, J 1.4 Hz, J 7.7 Hz, arom.), 7.69 (t, 1 H, J 7.8 Hz,
arom.), 4.07 (dd, 2 H, J 2.5 Hz, J 5.5 Hz, CH2), 3.14 (t, 1 H, J
2.5 Hz, CH); 13C NMR (100 MHz, DMSO-d6): d 164.12, 134.88,
134.71, 132.02, 130.89, 129.82, 118.22, 111.53, 80.76, 73.20, 28.66;
ES HRMS calcd for C11H8N2ONa ([M + Na]+) 207.05288, found
207.05275.
2,5-Difluoro-N-(prop-2-ynyl)benzamide (7.24) as a colourless
solid (84%): mp 73–74 ◦C; IR (KRS-5 disk) n 2009 cm-1; 1H NMR
(500 MHz, CDCl3): d 7.83–780 (m, 1 H, arom.), 7.19–7.10 (m, 2
H, arom.), 6.94 (s, 1 H, NH), 4.28 (m, 2 H, CH2), 2.30 (t, 1 H, J
2.5 Hz, CH); 13C NMR (100 MHz, DMSO-d6): d 160.5 (d, JCF 332
Hz), 155.2 (d, JCF 246 Hz), 124.7 (dd, JCF 7.5 Hz, JCF 17.5 Hz),
119.1 (dd, JCF 9.0 Hz, JCF 24.0 Hz), 118 (dd, JCF 8 Hz, JCF 25.5
Hz), 116.2 (d, JCF 25 Hz), 80.63, 73.08, 28.59; ES HRMS calcd for
C10H7NOF2Na ([M + Na]+) 218.03879, found 218.03875.
4-Nitro-N-(prop-2-ynyl)benzamide (7.16).47
3,5-Dinitro-N-(prop-2-ynyl)benzamide (7.17) as a colourless
◦
1
solid (91%): mp 132–133 C; IR (KRS-5 disk) n 2127 cm-1; H
NMR (300 MHz, CDCl3): d 9.19 (m, 1 H, arom.); 8.98 (m, 2 H,
arom.), 6.6 (broad s, 1 H, NH), 4.34 (dd, 2 H, J 2.5 Hz, J 5.2 Hz,
CH2), 2.37 (t, 1 H, J 2.5 Hz, CH); 13C NMR (100 MHz, DMSO-
d6): d 162.47, 148.76, 136.79, 128.15, 121.58, 80.87, 74.15, 29.57;
2,4-Difluoro-N-(prop-2◦-ynyl)benzamide (7.25) as a colourless
solid (93%): mp 70–71 C; IR (solid) n 2116 cm-1; 1H NMR
3668 | Org. Biomol. Chem., 2011, 9, 3658–3671
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(300 MHz, CDCl3): d 8.20–8.11 (m, 1 H, arom.), 7.01 (td, 1 H, J
6.6 Hz, J 8.9 Hz, arom.), 6.88 (ddd, 1 H, J 2.4 Hz, J 8.4 Hz, J
12.0 Hz), 6.83 (s, br, 1 H, NH), 4.27 (m, 2 H, CH2), 2.29 (t, 1 H,
J 2.6 Hz, CH); 13C NMR (100 MHz, CDCl3): d 164.95 (dd, JCF
13.4 Hz, JCF 256.0 Hz), 162.00, 161.00 (dd, JCF 12.6 Hz, JCF 250.4
Hz), 133.92 (d, JCF 10.5 Hz), 112.47 (d, JCF 21.0 Hz), 104.27 (t, JCF
107.2 Hz), 79.13, 71.87, 29.71; ES HRMS calcd for C10H7NOF2Na
([M + Na]+) 218.03879, found 218.03869.
CH2), 3.81 and 3.72 (2dd, total 1 H, J 4.5 Hz, J 10.5 Hz, CH2),
3.58 and 3.48 (2dd, total 1 H, J 5.8 Hz, J 10.5 Hz, CH2), 2.147
and 2.145 (2 s, total 3 H, OAc), 2.10 and 2.09 (2 s, total 3 H, OAc),
2.05 and 2.03 (2 s, total 3 H, OAc), 2.01 (s, 3 H, OAc), 1.75 (s, 1H,
OH); 13C NMR (125 MHz, CDCl3): d 170.59, 170.52, 170.26,
170.18, 170.17, 170.15, 170.10, 166.44, 165.87, 163.86, 144.59,
144.51, 130.11, 130.08, 129.47, 129.40, 123.89, 123.87, 115.73,
115.56, 97.11, 97.02, 70.15, 70.10, 69.01, 68.99, 68.02, 67.95, 67.92,
67.90, 67.47, 67.42, 66.74, 66.65, 61.71, 52.92, 52.68, 35.42, 20.79,
20.78, 20.73, 20.68, 20.64; ES HRMS calcd. for C27H33FN4O12Na
([M + Na]+) 647.19712, found 647.19642.
3,4,5-Trifluoro-N-(prop-2-ynyl)benzamide (7.26) as a colourless
◦
1
solid (93%): mp 122–123 C; IR (solid) n 2130 cm-1; H NMR
(300 MHz, CDCl3): d 7.46 (m, 2 H, arom.), 6.37 (s, 1 H, NH), 4.24
(dd, 2 H, J 2.5 Hz, J 5.2 Hz, CH2), 2.31 (t, 1 H, J 2.5 Hz, CH);
13C NMR (100 MHz, CDCl3): d 164.04, 151.15 (dd, JCF 7.7 Hz,
JCF 252.25 Hz), 142.16 (dt, 14.6 Hz, JCF 257.81 Hz), 111.86 (m),
78.72, 72.35, 30.07; ES HRMS calcd for C10H6NOF3Na ([M +
Na]+) 236.02937, found 236.02955.
3-(4-((3,4-Difluorobenzamido)methyl)-1H-1,2,3-triazol-1-yl)-2-
(R,S)-hydroxypropyl 2,3,4,6-tetra-O-acetyl-a-D-galactopyranoside
(13). To a clear, stirred solution of 3a (120 mg, 268 mmol) and
3,4-difluoro-N-(prop-2-ynyl)benzamide (7.28) (105 mg, 536 mmol)
in H2O–MeOH (1 : 10, 11 mL) aqueous 1 M sodium ascorbate
(80.4 mL, 80.4 mmol) and aqueous CuSO4·0.5H2O (0.05 M, 268 mL,
13.4 mmol) were added then stirred for 22 h at room temperature.
After removal of the solvent, the resulting residue was purified
by flash chromatography (EtOAc) to yield a diastereoisomeric
mixture 13 (154 mg, 89%) as a colourless foam: IR (KRS-5 disk)
n 3354, 3078, 2939, 1746, 1651, 1606, 1510, 1227, 1051, 775, 760;
1H NMR (500 MHz, CDCl3): d 7.81 (d, 1 H, J 2.4 H, triazole),
7.74–7.70 (m, 2 H, arom.), 7.61–7.60 (m, 1 H, NH), 7.21 (q, 1 H,
J 8.5 Hz, arom.) 5.46 (m, 1 H, H-4), 5.34 and 5.30 (2dd, total 1
H, J3,2 10.4 Hz, J3,4 3.3 Hz, H-3), 5.18–5.14 (m, 2 H, H-1, H-2),
4.67–4.65 (m, 2 H, CH2), 4.59–4.530 (m, 1 H), 4.47–4.41 (m, 1 H),
4.34–4.22 (m, 2 H), 4.10 (dd, 2 H, J 2.9 Hz, 6.5 Hz, CH2), 3.82 and
3.74 (2dd, total 1 H, J 4.6 Hz, J 10.6 Hz, CH2), 3.56 and 3.50 (2dd,
total 1 H, J 4.9 Hz, J 10.6 Hz, CH2), 2.16 (s, 3 H, OAc), 2.09 (s, 3
H, OAc), 2.04 and 2.02 (2 s, total 3 H, OAc), 2.01 (s, 3 H, OAc);
13C NMR (125 MHz, CDCl3): d 170.64, 170.57, 170.31, 170.30,
170.22, 170.20, 170.16, 165.39, 153.65, 153.54, 151.62, 151.52,
151.22, 151.12, 149.25, 149.15, 144.31, 144.27, 144.24, 144.21,
130.89, 123.77, 117.52, 117.38, 117.07, 116.92, 97.05, 96.93, 70.06,
70.02, 68.94, 68.91, 68.05, 67.98, 67.94, 67.91, 67.48, 67.43, 66.70,
66.61, 61.71, 53.13, 52.93, 35.24, 20.77, 20.67; ES HRMS calcd.
for C27H32F2N4O12Na ([M + Na]+) 665.18770, found 665.18676.
4-Fluoro-N-(prop-2-ynyl)benzamide (7.27).48
3,4-Difluoro-N-(prop-2◦-ynyl)benzamide (7.28) as a colourless
solid (92%): mp 53–54 C; IR (solid) n 2124 cm-1; 1H NMR
(300 MHz, CDCl3): d 7.67 (ddd, 1 H, J 2.2 Hz, J 7.5 Hz, J 10.5
Hz, arom.), 7.53 (m, 1 H, arom.), 7.23 (ddd, 1 H, J 7.6 Hz, J 8.6
Hz, J 9.7 Hz, arom.), 6.26 (s, 1 H, NH), 4.25 (dd, 2 H, J 2.6 Hz,
J 5.2 Hz, CH2), 2.30 (t, 1 H, J 2.6 Hz, CH); 13C NMR (100 MHz,
CDCl3): d 164.04, 151.15 (dd, JCF 9.9 Hz, JCF 252.9 Hz), 142.16
(dt, JCF 14.4 Hz, JCF 257.8 Hz), 129.55 (d, JCF 3.7 Hz), 111.87 (m),
78.72, 72.35, 30.07; ES HRMS calcd for C10H7NOF2Na ([M +
Na]+) 218.03879, found 218.03866.
General procedure for synthesis of polymeric heterobifunctional
compounds 5an, 5bn 6an, and 6bn
To a stirred solution of an azido-polymer 5a, 5b, 6a, or 6b (20 mg,
7.0 mmol) and an alkyne derivative 7.n (14 mmol) in DMSO–
H2O (3 : 2 by volume, 2.5 mL) aqueous sodium ascorbate (1 M,
84 mL, 84 mmol) was added followed by aqueous CuSO4·0.5H2O
(0.05 M, 168 mL, 8.4 mmol) at room temperature. The progress of
the reaction was monitored during 3–5 d by disapperance of the
characteristic N3 band in IR spectra. When completed the reaction
was quenched by addition of aqueous EDTA (0.5 M, 1 mL), and
the mixture was dialyzed extensively against deionized water using
dialysis tube with MWCO 12 000–14 000. Lyophilization of the
residue yielded the corresponding conjugate.
3-(4-((4-Fluorobenzamido)methyl)-1H-1,2,3-triazol-1-yl)-2-
(R,S)-hydroxypropyl a-D-galactopyranoside (10). To a stirred
solution of 12 (120 mg, 1.9 mmol) in MeOH (10 mL) at room
temperature was added aqueous NH4OH (1 mL). The reaction
mixture was stirred overnight at room temperature. After the
removal of solvent, the resulting residue was purified by column
chromatography on silica gel (EtOAc–acetone) to yield a mixture
of diastereoisomers 10 (68 mg, 78%) as a colourless foam: IR
(KRS-5 disk) n 3358 (br, s), 2931, 1640, 1604, 1559, 1505, 1456,
3-(4-((4-Fluorobenzamido)methyl)-1H-1,2,3-triazol-1-yl)-2-
(R,S)-hydroxypropyl 2,3,4,6-tetra-O-acetyl-a-D-galactopyranoside
(12). To a clear, stirred solution of 3a (120 mg, 268 mmol)
and 4-fluoro-N-(prop-2-ynyl)benzamide (7.27, 95 mg, 536 mmol)
in H2O–MeOH (1 : 10, 11 mL) aqueous 1 M sodium ascorbate
(80.4 mL, 80.4 mmol) and aqueous CuSO4·0.5H2O (0.05 M, 268 mL,
13.4 mmol) were added at room temperature. The reaction mixture
was stirred at room temperature for 72 h. After removal of the
solvent, the resulting residue was purified by flash chromatography
(EtOAc) to yield a diastereoisomeric mixture 12 (60%) as a
colourless foam: IR (KRS-5 disk) n 3367, 3058, 2934, 1748, 1650,
1605, 1541, 1503, 1373, 1233, 1055, 855, 768 cm-1; 1H NMR
(500 MHz, CDCl3): d 7.85–7.75 (m, 3 H, triazole, arom.), 7.15–
7.10 (m, 3 H, NH, arom.), 5.48–5.46 (m, 1 H, H-4), 5.35 and 5.30
(2dd, total 1 H, J3,2 10.6 Hz, J3,4 3.3 Hz, H-3), 5.20–5.14 (m, 2 H,
H-1, H-2), 4.70 (m, 2 H, CH2), 4.58–4.52 (m, 1 H, H-5), 4.47–4.40
(m, 1 H), 4.33–4.26 (m, 2 H), 4.10 (dd, 2 H, J 4.3 Hz, J 6.4 Hz,
1
1340, 1233, 1059, 853, 767 cm-1; H NMR (500 MHz, D2O): d
7.96 (s, 1 H, triazole), 7.81–7.75 (m, 2 H, arom.), 7.20 (t, 2 H, J
8.5 Hz, arom.), 4.94–4.92 (m, 1 H, H-1), 4.65–4.48 (m, 4 H, CH2),
4.33–4.28 (m, 1 H, CH), 3.96–3.94 (m, 1 H, H-4), 3.92–3.68 (m, 6
H, H-2, H-3, H-5, H-6a, H-6b, CH2), 3.55 (dd, 0.5 H, J 4.2 Hz,
J 10.6 Hz, CH2), 3.46 (dd, 0.5 H, J 6.0 Hz, J 10.7 Hz, CH2); 13
C
NMR (125 MHz, D2O): d 170.59, 166.70, 166.09, 164.71, 156.92,
152.10, 145.56, 130.59, 130.52, 130.36, 125.58, 125.52, 116.64,
116.46, 99.72, 99.55, 72.01, 71.95, 70.29, 70.27, 70.12, 69.64, 69.61,
69.59, 69.52, 69.28, 62.04, 53.64, 53.49, 35.87; ES HRMS calcd.
for C19H25FN4O8Na ([M + Na]+) 479.15486, found 479.15450.
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3-(4-((3,4-Difluorobenzamido)methyl)-1H-1,2,3-triazol-1-yl)-
2-(R,S)-hydroxypropyl a-D-galactopyranoside (11). The title
compound (78 mg, 90%, colourless foam) was obtained as a
mixture of diastereoisomers as described for 10 by deacetylation
of 13: IR (KRS-5 disk) n 3350 (br, s), 2931, 1648, 1606, 1558, 1512,
and incubated at room temperature overnight, then washed (6¥)
with PBST (0.05% Tween 20 in phosphate buffer saline, PBS).
Inhibitor at decreasing concentrations (starting at 20 mg mL-1,
dilution factor 3.16; 50 mL well-1) was mixed with cholera toxin
B-subunit conjugated to horseradish peroxidase (Sigma, 20 ng
mL-1; 50 mL well) and the mixtures were applied to the plates.
After incubation at room temperature for 2 h the plates were
washed (6¥) with PBST. 3,3:5,5-Tetramethylbenzidine solution
(TMB purchased from KPL, 100 mL well-1) was added. After
15 min incubation the reaction was stopped by addition of H3PO4
(100 mL well-1) and absorbance was measured at 450 nm.
1
1427, 1300, 1108, 1060, 777 cm-1; H NMR (500 MHz, D2O): d
8.05 (s, 1 H, triazole), 7.74–7.71 (m, 1 H, arom.), 7.65–7.63 (m, 1
H, arom.), 7.44–7.39 (m, 1 H, arom.), 5.02 (m, 1 H, H-1), 4.73–
4.57 (m, 4 H, CH2), 4.40–4.37 (m, 1 H, CH), 4.04–4.03 (m, 1 H,
H-4), 4.00–3.76 (m, 6 H, H-2, H-3, H-5, H-6a, H-6b, CH2), 3.64
(dd, 0.5 H, J 4.3 Hz, J 10.7 Hz, CH2), 3.54 (dd, 0.5 H, J 6.0 Hz, J
10.6 Hz, CH2); 13C NMR (125 MHz, D2O): d 169.42, 154.48,
154.38, 152.47, 152.37, 151.82, 151.72, 149.85, 149.75, 145.48,
131.30, 125.71, 125.66, 125.26, 125.23, 125.20, 125.17, 118.74,
118.60, 117.75, 117.60, 99.82, 99.65, 72.11, 72.05, 70.39, 70.36,
70.22, 69.75, 69.71, 69.69, 69.61, 69.37, 69.14, 53.74, 53.59, 35.99;
ES HRMS calcd. for C19H24F2N4O8Na ([M + Na]+) 497.1454,
found 497.1451.
Acknowledgements
This work was funded by Alberta Ingenuity Centre for Car-
bohydrate Science, a Grand Challenges Exploration grant to
TheraCarb from the Bill and Melinda Gates Foundation and
an Alberta Innovates R&D Industrial Associate Award to H.-A.
Tran.
N -b-D-Galactopyranosyl-(1→3)-2-acetamido-2-deoxy-b-D-
galactopyranosyl-(1→4)-[(5-amino-3,5-dideoxy-D-glycero-a-D-
galacto - non - 2 - ulosonic acid) - yl - (1→3)] - b - D - galacto-
pyranosyl-(1→4)-b-D-glucopyranosyl-undecyl-10-enylamide (9).
A tightly enclosed vial containing solution of GM1OS 8 obtained
as previously described24 (28 mg 0.028 mmol) in wet liquid
ammonia (~2 mL, ~20 mL water added) was incubated at 60 ◦C in
oil bath (caution: possible explosion, use protective gear!) for 24
h. The vial was cooled in dry ice-acetone and opened. Ammonia
was allowed to escape; then the mixture was taken up in water
and freeze-dried. To a solution of the residue in methanol (2 mL)
and Et3N (12 mL) 10-undecenoic acid NHS ester49 (3 eq., 24 mg)
in DCM (0.5 mL) was added slowly at room temperature. After
16 h the mixture was concentrated and purified by HPLC on
C18 column in gradient water–MeOH to give the title compound
(20.6 mg, 63%), [a]D +9.5 (c 1.05, water); IR (solid) n 3351, 2927,
2856, 1639, 1411, 1076 cm-1; 1H NMR (600 MHz, D2O, Gal1 and
Gal2 assignments belong to the middle and the terminal galactose
moieties correspondingly): d 5.94–5.88 (m, 1 H, CH CH2),
5.06–5.02 (m, 1 H, CH CH2), 4.98–4.95 (m, 2 H, J 9.1 Hz,
H-1Glc, CH CH2), 4.78 (d, 1 H, J 8.4 Hz, H-1GalNAc), 4.52 (d, 2
Notes and references
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,
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105.62, 103.42, 103.38, 102.51, 81.22, 79.99, 78.89, 78.02, 77.24,
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