Synthesis and affinity evaluation of a small library of bidentate cholera toxin ligands: Towards nonhydrolyzable ganglioside mimics

Article abstract of DOI:10.1002/chem.200902469

A small library of nonhydrolyzable mimics of GM1 ganglioside, featuring galactose and sialic acid as pharmacophoric carbohydrate residues, was synthesized and tested. All compounds were synthesized from readily available precursors using high-performance reactions, including click chemistry protocols, and avoiding O-glycosidic bonds. Some of the most active molecules also feature a point of further derivatization that can be used for conjugation with polyvalent aglycons. Their affinity towards cholera toxin was assessed by weak affinity chromatography, which allowed a systematic evaluation and selection of the best candidates. Affinity could be enhanced up to one or two orders of magnitude over the affinity of the individual pharmacophoric sugar residues.

Full text of DOI:10.1002/chem.200902469

FULL PAPER
DOI: 10.1002/chem.200902469
Synthesis and Affinity Evaluation of a Small Library of Bidentate Cholera
Toxin Ligands: Towards Nonhydrolyzable Ganglioside Mimics
[b]
Pavel Cheshev,[a] Laura Morelli,[a] Marco Marchesi,[a] Crtomir Podlipnik,
ˇ
Maria Bergstrçm,[c] and Anna Bernardi*[a]
Abstract: A small library of nonhydro-
lyzable mimics of GM1 ganglioside,
featuring galactose and sialic acid as
pharmacophoric carbohydrate residues,
was synthesized and tested. All com-
pounds were synthesized from readily
available precursors using high-perfor-
mance reactions, including click
chemistry protocols, and avoiding O-
glycosidic bonds. Some of the most
active molecules also feature a point of
further derivatization that can be used
for conjugation with polyvalent agly-
cons. Their affinity towards cholera
toxin was assessed by weak affinity
chromatography, which allowed a sys-
tematic evaluation and selection of the
best candidates. Affinity could be en-
hanced up to one or two orders of
magnitude over the affinity of the indi-
vidual pharmacophoric sugar residues.
Keywords: carbohydrates · cholera
toxin · combinatorial chemistry ·
glycosides · weak affinity chroma-
tography
Introduction
of protein–carbohydrate interactions. For medicinal chemis-
try, they may also provide key insights for the structure-
based design of glycomimetic drug leads.[2] A rational design
of galactose-based ligands for CT and LT has been reported
and recently reviewed.[3] A series of CT ligands designed to
mimic the three-dimensional structure of GM1 ganglioside
has been reported[4] and C-galactosides have been used as
scaffolds to prepare a small library of nonhydrolyzable in-
hibitors of binding.[5]
The B pentamer of CT (CTB) interacts with the soluble,
monovalent oligosaccharide portion of GM1 (Galb1-3Gal-
NAcb1-4(Neu5Aca2-3)Galb1-4Glc, GM1os, Scheme 1) with
a strong affinity (43 nm at room temperature, as measured
by isothermal calorimetry (ITC)).[6] The X-ray structure of
the CTB-GM1os complex[7] shows a “two-fingered grip” of
the sugar on the toxin, created by a sialic acid “thumb” and
a Galb1-3GalNAc “forefinger”. The terminal galactose resi-
due in the forefinger reaches a well-defined galactose bind-
ing pocket, which is lined by the indole side chain of Trp-88
and shielded from the solvent. The rest of the toxin binding
site is shallow and exposed to solvent. The sialic acid
(NeuAc) thumb interacts with a carboxylate binding region,
which includes one highly conserved crystallographic water
molecule. It has been shown by ITC that the individual “fin-
gers” interact very weakly with the toxin (with dissociation
constant in the high mm range),[6] so the high binding affinity
of the CTB–GM1os pair has been credited to structural pre-
organization of GM1os,[8] which allows a near lock-and-key
interaction with CTB.
Cholera toxin (CT) belongs to the AB5 bacterial toxins
family, named after the characteristic architecture compris-
ing a single catalytically active component, A, and a nontox-
ic receptor-binding pentamer of B subunits.[1] The B5 pen-
tamer is responsible for binding to GM1 ganglioside at the
surface of host cells; the complete AB5 holotoxin is required
for the toxic effects. The CT family includes enterotoxins
that are responsible for several disorders, from the relatively
mild travellerꢀs diarrhea (from E. coli heat-labile toxin, LT)
to the much more serious cholera. Complexes formed be-
tween gangliosides and AB5 toxins offer a paradigmatic
model for studying the structural and thermodynamic basis
[a] P. Cheshev, L. Morelli, M. Marchesi, A. Bernardi
Dipartimento di Chimica Organica e Industriale
Universitꢁ degli Studi di Milano
via Venezian 21
20133 Milano (Italy)
Fax : (+39)02-50314072
E-mail: anna.bernardi@unimi.it
ˇ
[b] C. Podlipnik
Faculty of Chemistry and Chemical Technology
University of Ljubljana, Ljubljana (Slovenia)
[c] M. Bergstrçm
School of Pure and Applied Natural Sciences
University of Kalmar, 391 82 Kalmar (Sweden)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902469.
Chem. Eur. J. 2010, 16, 1951 – 1967
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1951

Scheme 1. GM1os, some known CT ligands (ps-GM1, MNPG), and gen-
eral structure of the proposed library. The pharmacophoric sugar frag-
ments are highlighted in the boxes.
Scheme 2. General strategy for library assembly.
This analysis suggests that functional mimics of GM1 may
be designed by stringing together the two pharmacophoric
sugar fragments, galactose and sialic acid, while trying to
preserve the relative orientation adopted in GM1os. This
concept was implemented in our initial work by generating
pseudo-GM1 (psGM1, Scheme 1) structures that are close
mimics of GM1os.[4] Like other known inhibitors of CT
binding, such as meta-nitrophenylgalactoside (MNPG,
Scheme 1),[3] this molecule contains enzymatically labile O-
glycosidic linkages that limit their potential application in
vivo. Furthermore, the synthetic methods used to connect
the pharmacophoric sugar moeties in psGM1 are those of
traditional carbohydrate chemistry, which are often labori-
ous and low-yielding procedures. To develop metabolically
stable and synthetically more accessible mimics of GM1os,
we set our efforts towards the synthesis of a library of bi-
functional compounds of the general formula shown in
Scheme 1. The target molecules contain a galactose and a
sialic acid moiety connected through a linker. By taking ad-
vantage of the terminal alkyne of the linker and the ready
availablility of the sialic acid azide 26,[9] a triazole was used
to connect the NeuAc residue to the linker (Scheme 2)
through a Cu-catalyzed (click) triazole formation reaction.[10]
The library design (Scheme 2) relied on combinatorial selec-
tion of the alkyne linkers and the galactose-mimicking frag-
ments, with the following two constraints: 1) O-glycosides
were excluded to stabilize the constructs against the activity
of hydrolytic enzymes; 2) the functionalization of the galac-
tose ring (X in Schemes 1 and 2) was chosen to allow a
facile conjugation to the putative linkers. Selection of the
linker and identification of the best bidentate ligand was
achieved by synthesizing a small library of compounds and
testing their CTB affinity by weak affinity chromatography
(WAC). Herein we report our initial results.
Results and Discussion
Synthesis of the library: A library of bidentate ligands was
synthesized using linear alkyne linkers by following the
strategy outlined in Scheme 2. Four functionalized Gal frag-
ments were conjugated through an amide bond to linear
alkyne linkers of variable length to obtain linker-armed gal-
actose mimics. Incorporation of the sialic acid residue using
click chemistry was planned as the final step of the synthe-
sis, since Cu-catalyzed cycloaddition is known to tolerate a
wide range of substrates, solvents, and reaction conditions.
The four functionalized Gal fragments (Scheme 3) used
were: b-galactosyl amine 1, a- or b-C-aminoethyl galacto-
sides 2 and 3, respectively, and b-carboxylic acid 4. All of
these fragments can be connected by amide bonds to the
alkyne linkers to set the stage for the final “click” sialylation
step. b-Galactosyl amine 1 is a well known and readily avail-
able compound.[11] The remaining three C-galactose deriva-
tives were obtained from known, easily accessible a-C-allyl
galactoside[12] via a-C-galactosyl aldehyde 5[13] (Scheme 3).
The crucial step of the synthesis of b-C-galactosides 3 and
4 was the inversion of configuration of 5 to the b-C-galacto-
syl aldehyde 6, which was achieved by using l-proline catal-
ysis, as recently described.[14] However, scale up of this step
was complicated by proline-catalyzed autocondensation of 6
during work up. The problem was solved by using polyeth-
AHCTUNGTRENNUNG
ylene glycol (PEG)-supported proline[15] as the catalyst,
which can be precipitated from the reaction mixture before
work up. With this modification of the reported procedure,
yields improved from moderate to excellent and purification
issues reported in the original paper were resolved (we have
performed the reaction on up to 1.5 g of aldehyde 5 with
99% yield of 6).
Oxidation of 6 with 2,2,6,6-tetramethylpiperidine-N-oxyl
(TEMPO) and bis
ACHUTGTNRENNUG[acetoxyACHTUNGTNER(NUGN iodo)]benzene (BAIB) in a 1:1
mixture of acetonitrile and water,[16] afforded acid 4 in good
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Bidentate Cholera Toxin Ligands
FULL PAPER
butynylamine to afford alkynes
24
and
25,
respectively
(Scheme 5).
Finally, Cu-catalyzed (click)
cycloaddition of alkynes 15–25
with fully acetylated sialyl azide
26[9] under Sharpless reaction
conditions (CuSO4, sodium as-
corbate)
(Scheme 6). Complete depro-
tection of peracetylated cou-
was
performed[10]
AHCTUNGTRENNUNG
pling products 27–37 gave the
target bidentate adducts 38–48.
These compounds can be
grouped into three general fam-
ilies of ligands according to the
anomeric composition and con-
figuration of the galactose
moiety. For clarity, in the rest
of the text these families will be
identified as: b-Gal-N (com-
pounds 38–40), a-Gal-C (41–
Scheme 3. The functionalized Gal fragments used in this work.
43), and b-Gal-C (44–48).
As will be shown, compounds
yields (Scheme 4). Since direct reductive amination of a-
and b-C-galactosyl aldehydes 5 and 6 to the corresponding
amines 2 and 3 was complicated by an N-bisACTHNUGRTENUNGalkylation pro-
cess and proceeded with poor yield, a two-step procedure
via intermediate formation of azides 7 and 8 was used in-
stead (Scheme 4).
of the b-Gal-C family and, in particular, the pentynoic acid
derivative 44, turned out to be the most active members of
this library. Thus, the library was expanded to include mole-
cules featuring an additional branching point on the penty-
noic acid backbone. Such a functional group on the linker
could also act as a point of conjugation with a multivalent
aglycon to allow the synthesis
of multivalent ligands in a strat-
egy that has previously been
successfully employed against
CT.[18]
To introduce
a branching
point, we used commercially
available propargyl glycine 49
(Scheme 7) as the linker precur-
sor. Initial studies were per-
formed with racemic 49 to
obtain both possible isomers at
Scheme 4. Synthesis of 2–4. Reagents and conditions: a) TEMPO/BAIB, 1:1 CH3CN/H2O; b) i) diphenylphos-
phoryl azide (DPPA), 1208C, microwave (MW); ii) NaBH3ACHTNUTRGNEU(GN OAc); c) H2, Pd/C.
the same time; this approach
capitalized on the ability of
weak affinity chromatography
(WAC) to analyze mixtures of
For the synthesis of the linker-armed Gal fragments from
amines 1–3, three commercially available, linear, terminal
alkynoic acids 9–11 (from C5 to C7) were converted into the
corresponding pentafluorophenyl esters 12–14 and coupled
with either freshly prepared crude galactosylamine 1, or
aminoethyl galactosides 2 and 3 (Scheme 5). Attempts to
apply a direct Staudinger acylation procedure by treating
pentafluorophenyl esters 12–14 with azides 7 or 8[17] did not
lead to any significant improvement in overall yields.
compounds (see below). Starting from 49, the N-acetyl pen-
tafluorophenyl ester 50 was prepared and allowed to react
with b-aminoethyl galactoside 3 to give alkyne 51. The latter
underwent click cycloaddition with sialyl azide to give, after
deprotection, the target divalent ligand 52 as a 1:1 mixture
of two inseparable epimers.
Similarly, another group of eight compounds 56–59 (four
pairs of epimers at the linkerꢀs stereocenter) was prepared
according to Scheme 8 to evaluate the effect of lipophilic
substituents on the linker. Thus, the N-Boc-protected ligand
55 was prepared as previously described for 52, deprotected
Ligands with shorter linker chains were prepared by cou-
pling the b-galactosyl acid 4, with either propargylamine or
Chem. Eur. J. 2010, 16, 1951 – 1967
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A. Bernardi et al.
pressed as the retention factor, k’) should be directly related
to the affinity of the interaction.[22] The k’ value can be
transformed into the dissociation constant, Kd, of the ligand
by calibrating the retardation with a compound of known af-
finity. The Kd values of galactose (51 mm)[20] and GalbOMe
(15 mm)[6] have been determined by inhibition chromatogra-
phy and ITC, respectively. However, these values are too
low to be used as a reference in WAC because they fall
below the revelation threshold. Instead, meta-nitropenyl-a-
d-galactopyranoside (MNPG), a well-known CT ligand,[3]
was used to calibrate our experiments. Although MNPG
does not contain the critical NeuAc residue, X-ray data[23]
have revealed that the oxygen atoms of the nitro group dis-
places a conserved water molecule from the binding site to
À
make a hydrogen bond with a backbone N H in the toxin.
The increased entropy associated with the displacement of
this water into bulk solvent accounts for a large part of the
affinity gain of MNPG relative to galactose, which is esti-
mated to be one or two orders of magnitude greater, de-
pending on the methods adopted. The reported dissociation
constants for the MNPG–CTB complex vary in the range
from 1 to 0.2 mm.[3,20,24] For consistency, comparison with the
WAC-determined value (Table 1, entry 1) will be made
throughout this paper. The results of the WAC analysis of
the ligand library compared with MNPG are shown in
Table 1 (entries 1–12).
The galactosylamine derivatives 38–40 (Table 1, entries 2–
4) and the two amides 47 and 48 obtained from acid 4
(Table 1, entries 11 and 12) bound below the measurable
threshold at pH 5.5 and pH 7.0. In contrast, the affinity of li-
gands 41–46 (Table 1, entries 5–10) could be measured and
some interesting candidates were identified. The most active
ligand of this set, compound 44 (Table 1, entry 8), gave a k’
of 1.25 at pH 7, which corresponds to a Kd value of 1.3 mm,
thus matching the affinity of the reference MNPG. By vary-
ing the mobile phase in the WAC analysis, the pH depen-
Scheme 5. Synthesis of linker-armed Gal fragments 15–25. Reagents and
conditions: a) C6F5OH, dicyclohexylcarbodiimide (DCC), THF;
b) i) Et3N, DMF; ii) Ac2O/Py; c) Et3N, THF.
(trifluoroacetic acid (TFA)), and treated with either benzoyl
chloride or the pentafluorophenyl ester of phenylacetic acid
to afford, after deprotection, amides 56 and 57 as a pair of
inseparable isomers. Alternatively, acylation of the free
amine with either phenylisocyanate or benzylisocyanate, fol-
lowed by deacetylation, yielded the two ureas 58 and 59.
AHCTUNGTREGdNNNU ence of the interaction could be studied (Figure 1). Inter-
estingly, the interaction with ligands 41–46 was found to be
strongly pH dependent, with a maximum retardation at
pH 6. MNPG binding was less pH dependent and displayed
maximum affinity at pH 7. The retention factor (k’) and dis-
sociation constant (Kd) of all ligands at pH 6 and 7 are col-
lected in Table 1; the pH affinity dependence of 44 and of
MNPG is shown in Figure 1.
The Kd value of 44 at pH 6 was found to be 0.8 mm,
whereas MNPG had a Kd of 1.3 mm (Table 1, entries 8 and
1, respectively). The pH-dependent affinity of compounds
41–46 may be related to the interaction of their sialic acid
moiety with the His13 residue in the CTB binding site,
which should be optimal when the His side chain is proton-
ated. The X-ray structure of the CTB–MNPG complex is
known[23] and, as expected for this small ligand, no contact
between MNPG and His13 was observed.
Ligand ranking by weak affinity chromatography (WAC):
The affinity of the ligands for CT is often determined by
fluorescence titrations using the intrinsic fluorescence of
Trp-88 in the protein binding site.[19] This method could not
be used in this case because of interference of the triazole
moiety in the tested structures. WAC has previously been
shown to be an interesting alternative for the study of CT li-
gands[20] and it was found to be the method of choice in our
case. This approach is especially suitable for quantifying
transient binding events such as sugar–protein interac-
tions.[21] A HPLC column with immobilized recombinant
CTB was used and small amounts (0.1 mg) of the synthesized
ligands were injected. Because the concentrations of the in-
jected compounds were low, a linear binding isotherm might
be assumed and, under these conditions, the retardation (ex-
From the structural point of view, WAC analysis of 38–48
clearly shows some interesting trends. In particular, a group
of three compounds in the b-Gal-C family (44–46) and com-
pound 42 in the a-Gal-C family, displayed affinities one
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Bidentate Cholera Toxin Ligands
FULL PAPER
which, in turn, causes a signal
enhancement that can be best
appreciated in the difference
spectrum. STD experiments
were carried out in the pres-
ence of CTB (96 mm) in D2O
(80 mm
phosphate
buffer,
pH 7.8) at several ligand to pro-
tein ratios (from 6 to 245) and
different saturation times (from
0.5 to 3 s). Quantitative data
analysis and epitope mapping
were complicated by severe
signal overlap, nonetheless the
experiments showed clear sig-
nals corresponding to the galac-
tose (Gal-H2 at 3.33 ppm) and
sialic acid (NeuAc-H5 at d=
3.89 ppm) fragments, thus con-
firming that 44 operates as a bi-
dentate ligand. The binding af-
finity was also estimated by
STD experiments at different
ligand to protein ratios (from 6
to 250) at constant concentra-
tion of the protein (96 mm) in
D2O (80 mm phosphate buffer,
pH 7.8) with a saturation time
of 2 s. Plotting the STD amplifi-
cation factors[26] against the
concentration of the added
ligand for the proton with the
largest
STD
amplification
factor (Gal-H2) allowed us to
estimate an EC50 of 2.7 mm for
the CTB–44 complex (see Fig-
ure SI-1 in the Supporting In-
formation), which is consistent
with the WAC results at similar
pH.
Scheme 6. Synthesis of the bidentate adducts 38–48. Reagents and conditions: a) CuSO4/Na ascorbate;
b) NaOH, MeOH/H2O.
On the basis of the above
findings, the propargyl glycine
order of magnitude higher than the revelation threshold.
These values may be assumed to result from simultaneous
interaction of the two sugar fragments with the toxin, which
apparently is optimally allowed in this series by the frame-
work of ligand 44 (C5 linker chain from b-Gal-C frame-
work).
As mentioned above, further characterization of the inter-
action of 44 with CTB by fluorescence spectroscopy was
precluded by the interference of the triazole moiety, which
absorbs in the same range as CTB Trp-88, and, therefore, fil-
ters the excitant radiation. However, saturation transfer dif-
ference (STD) NMR spectroscopy[25] experiments allowed
the observation of binding events between CT and ligand
44. In STD experiments, irradiation of the protein is fol-
lowed by transfer of magnetization to the ligand protons,
derivatives 52–59 were synthesized to obtain compounds
with the same framework as 44, but capable of further deri-
vatization and conjugation to multivalent aglycons. Starting
from racemic propargyl glycine, epimer pairs could be syn-
thesized and their CTB affinity simultaneously evaluated by
WAC. As an example, WAC analysis of the epimeric mix-
ture of 52 is shown in Figure 2. The analysis clearly revealed
two peaks, corresponding to the two epimers of 52. One of
the two isomers demonstrated some inhibition activity (was
retarded on the column), although it was lower than that of
the simple linear ligand 44, while the second eluted close to
the nonretarded void fraction. The corresponding evaluated
retention factors and dissociation constants are reported in
Table 1 (Entries 13 and 14. For the configurational assign-
ment in the Table, see below.)
Chem. Eur. J. 2010, 16, 1951 – 1967
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1955

A. Bernardi et al.
52, to approach the potency of
the underivatized ligand 44.
Among the compounds tested,
one of the epimers of phenyl-
AHCTUNGTERGaNNUN cetamide (57) and one of the
phenylureas (58) were found to
be the most active ligands, with
Kd values of 1.2 and 0.8 mm, re-
spectively. When the two R and
S isomers of 58 were synthe-
sized separately, starting from
enantiomerically pure d- or l-
propargyl glycine, WAC analy-
sis clearly showed that the R
isomer of 58 had higher affinity
than the corresponding
S
isomer (Table 1). This, in fact,
had been suggested by docking
models obtained by using
Glide,[28] which showed that the
R epimer of the side-chain ste-
Scheme 7. Reagents and conditions: a) i) Ac2O; ii) C6F5OH, DCC, THF; b) Et3N, THF; c) i) CuSO4/sodium as-
corbate; ii) NaOH, MeOH/H2O.
The loss of affinity upon branching of the linker suggests
that the presence of the NHAc substituent disfavors the
conformation required to allow simultaneous contact of the
two pharmacophoric sugars with the toxin. However, this
unfavorable factor could be offset by additional interactions
between the linker side chain and the toxin binding site.
Since the CTB binding site is known to possess a lipophilic
patch near the sialic acid side-chain binding region,[27] the
contribution of more lipophilic side chains was explored by
testing ligands 56–59 (Table 1, entries 15–21), which showed
similar pH dependence to the parent compound 44. As can
be seen from Table 1, the affinity of most structures im-
proved significantly compared with the N-acetyl derivative
reocenter should direct the N-substituent towards the lipo-
philic region of the binding site (Figure 3).
Conclusion
A modular approach to a library of new glycomimetic CT li-
gands has been developed. The cornerstone of this approach
consists of tethering carbohydrate epitopes that are known
to interact with the CTB binding site, galactose, and sialic
acid, to a properly designed linker through nonhydrolyzable,
non-O-glycosidic bonds. The Gal epitope was introduced as
one of four simple C- or N-galactosides. The NeuAc residue
Scheme 8. Synthesis of functionalized ligands 56–59. Reagents and conditions: a) i) Boc2O (Boc=tert-butyloxycarbonyl); ii) C6F5OH, DCC, THF;
b) Et3N, THF; c) i) CuSO4/sodium ascorbate, 26; d) CF3COOH/CH2Cl2, followed by the appropriate reagent i–iv; e) NaOH, MeOH/H2O. Bn =benzyl.
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Bidentate Cholera Toxin Ligands
FULL PAPER
Table 1. Retention factor (k’) and dissociation constant (Kd) of CT li-
gands at 238C determined with WAC at pH 6 and 7.
pH 6
Kd [mm]
pH 7
Kd [mm]
Entry
Ligand
k’
k’
1
2
3
4
5
6
7
8
9
MNPG[a]
38
39
40
41
42
43
44
1.28
1.3
>30
>30
>30
9.9
4.4
20
0.8
2.2
6.6
1.58
1.1[b]
>30
>30
>30
19
9.1
31
1.3
4.6
12
<0.05
<0.05
<0.05
0.17
0.38
0.09
2.17
0.76
0.25
<0.05
<0.05
<0.05
0.09
0.19
0.05
1.25
0.37
0.14
45
46
10
11
12
13
14
15
16
17
18
19
20
21
47
<0.05
<0.05
0.70
0.07
1.10
1.35
1.19
2.20
1.39
>30
>30
2.4
26
1.5
<0.05
<0.05
0.47
<0.05
0.71
0.86
0.86
1.43
0.93
>30
>30
3.6
>30
2.4
48
(R)-52[c]
(S)-52[c]
A
(R)-57[c]
(S)-57[c]
(R)-58[c]
(S)-58[c]
(R)-59[c]
(S)-59[c]
1.2
1.4
0.8
1.2
0.9
1.4
2.0
2.0
1.2
1.8
1.4
2.1
1.86
1.18
1.23
0.80
[a] meta-Nitrophenyl-a-d-galactopyranoside. [b] From ref. [20]. [c] Con-
figuration of the linkerꢀs stereocenter.
Figure 3. Docked conformations of ligands (R)-57 (top) and (R)-58
(bottom) in the CTB binding site (the stereochemical descriptor refers to
the linker stereocenter).
was connected through a triazole spacer, starting from the
known sialyl azide. Simple linear linkers were used to con-
nect the sugar fragments. The affinity of the bidentate li-
gands was evaluated by WAC[20] with immobilized CTB. The
results showed that only some appropriate combinations of
fragments led to a measurable improvement of affinity over
the individual epitopes. In particular, a group of molecules
was identified that displayed affinities at least one order of
magnitude higher than galactose. Such values may be as-
sumed to result from simultaneous interaction of the two
sugar fragments with the toxin.
~
Figure 1. Affinity (k’) of 44 and MNPG versus pH. : 44; &: MNPG.
Although docking studies could, in principle, have been
used to select favorable conformational properties of the
linkers and to identify ligands capable of engaging both
binding areas in the toxin, in practice, the nature of CTB
binding site, which is large and shallow in the area that
should be covered by the linker (see Figure 3), does not
lend itself to reliable predictive use of computational
models. Indeed, initial docking experiments conducted by
using Glide[28] software did not reveal significant differences
in docking scores between similar structures containing the
Gal residue in the form of N- or C-glycoside in either a- or
b-anomeric configuration, albeit with some preference for
the latter. The linear linker developed in this study can now
be used as a platform for rational ligand design, and be di-
rected towards the stabilization of the bound conformation
Figure 2. WAC diagram for compounds 52 (1:1 isomers, dotted line) and
44 (solid line) at pH 6.
Chem. Eur. J. 2010, 16, 1951 – 1967
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1957

A. Bernardi et al.
AVANCE-400 instrument; samples were measured in CDCl3 at room
temperature, unless otherwise specified. Chemical shifts (d) for 1H and
13C NMR spectra are expressed in ppm relative to internal tetramethylsi-
lane as standard. Assignments were aided by homo- and heteronuclear
two-dimensional experiments. Common mass spectra were obtained with
a Bruker ion-trap Esquire 3000 plus apparatus (ESI ionization), exact
mass spectra were registered with a Bruker Daltonics ICR-FTMS APEX
II spectrometer (ESI ionization). TLC was carried out with precoated
Merck F254 silica gel plates. TLC detection was performed by treatment
with phosphomolybdic acid, ninhydrin, potassium permanganate, or va-
nillin. Automated flash direct and reverse-phase chromatography were
carried out on a Biotage SP1 system equipped with Biotage SNAP car-
tridges.
of the ligand, thus increasing its preorganization and, ulti-
mately, its affinity for CT.
All compounds reported herein could be synthesized from
precursors that are readily available on a large scale by
using high-performance reactions, including click chemistry
protocols. Previously known artificial CT ligands basically
fall into two classes: the MNPG derivatives introduced by
the group of Fan[3] and the structural mimics of the GM1os
reported by our group (Scheme 1).[4] Both types contain en-
zymatically labile O-glycosidic linkages. Several of the non-
hydrolyzable ligands described herein demonstrated reason-
ably high CT affinities that were similar to or higher than
the affinity of MNPG.[20,24] They are therefore among the
most active monovalent CT ligands that do not closely re-
produce the GM1 structure. Some of the most active mole-
cules identified herein also feature a point of further deriva-
tization that can be used for conjugation with polyvalent
aglycons. Even though there was either no, or negligible, af-
finity gained by linker derivatization, this approach opens
the way to the creation of polyvalent constructs that may be
used to block the toxin in a therapeutically relevant con-
text.[18]
Compound 6: l-Proline PEG conjugate (2.27 g, ꢀ0.1 equiv)[15] was added
to
a stirred solution of the corresponding a-aldehyde 5 (1.58 g,
4.22 mmol)[13] in MeOH (11 mL) at 08C. The mixture was sonicated and
then subjected to microwave irradiation for 4 h at a controlled tempera-
ture of 508C (constant power 13 W, cooling by compressed air). The reac-
tion was monitored by 1H NMR (H1Gala: m, d=4.86 ppm; H1Galb: m, d=
4.00 ppm). The reaction mixture was allowed to cool to RT then the sol-
vent was evaporated and solid residue was purified by automated flash
chromatography to afford pure b-anomer 6 (1.57 g, 99%); analytical data
corresponded to those reported in the literature.[14] 1H NMR (400 MHz,
CDCl3): d=9.72 (t, J=3 Hz, 1H; -COH), 5.41 (t, J=3 Hz, 1H; H-4),
5.28 (dd, J1 =5, J2 =9 Hz, 1H; H-2),
5.17 (dd, J1 =3, J2 =9 Hz, 1H; H-3),
4.03–4.14 (m, 2 H; H-5, H-6a), 4.03
(ddd, J1 =9.6, J2 =8, J3 =4 Hz, 1H; H-
1), 3.95 (t, J=6.5 Hz, 1H; H-6b), 2.78
(ddd, J1 =19, J2 =9.6, J3 =2 Hz, 1H;
GalCH2a), 2.60 (ddd, J1 =19, J2 =4,
Interesting findings, such as pH dependence of the bind-
ing affinity of the ligands and the structural preference of
the CTB binding site towards one of the two diastereomeric
forms of the ligand, were assessed with WAC, which will be
useful for further development of inhibitors of CT binding.
J3 =2 Hz,
1H;
GalCH2b),
2.01–
2.20 ppm (m, 12H; 4ꢅCH3CO); MS
(ESI): 414.9 [M++K].
Compound 7: Acetic acid (100 mL, 1.76 mmol) and NaBH4 (63 mg,
1.76 mmol) were added to a stirred solution of aldheyde 5 (332 mg,
0.88 mmol)[13] in anhydrous THF (10 mL). The reaction mixture was vigo-
rously stirred at RT for ꢀ2 h until reduction was complete. The reaction
was quenched with acetic acid (200 mL) and the solvent was evaporated.
The excess acetic acid was removed by co-evaporation with toluene and
the solid residue was filtered through silica gel (hexane/EtOAc 1:3) to
give the alcohol intermediate, which was subsequently dissolved in anhy-
drous DMF (2.5 mL). Diphenyl phosphoryl azide (266 mL, 1.23 mmol,
Experimental Section
General: WAC was performed essentially as described previously.[20] In
short, recombinant CTB (SBL vaccines, Stockholm, Sweden) was immo-
bilized with reductive amination to aldehyde-derivatized Nucleosil silica
(10 mm, 300 ꢃ; Macherey–Nagel, Dꢄren, Germany) and packed into a
50ꢅ2.1 mm column. The number of binding sites on the column was de-
termined with frontal chromatography to be 261 nmol at pH 7. All chro-
matography experiments were performed on an Agilent 1100 series
HPLC system (Agilent Technologies, Santa Clara, CA, USA); the mobile
phase was 0.15m sodium chloride with 10 mm sodium phosphate (ad-
justed to pH 5.5, 6.0, 6.5, or 7.0 as indicated in the text); the flow rate
was 0.1 mLminÀ1 and the temperature was 238C; the injection volume
was 5 mL and the concentration of each sample was 20 mgmLÀ1. Detec-
tion was performed at 220 nm and the detector signal was collected with
an Agilent ChemStation chromatography system. The retention factor
(k’) was calculated from the retention time (Tr) and the mobile phase
hold up time, also called the void time (Tm), according to the equation:
k’=(TrÀTm)/Tm. The void time of the column was determined to be
1.55 min. Dissociation constants (Kd) of the ligands were calculated from
1.4 equiv) and 1,8-diazabicycloACTHNUTRGNE[NUG 5.4.0]undec-7-ene (184 mL, 1.23 mmol,
1.4 equiv) were added while stirring. The mixture was then subjected to
microwave irradiation for 20 min at 1208C (dynamic control) at 200 W
power. The solvent was evaporated and the mixture was purified by flash
chromatography (hexane/EtOAc) to afford the azide 7 (247 mg, 70%).
[a]D =+36.45 (c=0.92 in CHCl3); 1H NMR (400 MHz, CDCl3): d=5.45
(t, J1 =J2 =3 Hz, 1H; H-4), 5.29 (dd, J1 =5, J2 =9 Hz, 1H; H-2), 5.21 (dd,
J1 =3, J2 =9 Hz, 1H; H-3), 4.31–4.40 (m, 2H; H-1, H-6a), 4.07–4.16 (m,
2H; H-5, H-6a), 3.35–3.50 (m, 2H; CH2N3), 2.09–2.15 (m, 12H; 4ꢅ
CH3CO), 1.87–2.00 (m, 1H; Gal-CH2a), 1.63–1.78 ppm (m, 1H; Gal-
CH2b); 13C NMR (100.6 MHz, CDCl3):
d=170.6, 170.0, 169.8, 169.7 (4ꢅ
CH3CO), 69.0 (C-5), 68.8 (C-1), 68.3
(C-2), 67.9 (C-3), 67.2 (C-4), 61.2 (C-
6), 47.7 (CH2N3), 26.0 (Gal-CH2),
the following equation: Kd =Btot/ACTHGUNRTENNU(G k’*Vm) in which Btot is the number of
binding sites in the column and Vm is the void volume.[21,22] It seems rea-
sonable to assume that Btot and Vm are constant under the conditions
studied and the equation can be simplified to Kd =const/k’. By using this
relationship, the affinity (Kd) of any compound can easily be calculated
from the k’ by comparing the retardation with a compound of known af-
finity.
20.74, 20.66 ppm (4ꢅCH3CO); MS
(ESI): m/z: 423.9 [M++Na].
Compound 8: Acetic acid (206 mL, 3.606 mmol) and NaBH4 (137 mg,
3.606 mmol) were added to a stirred solution of the aldheyde 6 (672 mg,
1.8 mmol) in anhydrous THF (10 mL). The reaction mixture was stirred
for ꢀ2 h until reduction was complete, then quenched with acetic acid
(200 mL). The same protocol described above yielded the intermediate al-
cohol, which was subsequently dissolved in anhydrous DMF (5 mL). Di-
phenyl phosphoryl azide (544 mL, 2.52 mmol, 1.4 equiv) and 1,8-
Solvents were dried by standard procedures: dichloromethane, methanol
and triethylamine (TEA) were dried over calcium hydride; THF was
dried on sodium; anhydrous DMF was purchased from Fluka. Reactions
requiring anhydrous conditions were performed under nitrogen. Micro-
wave-assisted reactions were performed on a CEM Discover instrument.
1H and 13C NMR spectra were recorded at 400 MHz on a Bruker
diazabicycloACTHNUTRGNEUG[N 5.4.0]undec-7-ene (376 mL, 2.52 mmol, 1.4 equiv) were added
1958
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ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 1951 – 1967

Bidentate Cholera Toxin Ligands
FULL PAPER
while stirring. The mixture was then subjected to microwave irradiation
for 20 min at 1208C (dynamic control) at 200 W power. The solvent was
evaporated and the mixture was purified by flash chromatography
(hexane/EtOAc) to afford the azide 8 (560 mg, 77.3%). [a]D =+1.8 (c=1
in CHCl3); 1H NMR (400 MHz, CDCl3): d=5.41 (dd, J1 =1, J2 =3 Hz,
1H; H-4), 5.07 (t, J=10 Hz, 1H; H-2), 5.01 (dd, J1 =3, J2 =10 Hz, 1H;
H-3), 4.16–4.46 (m, 2H; H-6), 3.86 (brt, J=7 Hz, 1H; H-5), 3.69 (td, J1 =
4, J2 =9 Hz, 1H; H-1), 3.41 (dt, J=6 Hz, 2H; CH2N3), 1.92–2.15 (m,
12H; 4ꢅCH3CO), 1.74–1.82 ppm (m, 2H; CH2CH2N3); 13C NMR: d=
170.5, 170.2, 170.1, 169.9 (4ꢅCH3CO),
2), 5.07 (dd, J1 =3, J2 =10 Hz, 1H; H-3), 4.05–4.17 (m, 2H; H-6), 3.85–
3.95 (m, 2H; H-5, H-1), 2.63 (dd, J1 =6.1, J2 =16 Hz, 2 H; CH2COOH),
1.98–2.21 ppm (m, 12H; 4ꢅCH3CO).
General procedure for the synthesis of 12–14 and 50: 2,3,4,5,6-Pentafluoro-
phenol (1.5 equiv) was added to a solution of alkynoic acid (0.5m) in an-
hydrous THF at RT followed, after 15 min, by dicyclohexylcarbodiimide
(1.5 equiv). The resulting suspension was vigorously stirred overnight at
RT, diluted with ethyl acetate, and filtered through Celite. The filtrate
was concentrated and purified by flash chromatography (hexane/EtOAc
gradient) to afford pure pentafluorophenyl ester.
75.0 (C-1) , 74.3 (C-5), 72.0 (C-3), 69.2
(C-2), 67.7 (C-4), 61.7 (C-6), 47.1
(CH2N3), 31.0 (CH2CH2N3), 20.8, 20.7,
Compound 53: Anhydrous Et3N (500 mL) and Boc2O (308 mg,
1.41 mmol) were added to a stirred suspension of C-propargylglycine
(133 mg, 1.18 mmol) in MeOH (3.2 mL). During the course of reaction
20.6 ppm (4ꢅCH3CO); MS (ESI):
A
m/z: 423.9 [M++Na].
cating completion of carbamate formation. The solvent was evaporated,
the resulting solid was dried in vacuo and the crude carbamate was con-
verted into its pentafluorophenyl ester as described in the general proce-
dure to afford 53 (440 mg, 98%).
Compound 2: Acetic acid (500 mL) and a catalytic amount of Pd/C was
added to a stirred solution of the azide 7 (636 mg, 1.6 mmol) in a mixture
of MeOH/H2O (5:1, 30 mL). The reaction vessel was filled with hydrogen
and the reaction mixture was vigorously stirred at RT for ꢀ2 h until the
reduction was complete. The reaction mixture was filtered through Celite
and the resulting solution was evaporated and dried in vacuo to afford
1H NMR (400 MHz, CDCl3): d=5.37
(d, J=8 Hz, 1H; NH), 4.75–4.90 (m,
1H; CHNH), 2.97 (brd, J=17 Hz,
1H; H-3a), 2.85 (ddd, J1 =3, J2 =5,
amine
2 as the acetate salt (834 mg, 100%). [a]D =+25.4 (c=1 in
MeOH); 1H NMR (400 MHz, CD3Cl3): d=5.39 (t, J1 =J2 =3 Hz, 1H; H-
4), 5.25 (dd, J1 =3, J2 =8 Hz, 1H; H-3), 5.19 (dd, J1 =5, J2 =8 Hz, 1H; H-
2), 4.42–4.52 (m, 1H; H-6a), 4.30–4.40 (m, 1H; H-1), 4.20 (dt, J1 =3, J2 =
8 Hz, 1H; H-5), 4.08 (dd, J1 =4, J2 =8 Hz, 1H; H-6b), 2.98–3.10 (m, 2H;
CH2NH2), 1.97–2.19 (m, 13H; Gal-CH2a, 4ꢅCH3CO), 1.93 (s, 3H;
CH3COOÀ), 1.78–1.88 ppm (m, 1H; Gal-CH2b); 13C NMR (100.6 MHz,
CDCl3): d=172.4, 171.6, 171.3, 171.2 (4ꢅCH3CO), 70.8 (C-5), 70.5 (C-1),
69.6 (C-2), 68.93 (C-3), 68.94 (C-4),
J3 =17 Hz, 1H; H-3b), 2.15 (brs, 1H;
ꢁ
C CH), 1.48 ppm (s, 9H; tBu).
General procedure for the synthesis of 15–17: 2,3,4,5,6-Pentafluorophenyl
akynoate (1.05 mmol) was added to a stirred solution of galactosylamine
1 (180 mg, 1.0 mmol) in DMF (3 mL). The reaction mixture was stirred
overnight at RT then pyridine (3 mL, 37.1 mmol) and acetic anhydride
(1 mL, 10.60 mmol) were added to the reaction mixture and stirring was
continued at RT overnight. The resulting solution was diluted with
EtOAc (30 mL), washed with 1m HCl (15 mL), water (15 mL), and a sa-
turated aqueous solution of NaHCO3 (15 mL). Organic phase was dried
over Na2SO4, filtered, and concentrated. The crude product was purified
by flash chromatography (hexane/EtOAc).
62.2 (C-6), 38.0 (CH2NH2), 25.8 (Gal-
CH2), 23.4 (CH3COOÀ), 20.7, 20.6,
20.6, 20.5 ppm (4ꢅCH3CO); HRMS
(FT-ICR, ESI): m/z calcd for
C16H26NO9:
376.16021
[M++H]:
Compound 15: Yield: 162 mg (0.379 mmol, 38%); 1H NMR (400 MHz,
CDCl3): d=6.49 (d, J=1 Hz, 1H;
found: 376.16054.
Compound 3: A catalytic amount of Pd/C was added to a stirred solution
of the azide 8 (136.4 mg, 0.340 mmol) in a mixture of MeOH/H2O/AcOH
(25:5:3, 10 mL). The reaction vessel was filled with hydrogen and the re-
action mixture was vigorously stirred at RT for ꢀ2 h until reduction was
complete. The reaction mixture was filtered through Celite and the re-
sulting solution was evaporated and dried in vacuo to afford amine 3 as
the acetate salt (148 mg, 100%). [a]D =+4.1 (c=1 in CHCl3); 1H NMR
(400 MHz, CD3OD): d=5.40 (dd, J1 =1, J2 =3 Hz, 1H; H-4), 5.09 (dd,
J1 =3, J2 =10 Hz, 1H; H-3), 5.00 (t, J=10 Hz, 1H; H-2), 4.03–4.13 (m,
3H; H-5, H-6), 3.69 (td, J1 =3, J2 =10 Hz, 1H; H-1), 3.07 (td, J1 =3, J2 =
7 Hz, 2H; CH2NH2), 1.88–2.11 (m, 13H; 4ꢅCH3CO, CH2bCH2N3), 1.75–
1.85 ppm (m, 1H; CH2aCH2N3); 13C NMR: d=172.3, 172.0, 171.8, 171.6
(4ꢅCH3CO), 77.0 (C-1), 74.9 (C-5),
NH), 5.44 (d, J=2 Hz, 1H; H-4), 5.26
(t, J=9 Hz, 1H; H-1), 5.07–5.15 (m,
2H; H-2, H-3), 4.00–4.15 (m, 3H; H-5,
H-6a, H-6b), 2.35–2.60 (m, 4H;
ꢁ
(CH2)2), 1.95–2.20 ppm (m, 13H; C
CH, 4ꢅCH3CO).
Compound 16: Yield: 156 mg (0.353 mmol, 35%); 1H NMR (400 MHz,
CDCl3): d=6.49 (d, J=1 Hz, 1H; NH), 5.43 (d, J=2 Hz, 1H; H-4), 5.24
(t, J=9 Hz, 1H; H-1), 5.07–5.15 (m, 2H; H-2, H-3), 4.00–4.15 (m, 3H;
ꢁ
H-5, H-6a, H-6b), 1.70–2.40 ppm (m, 17H; (CH2)3, C CH, 4ꢅCH3CO).
72.7 (C-3), 69.5 (C-4), 69.4 (C-2), 62.3
(C-6), 37.6 (CH2N3), 22.9 (CH2CH2N3),
20.8, 20.7, 20.6 ppm (4ꢅCH3CO);
HRMS (FT-ICR, ESI): m/z calcd for
C16H26NO9: 376.16021 [M++H]; found:
Compound 17: Yield: 126 mg (0.277 mmol, 28%); 1H NMR (400 MHz,
CDCl3): d=6.25 (d, J=1 Hz, 1H; NH), 5.43 (d, J=2 Hz, 1H; H-4), 5.25
(t, J=9 Hz, 1H; H-1), 5.05–5.15 (m, 2H; H-2, H-3), 4.00–4.17 (m, 3H;
376.16059.
Compound 4: TEMPO (25 mg, 0.160 mmol) and BAIB (565 mg,
1.75 mmol) were added to
a solution of b-aldheyde 6 (300 mg,
0.797 mmol) in MeCN/H2O (1:1, 3 mL). The reaction mixture was stirred
for ꢀ3 h until oxidation was complete. The reaction was quenched with
an aqueous solution of Na2S2O3 (2 mL, 1m) then diluted with CH2Cl2
(15 mL) and filtered through Celite. The solvents were evaporated and
the resulting solid was purified by automated flash chromatography
(MeOH/CH2Cl2, 0–25%) to afford
ꢁ
H-5, H-6a, H-6b), 1.95–2.35 (m, 15H; NHC(O)CH2, C CH, 4ꢅCH3CO),
1.50–1.90 ppm (m, 6H; (CH2)3).
pure acid 4 (311 mg, 100%), the ana-
lytical data for which corresponded to
those reported.[29] 1H NMR (400 MHz,
CDCl3): d=5.45 (dd, J1 =1, J2 =3 Hz,
1H; H-4), 5.16 (t, J1 =10 Hz, 1H; H-
General procedure for the synthesis of 18–23, 51, and 54: Alkynoic acid
2,3,4,5,6-pentafluorophenyl ester (1.2–1.5 equiv) and triethylamine
(3 equiv) were added to a stirred solution of aminoethylgalactoside 2 or 3
Chem. Eur. J. 2010, 16, 1951 – 1967
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1959

A. Bernardi et al.
(acetate salt; 0.5m) in THF. The reaction mixture was stirred at RT over-
night, then the solvent was evaporated, and resulting solid was purified
by flash chromatography (hexane/EtOAc).
3.60 (m, 1H;CH2bNC(O)), 3.43 (dt, J1 =10.4, J2 =3.0 Hz, 1H; H-1), 3.15–
3.25 (m, 1H; CH2aNC(O)), 2.42–2.52 (m, 2H; HNC(O)CH2CH2CCH),
ꢁ
2.20–2.30 (m, 2H; HNC(O)CH2CH2CCH), 1.93–2.13 (m, 13H; C CH,
Compound 18: The reaction of a-aminoethylgalactoside
2 (30 mg,
4ꢅCH3CO), 1.57–1.85 ppm (m, 2H; CH2CH2NC(O)).
0.069 mmol, N-acetate form) with pentafluorophenyl ester 12 (22 mg,
0.083 mmol) afforded alkyne 18 (25 mg, 0.055 mmol, 80%). 1H NMR
(400 MHz, CDCl3): d=6.18 (brt, J=6.5 Hz, 1H; NH), 5.38 (t, J=3 Hz,
1H; H-4), 5.05–5.15 (m, 2H; H-2, H-3), 4.50 (dd, J1 =8.3, J2 =11 Hz, 1H;
H-6b), 4.20 (dt, J1 =3, J2 =11 Hz, 1H; H-5), 4.05–4.10 (m, 1H; H-1), 3.95
(dd, J1 =11, J2 =5 Hz, 1H; H-6a), 3.38–3.50 (m, 1H; CH2bNC(O)), 3.08–
3.18 (m, 1H; CH2aNC(O)), 2.45–2.55 (m, 2H; HNC(O)CH2CH2CCH),
ꢁ
2.40–2.50 (m, 2H; HNC(O)CH2CH2CCH), 1.95–2.07 (m, 13H; C CH,
4ꢅCH3CO), 1.75–1.95 ppm (m, 2H; CH2CH2NC(O)).
Compound 22: The reaction of b-aminoethylgalactoside
3 (30 mg,
0.069 mmol, N-acetate form) with pentafluorophenyl ester 13 (23 mg,
0.083 mmol) afforded alkyne 22 (29 mg, 0.062 mmol, 90%). 1H NMR
(400 MHz, CDCl3): d=5.90 (brt, J=6.5 Hz, 1H; NH), 5.38 (d, J=3.3 Hz,
1H; H-4), 5.02 (t, J=10.4 Hz, 1H; H-2), 4.94 (dd, J1 =10.4, J2 =3.3 Hz,
1H; H-3), 4.00–4.15 (m, 2H; H-6), 3.81 (t, J=6.2 Hz, 1H; H-5), 3. 50–
3.58 (m, 1H; CH2bNC(O)), 3.43 (dt, J1 =10.4, J2 =3.0 Hz, 1H; H-1), 3.15–
3.25
(m,
1H;
CH2aNC(O)),
2.17–2.30
(m,
4H;
HNC(O)CH2CH2CH2CCH), 1.90–2.15 (m, 12H; 4ꢅCH3CO), 1.80 (t, J=
ꢁ
6.5 Hz, 1H; C CH), 1.52–1.82 ppm (m, 4H; CH2CH2CH2,
CH2CH2NC(O)).
Compound 19: The reaction of a-aminoethylgalactoside
2 (32 mg,
0.073 mmol, N-acetate form) with pentafluorophenyl ester 13 (31 mg,
0.111 mmol) afforded alkyne 19 (27 mg, 0.058 mmol, 78%). 1H NMR
(400 MHz, CDCl3): d=6.10 (brt, J=6.5 Hz, 1H; NH), 5.38 (t, J=3 Hz,
1H; H-4), 5.10–5.20 (m, 2H; H-2, H-3), 4.57 (dd, J1 =8.3, J2 =11 Hz, 1H;
H-6b), 4.23 (dt, J1 =3, J2 =11 Hz, 1H; H-5), 4.10–4.15 (m, 1H; H-1), 4.00
(dd, J1 =11, J2 =5 Hz, 1H; H-6a), 3.40–3.50 (m, 1H; CH2bNC(O)), 3.15–
3.22 (m, 1H; CH2aNC(O)), 2.18–2.40 (m, 4H; HNC(O)CH2CH2CCH),
ꢁ
1.95–2.15 (m, 13H; C CH, 4ꢅCH3CO), 1.65–1.95 ppm (m, 4H;
CH2CH2CH2, CH2CH2NC(O)).
Compound 23: The reaction of b-aminoethylgalactoside
3 (23 mg,
0.053 mmol, N-acetate form) with pentafluorophenyl ester 14 (15 mg,
0.051 mmol) afforded alkyne 23 (12 mg, 0.025 mmol, 47%). 1H NMR
(400 MHz, CDCl3): d=5.90 (brt, J=6.5 Hz, 1H; NH), 5.45 (d, J=3.3 Hz,
1H; H-4), 5.13 (t, J=10.0 Hz, 1H; H-2), 5.03 (dd, J1 =3.3, J2 =10.0 Hz,
1H; H-3), 4.15 (ddd, J1 =6.9, J2 =11.0, J3 =17.0 Hz, 2H; H-6), 3.90 (t, J=
6.9 Hz, 1H; H-5), 3.57–3.65 (m, 1H; CH2bNC(O)), 3.51 (dt, J1 =10.0, J2 =
6.6 Hz, 1H; H-1), 3.23–3.33 (m, 1H; CH2aNC(O)), 2.20–2.29 (m, 4H;
HNC(O)CH2ACHTNUTRGNENUG(CH2)2CH2CCH), 2.0–2.19 (m, 12H; 4ꢅCH3CO), 1.98 (t,
ꢁ
J=2.7 Hz, 1H; C CH), 1.53–1.90 ppm (m, 4H; CH
CH2CH2NC(O)).
2ACHTUGNTERN(NUNG CH2)2CH2,
Compound 20: The reaction of a-aminoethylgalactoside
2 (47 mg,
0.108 mmol, N-acetate form) with pentafluorophenyl ester 14 (38 mg,
0.130 mmol) afforded alkyne 20 (41 mg, 0.085 mmol, 79%). 1H NMR
(400 MHz, CDCl3): d=6.08 (brt, J=6.5 Hz, 1H; NH), 5.40 (t, J=3.2 Hz,
1H; H-4), 5.20 (dd, J1 =3.2, J2 =7.9 Hz, 1H; H-3), 5.14 (dd, J1 =7.9, J2 =
4.0 Hz, 1H; H-2), 4.66–4.60 (m, 1H; H-6b), 4.24 (dt, J1 =3.6, J2 =11.1 Hz,
1H; H-5), 4.13 (dt, J1 =4.0, J2 =8.1 Hz, 1H; H-1), 4.00 (dd, J1 =4.4, J2 =
11.1 Hz, 1H; H-6a), 3.40–3.50 (m, 1H; CH2bNC(O)), 3.14–3.21 (m, 1H;
CH2aNC(O)), 2.16–2.30 (m, 4H; HNC(O)CH
(m, 12H; 4ꢅCH3CO), 1.95 (t, J=2.6 Hz, 1H;C CH), 1.50–1.90 ppm (m,
6H; CH2A(CH2)2CH2, CH2CH2NC(O)).
2ACHTUNGTRENNUN(G CH2)2CH2CCH), 2.02–2.14
ꢁ
Compound 51: The reaction of b-aminoethylgalactoside 3 (40 mg,
0.092 mmol, N-acetate form) with pentafluorophenyl ester 50 (30 mg,
0.093 mmol) afforded alkyne 51 (20 mg, 0.039 mmol, 43%) together with
the byproduct from O- to N-acetyl transfer in 3 (19 mg, 47%). 1H NMR
(400 MHz, CDCl3): d=6.52, 6.37 (2H; 2ꢅNH), 5.36 (d, J=3.3 Hz, 1H;
H-4), 5.03 (t, J=9.8 Hz, 1H; H-2), 4.94 (dd, J1 =3.4, J2 =10.0 Hz, 1H; H-
3), 4.40–4.50 (m, 1H; CHNHAc), 3.97–4.17 (m, 2H; H-6), 3.75–3.85 (m,
1H; H-5), 3.49–3.58 (m, 1H; CH2bNC(O)), 3.43 (dt, J1 =10.0, J2 =3.4 Hz,
CHTUNGTRENNUNG
ꢁ
1H; H-1), 3.21–3.31 (m, 1H; CH2aNC(O)), 2.47–2.73 (m, 2H; CH2C
ꢁ
CH), 1.86–2.13 (m, 13H; 4ꢅCH3CO, C CH), 1.61–1.82 ppm (m, 2H;
CH2CH2NC(O)).
Compound 21: The reaction of b-aminoethylgalactoside
3 (30 mg,
0.069 mmol, N-acetate form) with pentafluorophenyl ester 12 (22 mg,
0.083 mmol) afforded alkyne 21 (29 mg, 0.064 mmol, 93%). 1H NMR
(400 MHz, CDCl3): d=6.0 (brt, J=6.5 Hz, 1H; NH), 5.40 (d, J=3.3 Hz,
1H; H-4), 5.02 (t, J=10.4 Hz, 1H; H-2), 4.93 (dd, J1 =10.4, J2 =3.3 Hz,
1H; H-3), 3.98–4.14 (m, 2H; H-6), 3.82 (t, J=6.2 Hz, 1H; H-5), 3.50–
1960
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 1951 – 1967

Bidentate Cholera Toxin Ligands
FULL PAPER
Compound 54: The reaction of the pentafluorophenyl ester 53 (234 mg,
0.619 mmol) with b-aminoethylgalactoside 3 (N-acetate salt, 224.5 mg,
0.516 mmol), afforded alkyne 54 (178 mg, 60%). 1H NMR (400 MHz,
CDCl3): d=6.59 (s, 0.5H; CH2NH), 6.53 (t, J=6 Hz, 0.5H; CH2NH),
5.42 (brs, 1H; H-4), 5.28 (s, 1H; NHBoc), 5.08 (t, J1 =10 Hz, 1H; H-2),
4.99 (dd, J1 =3, J2 =10 Hz, 1H; H-3), 4.20–4.31 (m, 1H; CHNH), 4.06–
4.18 (m, 2H; H-6), 3.86 (qd, J1 =1, J2 =6 Hz, 1H; H-5), 3.44–3.64 (m,
2H; H-1, CH2CH2aNH), 3.25–3.40 (m, 1H; CH2CH2bNH), 2.71–2.83 (m,
protected from light and the reaction mixture was vigorously stirred for
2–3 h. The solvent was then evaporated and the solid residue was extract-
ed with CH2Cl2 (15 mL). The combined organic layer was concentrated
and purified by flash chromatography.
Compound 27: The reaction of alkyne 15 (56 mg, 0.131 mmol) with 26
following the general procedure described above afforded compound 27
(100 mg, 0.106 mmol, 81%). [a]D =+9.9 (c=1 in CHCl3); 1H NMR
(400 MHz, CD3OD): d=8.00 (s, 1H; H-Triaz), 5.39–5.54 (m, 3H; H-
4Gal, H-7Neu, H-8Neu), 5.33 (d, J=9 Hz, 1H; H-1Gal), 5.23 (dd, J1 =10,
J2 =4 Hz, 1H; H-3Gal), 5.17 (t, J=9 Hz, 1H; H-2Gal), 5.09 (dt, J1 =4.5,
J2 =11 Hz, 1H; H-4Neu), 4.45–4.00 (m, 7H; H-9Neu, H-5Neu, H-6Neu,
H-5Gal, H-6Gal), 3.85 (s, 3H; COOCH3), 3.34 (dd, J1 =13.0, J2 =4.2 Hz,
1H; H-3eqNeu), 3.01 (brs, 2H; CH2CH2C(O)NH), 2.52–2.67 (m, 3H; H-
3axNeu, CH2CH2C(O)NH), 1.90–2.20 ppm (m, 27H; 9ꢅCH3CO);
13C NMR (400 MHz, CD3OD): d=168.0–172.0 (11ꢅC=O), 137.5 (C-
Triaz), 124.9 (CH-Triaz), 79.2, 74.0, 73.0, 71.6, 69.1, 69.0, 68.5, 67.8, 67.6
(C-1Gal, C-2Gal, C-3Gal, C-4Gal, C-5Gal, C-4Neu, C-6Neu, C-7Neu, C-
8Neu), 63.3, 62.0 (C-6Gal, C-9Neu), 53.9 (COOCH3), 49.0 (C-5Neu), 35.5
ꢁ
ꢁ
1H; CH2aC CH), 2.55–2.68 (m, 1H; CH2bC CH), 1.94–2.18 (m, 13H; 4ꢅ
CH3CO, C CH), 1.61–1.92 ppm (m, 2H; CH2CH2NH); 13C NMR: d=
ꢁ
170.6–170.0 (4ꢅCH3CO, CH2NHCO), 77.4 (C-1), 74.7 (C-5), 71.7 (C-3),
69.1 (C-2), 67.9 (C-4), 62.0 (C-6), 53.2 (CHNH), 36.6 (CH2NH), 31.0
ꢁ
(CH2CH2NH), 28.4 (tBu), 22.8 (CH2C CH), 20.7–20.9 ppm (4ꢅCH3CO).
(C-3Neu,
CH2CH2C(O)NH); HRMS (FT-ICR, ESI): m/z calcd for C39H53N5O22
966.307995
[M+Na+]; found: 966.30482.
CH2CH2C(O)NH),
21.6–21.9 ppm
(9ꢅCH3CO,
:
AHCTUNGTRENNUNG
Compound 24: The pentafluorophenyl ester of b-caboxymethylgalacto-
side 4 (127 mg, 0.229 mmol) prepared as described above, was treated
with propargylamine hydrochloride (21 mg, 0.229 mmol) in THF in the
presence of triethylamine (3 equiv). The reaction mixture was stirred at
RT overnight, then the solvent was evaporated, and the resulting solid
was purified by flash chromatography (hexane/EtOAc) to afford alkyne
24 (83 mg, 0.194 mmol, 85%). 1H NMR (400 MHz, CDCl3): d=6.26 (t,
J=4 Hz, 1H; NHCH2), 5.53 (brd, J=
Compound 28: The reaction of alkyne 16 (90 mg, 0.204 mmol) with 26
following the general procedure described above afforded 28 (152 mg,
0.159 mmol, 78%). [a]D =+12.4 (c=1 in CHCl3); 1H NMR (400 MHz,
CD3OD): d=7.95 (s, 1H; H-Triaz), 5.37–5.50 (m, 3H; H-4Gal, H-7Neu,
H-8Neu), 5.33 (d, J=9 Hz, 1H; H-1Gal), 5.23 (dd, J1 =10, J2 =4 Hz, 1H;
H-3Gal), 5.18 (t, J=9 Hz, 1H; H-2Gal), 5.11 (dt, J1 =4.5, J2 =11 Hz, 1H;
H-4Neu), 4.00–4.45 (m, 7H; H-9Neu, H-5Neu, H-6Neu, H-5Gal, H-
6Gal), 3.85 (s, 3H; COOCH3), 3.37 (dd, J1 =13.0, J2 =4.5 Hz, 1H; H-
3 Hz, 1H; H-4), 5.09 (t, J1 =10 Hz,
1H; H-2), 5.02 (dd, J1 =3, J2 =10 Hz,
1H; H-3), 4.00–4.15 (m, 4H; H-6,
ꢁ
CH2C CH), 3.83–3.96 (m, 2H; H-1,
H-5), 2.40–2.50 (m, 2H; CH2C(O)),
ꢁ
2.22 (t, J=2 Hz, 1H; CH2C CH),
1.93–2.18 ppm (m, 12H; 4ꢅCH3CO).
3
eqNeu), 2.75 (t, J=7.5 Hz, 2H; CH2CH2C(O)NH), 2.58 (t, J=12.5 Hz,
Compound 25: 1,5-DiazabicycloACTHNUTRGENUGN[5.4.0]undec-5-ene (DBU; 125 mL,
1H; H-3axNeu), 2.30 (t, J=7.5 Hz, 1H; CH2CH2CH2C(O)NH), 1.80–
2.25 ppm (m, 29H; 9ꢅCH3CO, CH2CH2CH2C(O)NH); 13C NMR
(400 MHz, CD3OD): d=168.0–174.0 (11ꢅC=O), 147.5 (C-Triaz), 120.2
(CH-Triaz), 77.8, 73.8, 72.0, 71.5, 68.9, 68.3, 68.0, 67.6, 66.8 (C-1Gal, C-
2Gal, C-3Gal, C-4Gal, C-5Gal, C-4Neu, C-6Neu, C-7Neu, C-8Neu), 61.9,
61.2 (C-6Gal, C-9Neu), 53.2 (COOCH3), 48.5 (C-5Neu), 35.5 (C-3Neu),
37.4 (CH2CH2CH2C(O)NH), 25.0 (CH2C(O)NH), 24.2 (CH2CH2CH2),
19.0–21.3 ppm (9ꢅCH3CO, CH2CH2C(O)NH); HRMS (FT-ICR, ESI):
m/z calcd for C40H55N5O22: 980.323645 [M+Na+]; found: 980.32300.
0.890 mmol) and DPPA (192 mL, 0.890 mmol) were added to a solution
of 4-butynol (69 mg, 0.99 mmol) in anhydrous DMF (300 mL). The reac-
tion mixture was stirred at RT until consumption of the alcohol was com-
plete. The mixture was then heated to 1208C and stirred for ꢀ2 h, until
disappearance of the intermediate phosphate was observed. The crude
azide solution was cooled to RT, PMe3 (0.988 mmol, 1m in toluene) was
added, and the reaction was stirred until no further formation of N2 was
observed. The crude amine solution was added by means of a cannula
into a second vessel containing the pentafluorophenyl ester derivative of
b-caboxymethylgalactoside 4 (55 mg, 0.0988 mmol). The reaction was
stirred overnight under a nitrogen atmosphere, then solvent was evapo-
rated, and the crude material was purified by flash chromatography
(hexane/EtOAc) to afford alkyne 25 (15.4 mg, 0.0349 mmol, 35% yield
overall from 4-butynol). 1H NMR (400 MHz, CDCl3): d=6.37 (t, J=
6 Hz, 1H; NHCH2), 5.45 (dd, J=1, J=3 Hz, 1H; H-4Gal), 5.11 (t, J=
10 Hz, 1H; H-2Gal), 5.04 (dd, J1 =3, J2 =10 Hz, 1H; H-3Gal), 4.00–4.20
(m, 2H; H-6Gal), 3.84–3.96 (m, 2H; H-1Gal, H-5Gal), 3.33–3.48 (m, 3H;
H-9, H-12), 2.40–2.50 (m, 2H; H-7, H-10), 1.94–2.19 ppm (m, 12H; 4ꢅ
CH3CO).
Compound 29: The reaction of alkyne 17 (10 mg, 0.022 mmol) with 26
following the general procedure described above afforded crude 29
(16 mg, 0.016 mmol), which was not further purified but subsequently de-
protected to ligand 40.
General procedure for the synthesis of 27–37, and 55 (click cycloaddi-
tion): Aqueous sodium ascorbate (1.1 equiv, 1m) and an aqueous solution
of CuSO4 (0.11 equiv, 0.3m) were added to a solution of alkyne (0.1m)
and sialic acid azide 26[8] (1.1 equiv) in methanol. The reaction vessel was
Chem. Eur. J. 2010, 16, 1951 – 1967
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1961

A. Bernardi et al.
Compound 30: The reaction of alkyne 18 (23 mg, 0.050 mmol) with 26
following the general procedure described above afforded 30 (31 mg,
0.032 mmol, 63%). 1H NMR (400 MHz, CDCl3): d=7.71 (s, 1H; H-
Triaz), 6.45 (br s, 1H; NH), 5.28–5.40 (m, 3H; H-4Gal, H-7Neu, H-
8Neu), 5.25 (d, J=9.8 Hz, 1H; AcNH), 5.00–5.17 (m, 3H; H-4Neu, H-
2Gal, H-3Gal), 4.46–4.50 (m, 1H; H-6bGal), 3.90–4.30 (m, 7H; H-9Neu,
H-5Neu, H-6Neu, H-1Gal, H-5Gal, H-6aGal), 3.71 (s, 3H; COOCH3),
3.28–3.46 (m, 2H; CH2bNC(O), H-3eqNeu), 3.24–3.06 (m, 1H;
CH2aNC(O)), 2.49–2.71 (m, 5H; H-3axNeu, CH2CH2C(O)NH), 1.80–2.15
(m, 27H; 9ꢅCH3CO), 1.60–1.75 (m, 1H; CH2bCH2NC(O)), 1.50–
1.60 ppm (m, 1H; CH2aCH2NC(O)).
Compound 33: The reaction of alkyne 21 (25 mg, 0.055 mmol) with 26
following the general procedure described above afforded 33 (36 mg,
0.037 mmol, 68%). 1H NMR (400 MHz, CDCl3): d=7.80 (s, 1H; H-
Triaz), 6.28 (br s, 1H; NH), 5.26–5.41 (m, 3H; H-4Gal, H-7Neu, H-
8Neu), 5.08–5.12 (m, 1H; H-4Neu), 5.03 (t, J=9.6 Hz, H-2Gal), 4.95 (dd,
J1 =3.4, J2 =10 Hz, 1H; H-3Gal), 4.17–4.28 (m, 2H; H-9bNeu, H-6Neu),
3.95–4.10 (m, 4H; H-5Neu, H-9aNeu, H-6Gal), 3.84 (t, J=6.5 Hz, 1H;
H-5Gal), 3.71 (s, 3H; COOCH3), 3.31–3.55 (m, 3H; H-1Gal,
CH2bNC(O), H3eqNeu), 3.20–3.30 (m, 1H; CH2aNC(O)), 2.49–2.80 (m,
3H; H-3axNeu, CH2CH2C(O)NH), 1.80–2.25 (m, 29H; 9ꢅCH3CO,
CH2CH2C(O)NH), 1.50–1.80 ppm (m, 2H; CH2CH2NC(O)).
Compound 34: The reaction of alkyne 22 (25 mg, 0.053 mmol) with 26
following the general procedure described above afforded 34 (34 mg,
0.034 mmol, 65%). H NMR (400 MHz, CDCl3): d=6.30 (br s, 1H; NH),
5.25–5.42 (m, 3H; H-4Gal, H-7Neu, H-8Neu), 5.08–5.12 (m, 1H; H-
4Neu), 5.03 (t, J=9.6 Hz, H-2Gal), 4.93 (dd, J1 =3.4, J2 =10 Hz, 1H; H-
3Gal), 4.17–4.28 (m, 2H; H-9bNeu, H-6Neu), 3.95–4.11 (m, 4H; H-5Neu,
H-9aNeu, H-6Gal), 3.81 (t, J=6.7 Hz, 1H; H-5Gal), 3.72 (s, 3H;
COOCH3), 3.30–3.52 (m, 3H; H-1Gal, CH2bNC(O), H-3eqNeu), 3.20–3.30
Compound 31: The reaction of alkyne 19 (27 mg, 0.058 mmol) with 26
following the general procedure described above afforded 31 (51 mg,
0.052 mmol, 90%). 1H NMR (400 MHz, CDCl3): d=7.70 (s, 1H; H-
Triaz), 6.30 (br s, 1H; NH), 5.36–5.48 (m, 3H; H-4Gal, H-7Neu, H-
8Neu), 5.33 (d, J=9.0 Hz, 1H; AcNH), 5.11–5.23 (m, 3H; H-4Neu, H-
2Gal, H-3Gal), 4.45 (t, J=7.5 Hz, 1H; H-6bGal), 4.23–4.37 (m, 3H; H-
9Neu, H-5Gal), 4.00–4.19 (m, 4H; H-5Neu, H-6Neu, H-1Gal, H-6aGal),
3.70 (s, 3H; COOCH3), 3.39–3.53 (m, 2H; CH2bNC(O), H-3eqNeu), 3.12–
3.30 (m, 1H; CH2aNC(O)), 2.68–2.90 (m, 2H; CH2CH2CH2C(O)NH),
2.68 (t, J=12 Hz, 1H; H-3axNeu), 2.28 (t, J=7 Hz, 2H;
CH2CH2C(O)NH), 1.60–2.25 ppm (m, 31H; 9ꢅCH3CO, CH2CH2NC(O),
CH2CH2CH2).
1
(m,
1H;
CH2aNC(O)),
2.49–2.80
(m,
(m,
3H;
33H;
H-3axNeu,
9ꢅCH3CO,
CH2CH2CH2C(O)NH),
1.50–2.27 ppm
CH2CH2CH2C(O)NH, CH2CH2NC(O)).
Compound 35: The reaction of alkyne 23 (9 mg, 0.019 mmol) with 26 fol-
lowing the general procedure described above afforded 35 (13 mg,
0.013 mmol, 70%). 1H NMR (400 MHz, CDCl3): d=6.45 (br s, 1H; NH),
5.23–5.42 (m, 3H; H-4Gal, H-7Neu, H-8Neu), 5.08–5.12 (m, 1H; H-
4Neu), 5.03 (t, J=9.5 Hz, H-2Gal), 4.91 (dd, J1 =3.4, J2 =10 Hz, 1H; H-
3Gal), 4.20–4.32 (m, 2H; H-9bNeu, H-6Neu), 3.95–4.13 (m, 4H; H-5Neu,
H-9aNeu, H-6Gal), 3.81 (t, J=6.7 Hz, 1H; H-5Gal), 3.70 (s, 3H;
COOCH3), 3.30–3.52 (m, 3H; H-1Gal, CH2bNC(O), H-3eqNeu), 3.20–3.30
Compound 32: The reaction of alkyne 20 (31 mg, 0.064 mmol) with 26
following the general procedure described above afforded 32 (56 mg,
0.056 mmol, 87%). 1H NMR (400 MHz, CDCl3): d=7.61 (s, 1H; H-
Triaz), 6.13 (br s, 1H; NH), 5.25–5.42 (m, 4H; AcNH, H-4Gal, H-7Neu,
H-8Neu), 5.04–5.16 (m, 3H; H-4Neu, H-2Gal, H-3Gal), 4.45 (t, J=8 Hz,
1H; H-6bGal), 4.14–4.29 (m, 3H; H-9Neu, H-5Gal), 3.90–4.12 (m, 4H;
H-5Neu, H-6Neu, H-1Gal, H-6aGal), 3.71 (s, 3H; COOCH3), 3.31–3.44
(m, 2H; CH2bNC(O), H-3eqNeu), 3.12 (m, 1H; CH2aNC(O)), 2.68 (t, J=
(m, 1H; CH2aNC(O)), 2.47–2.80 (m, 3H; H-3axNeu, CH HCTUNGTREN(NUNG CH2)2C(O)NH),
2A
1.50–2.25 ppm
(m,
35H;
9ꢅCH3CO,
(CH2)3CH2C(O)NH,
CH2CH2NC(O)).
7.5 Hz, 2H; CH
2.17 (t, J=7.5 Hz, 2H; (CH2)3CH2C(O)NH), 1.60–2.13 ppm (m, 33H; 9ꢅ
CH3CO, CH2CH2NC(O), CH2A(CH2)2CH2).
2ACHTUNGTRENNUNG(CH2)2CH2C(O)NH), 2.60 (t, J=12 Hz, 1H; H-3axNeu),
CTHUNGTRENNUNG
Compound 36: The reaction of alkyne 24 (84.2 mg, 0.197 mmol) with 26
following the general procedure described above afforded 36 (136.1 mg,
0.147 mmol, 75%). 1H NMR (400 MHz, CDCl3): d=7.85 (s, 1H; CH-
1962
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 1951 – 1967

Bidentate Cholera Toxin Ligands
FULL PAPER
Triaz), 6.80 (br s, 1H; NHCH2), 5.31–5.51 (m, 3H; H-4Gal, H-8Neu, H-
7Neu), 4.96–5.20 (m, 3H; H-4Neu, H-2Gal, H-3Gal), 4.47–4.67 (m, 2H;
H-6Neu, H-9aNeu), 4.21–4.36 (m, 2H; H-5Neu, H-9bNeu), 3.90–4.17 (m,
6H; H-1Gal, H-5Gal, H-6Gal, NHCH2-Triaz), 3.78 (s, 3H;
CH3OCONeu), 3.40 (dd, J1 =4, J2 =13 Hz, H-3eqNeu), 2.65 (t, J=12 Hz,
1H; H-3axNeu), 2.48 (brs, 2H; Gal-CH2-), 1.80–2.20 ppm (m, 27H; 8ꢅ
CH3CO, CH3CONH).
Typical deprotection procedure for synthesis of 38–48, 52 and 56–59:
Sodium methoxide in methanol (1m, 4 equiv) was added to a solution of
acetylated ligand in anhydrous MeOH (ꢀ0.05m), and the reaction mix-
ture was stirred for 30 min at RT. Water (ꢀ6 equiv) was added and the
reaction was stirred for a further 20 min at RT, then neutralized with
Amberlyte [H+], filtered and purified by reverse-phase (C-18) automatic
chromatography using a water/methanol gradient.
Ligand 38: Deprotection of 27 (30 mg, 0.032 mmol) following the general
procedure described above afforded ligand 38 (18 mg, 0.030 mmol, 95%).
[a]D =+3.2 (c=0.8 in MeOH); 1H NMR (D2O, 400 MHz): d=7.90 (s,
1H; H-triaz), 4.77 (d, J=9 Hz, 1H; H-1Gal), 3.40–3.95 (m, 13H; H-
4Neu, H-5Neu, H-6Neu, H-7Neu, H-8Neu, 2ꢅH-9Neu, H-2Gal, H-3Gal,
H-4Gal, H-5Gal, 2ꢅH-6Gal), 3.11 (dd, J1 =12.6, J2 =4.0 Hz, 1H; H-
3
eqNeu), 2.94 (t, J=7.00 Hz, 2H; CH2CH2C(O)NH), 2.60 (t, J=7.00 Hz,
2H; CH2CH2C(O)NH), 2.11 (t, J=11 Hz, 1H; H-3axNeu), 1.90 ppm (s,
3H; CH3C(O)NH); 13C NMR (D2O, 400 MHz): d=176.4, 175.0 (C=O),
146.2 (C-Triaz), 120.9 (CH-triaz), 90.8 (C-2Neu), 79.7 (C-1Gal), 76.8,
74.0, 73.4, 71.2 , 69.3, 68.3, 68.2, 68.1, 68.0 (C-2Gal, C-3Gal, C-4Gal, C-
5Gal, C-4Neu, C-6Neu, C-7Neu, C-8Neu), 62.5, 60.9 (C-6Gal, C-9Neu),
51.5 (C-5Neu), 39.6 (C-3Neu), 34.9 (CH2CH2C(O)NH), 22.1
(NHCOCH3,), 20.7 ppm (CH2CH2C(O)NH); HRMS (FT-ICR, ESI): m/z
calcd for C22H35N5O14: 593.218055 [M+]; found: 593.21307.
Compound 37: The reaction of alkyne 25 (13.1 mg, 0.0297 mmol) with 26
following the general procedure described above afforded 37 (8.0 mg,
0.0132 mmol, 45%). 1H NMR (400 MHz, CDCl3): d=7.77 (s, 1H; CH-
Triaz), 6.79 (t, J=6 Hz, 1H; NHCH2), 5.40–5.48 (m, 2H; H-4Gal, H-
8Neu), 5.37 (dd, J1 =2, J2 =9 Hz, 1H, H-7Neu), 5.18 (dt, J1 =4, J2 =
12 Hz, 1H; H-4Neu), 5.10 (t, J=10 Hz, 1H; H-2Gal), 5.05 (dd, J1 =3,
J2 =10 Hz, 1H; H-3Gal), 4.28–4.35 (m, 2H; H-6Neu, H-9aNeu), 4.03–
4.15 (m, 4H; H-6Gal, H-5Neu, H-9bNeu), 3.91–3.99 (m, 2H; H-1Gal, H-
5Gal), 3.80 (s, 3H; CH3OCONeu), 3.55–3.72 (m, 2H; CHNH), 3.43 (dd,
J1 =4, J2 =13 Hz, H-3eqNeu), 2.93 (t, J=6 Hz, 2H; CHCH2-Triaz), 2.66
(dd, J1 =12, J2 =13 Hz, 1H; H-3axNeu), 2.43 (brs, 2H; Gal-CH2), 1.95–
2.20 (m, 24H; 8ꢅCH3CO), 1.91 ppm (s, 3H; CH3CONH).
Ligand 39: Deprotection of 28 (68 mg, 0.071 mmol) following the general
procedure described above afforded ligand 39 (42 mg, 0.069 mmol, 97%).
[a]D =+6.8 (c=0.7 in MeOH); 1H NMR (D2O, 400 MHz): d=7.95 (s,
1H; H-triaz), 4.77 (d, J=9 Hz, 1H; H-1Gal), 3.40–3.95 (m, 13H; H-
4Neu, H-5Neu, H-6Neu, H-7Neu, H-8Neu, 2ꢅH-9Neu, H-2Gal, H-3Gal,
H-4Gal, H-5Gal, 2ꢅH-6Gal), 3.11 (d, J=9 Hz, 1H; H-3eqNeu), 2.61–2.68
(m, 2H; CH2ACHTNUGTRNE(UNG CH2)2C(O)NH), 1.98–2.24 (m, 2H; (CH2)2CH2C(O)NH),
2.11 (t, J=11.5 Hz, 1H; H-3axNeu), 1.80–1.95 ppm (m, 5H; NHC(O)CH3,
CH2CH2CH2); 13C NMR (D2O, 400 MHz): d=177.3, 175.0 (C=O), 120.0
(CH-triaz), 79.8 (C-1Gal), 76.7, 74.1, 73.4, 71.2, 70.4, 69.3, 68.9, 68.1, 67.9,
66.7 (C-2Gal, C-3Gal, C-4Gal, C-5Gal, C-4Neu, C-5Neu, C-6Neu, C-
7Neu, C-8Neu), 62.8, 60.9 (C-6Gal, C-9Neu), 51.5 (C-5Neu), 39.5 (C-
3Neu), 34.4 ((CH2)2CH2C(O)NH), 24.4 (CH2CH2CH2C(O)NH), 24.0
Compound 55: The reaction of alkyne 54 (100.0 mg, 0.175 mmol) with 26
following the general procedure described above afforded 55 (190.0 mg,
0.175 mmol, 99.7%). 1H NMR (400 MHz, CDCl3): d=7.81 (s, 1H; CH-
Triaz), 6.97 (br s, 1H; NHCH2), 5.89 (s, 1H; NHBoc), 5.32–5.46 (m, 2H;
H-4Gal, H-7Neu, H-8Neu), 5.16 (dt, J1 =4, J2 =12 Hz, 1H; H-4Neu), 5.07
(t, J=10 Hz, 1H; H-2Gal), 5.00 (dd, J1 =3, J2 =10 Hz, 1H; H-3Gal),
4.45–4.52 (m, 1H; H-10), 4.25–4.29 (m, 2H; H-6Neu, H-9aNeu), 4.01–
4.17 (m, 4H; H-6Gal, H-5Neu, H-9bNeu), 3.90 (t, J=6 Hz, 1H; H-5Gal),
3.78 (s, 3H; CH3OCONeu), 3.35–3.55 (m, 3H; H-1Gal, H-3eqNeu,
CH2CH2aNH), 3.25–3.35 (m, 2H; CH2CH2bNH, CHCH2-Triaz), 2.60 (dd,
J1 =12, J2 =13 Hz, 1H; H-3axNeu), 1.93–2.20 (m, 24H; 8ꢅCH3CO), 1.90
(s, 3H; CH3CONH), 1.60–1.80 ppm (m, 2H; Gal-CH2); 13C NMR
(400 MHz, CDCl3): d=167.5–168.7 (8ꢅCH3CO, CH2CH2NHCO, C-1Neu,
CH3CONHNeu), 164.0 (NHCOO-tBu), 72.0 (C-5Gal), 71.5 (C-6Neu),
69.7 (C-3Gal), 66.8 (C-2Gal), 66.1 (C-4Neu), 65.9 (C-4Gal), 65.4 (C-
8Neu), 64.6 (C-7Neu), 60.0 (C-9Neu), 59.3 (C-6Gal), 51.8
(CH2ACHTNUGTRNE(UGN CH2)2C(O)NH), 21.0 ppm (NHCOCH3); HRMS (FT-ICR, ESI):
m/z calcd for C23H37N5O14: 607.233705 [M+]; found: 607.22861.
(CH3OCONeu), 51.5 (CACHTUNGTRENNUNG(CH3)3), 51.4 (CHNH), 47.0 (C-5Neu), 33.8 (2C,
C-3Neu, CH2NH), 28.5 (Gal-CH2), 26.0 (tBu), 25.8 (CHCH2Triaz), 20.9
(CH3CONHNeu), 18.7–18.9 ppm (8ꢅCH3CO).
Ligand 40: Deprotection of crude 29 following the general procedure de-
scribed above afforded ligand 40 (9 mg, 0.014 mmol, 66% from 17 (two
steps)). [a]D =++22.3 (c=0.3 in H2O); H NMR (D2O, 400 MHz): d=7.95
1
(s, 1H; H-triaz), 5.11 (d, J=7 Hz, 1H; H-1Gal), 3.37–4.14 (m, 13H; H-
4Neu, H-5Neu, H-6Neu, H-7Neu, H-8Neu, 2ꢅH-9Neu, H-2Gal, H-3Gal,
H-4Gal, H-5Gal, 2ꢅH-6Gal), 3.08 (d, J=8.7 Hz, 1H; H-3eqNeu), 2.56–
2.63
(m,
2H;
CH
(CH2)3C(O)NH),
2.15–2.26
(m,
2H;
(CH2)3CH2C(O)NH), 2.08 (t, J=12 Hz, 1H; H-3axNeu), 1.90 (s, 1H;
NHC(O)CH3), 1.38–1.62 ppm (m, 4H; (CH2)2); 13C NMR (D2O,
Chem. Eur. J. 2010, 16, 1951 – 1967
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1963

A. Bernardi et al.
400 MHz): d=177.1, 175.2 (C=O), 121.4 (CH-triaz), 79.0 (C-1Gal), 76.7,
74.3, 73.1, 71.1, 70.7, 68.9, 68.7, 67.8, 67.6, 66.8 (C-2Gal, C-3Gal, C-4Gal,
C-5Gal, C-4Neu, C-5Neu, C-6Neu, C-7Neu, C-8Neu), 62.7, 61.1 (C-6Gal,
C-9Neu), 51.6 (C-5Neu), 39.4 (C-3Neu), 34.6 ((CH2)3CH2C(O)NH), 25.0
(CH2CH2CH2); HRMS (FT-ICR, ESI): m/z calcd for C25H41N5O14
634.257180 [MÀH]À; found: 634.25653.
:
Ligand 43: Deprotection of 32 (37 mg, 0.037 mmol) following the general
procedure described above afforded ligand 43 (17 mg, 0.026 mmol, 71%).
[a]D =+41.9 (c=0.49 in MeOH); 1H NMR (D2O, 400 MHz): d=7.92 (s,
1H; H-triaz), 4.00–4.05 (m, 1H; H-1Gal), 3.54–3.99 (m, 13H; H-4Neu,
H-5Neu, H-6Neu, H-7Neu, H-8Neu, 2ꢅH-9Neu, H-2Gal, H-3Gal, H-
4Gal, H-5Gal, 2ꢅH-6Gal), 3.15–3.28 (m, 3H; H-3eqNeu, CH2NHC(O)),
(CH
N
24.2
(CH
G
21.1 ppm
(NHCOCH3); HRMS (FT-ICR, ESI): m/z calcd for C24H39N5O14
[MÀH]À: 620.241530 found: 620.24059.
2.69 (t, J=7.1 Hz, 2H; CH
CH2CH2C(O)NH, H-3axNeu), 2.01 (s,1H; NHC(O)CH3), 1.50–1.94 ppm
(m, 6H; CH2A
(CH2)2CH2, CH2CH2NHC(O)); 13C NMR (D2O, 400 MHz):
2ACHTUGNRTEN(NUNG CH2)2C(O)NH), 2.15–2.25 (m, 3H;
CTHUNGTRENNUNG
d=176.0, 174.3 (C=O), 120.0 (CH-triaz), 72.0 (C-1Gal), 72.9, 70.8, 70.2,
68.5, 67.8, 67.0, 66.9 (C-2Gal, C-3Gal, C-4Gal, C-5Gal, C-4Neu, C-5Neu,
C-6Neu, C-7Neu, C-8Neu), 61.5, 60.0 (C-6Gal, C-9Neu), 50.4 (C-5Neu),
38.5 (C-3Neu), 35.0 (CH2NHC(O)), 34.3 (CH2CH2C(O)NH), 26.8, 23.5
(CH2ACHTUNGRTENN(UG CH2)2CH2), 23.1 (CH2AHCTUNGTRENN(GUN CH2)3C(O)NH), 22.3 (CH2CH2C(O)NH),
20.9 ppm (NHCOCH3); HRMS (FT-ICR, ESI): m/z calcd for
Ligand 41: Deprotection of 30 (23 mg, 0.024 mmol) following the general
procedure described above afforded ligand 41 (14 mg, 0.023 mmol, 95%).
[a]D =+40.9 (c=0.2 in MeOH); 1H NMR (D2O, 400 MHz): d=7.87 (s,
1H; H-triaz), 3.95–4.00 (m, 14H; H-4Neu, H-5Neu, H-6Neu, H-7Neu, H-
8Neu, 2ꢅH-9Neu, H-1Gal, H-2Gal, H-3Gal, H-4Gal, H-5Gal, 2ꢅH-
6Gal), 3.03–3.15 (m, 3H; H-3eqNeu, CH2NHC(O)), 2.87 (t, J=7.1 Hz,
2H; CH2CH2C(O)NH), 2.44 (t, J=7.1 Hz, 2H; CH2CH2C(O)NH), 2.08
(m, 1H; H-3axNeu), 1.90 (s,1H; NHC(O)CH3), 1.50–1.75 ppm (m, 2H;
CH2CH2NHC(O)); 13C NMR (D2O, 400 MHz): d=177.4, 174.8 (C=O),
120.0 (CH-triaz), 72.9, 72.1, 70.8, 70.2, 70.7, 68.5, 67.8, 66.9 (C-1Gal, C-
2Gal, C-3Gal, C-4Gal, C-5Gal, C-4Neu, C-5Neu, C-6Neu, C-7Neu, C-
8Neu), 61.6, 59.9 (C-6Gal, C-9Neu), 50.4 (C-5Neu), 38.4 (C-3Neu), 35.0
(CH2NHC(O)), 34.3 (CH2CH2C(O)NH), 23.0 (CH2CH2C(O)NH),
20.9 ppm (NHCOCH3); HRMS (FT-ICR, ESI): m/z calcd for
C24H39N5O14: 620.241530 [MÀH]À; found: 620.24069.
C26H43N5O14: 648.272830 [MÀH]À; found: 648.27190.
Ligand 44: Deprotection of 33 (25 mg, 0.026 mmol) following the general
procedure described above afforded ligand 44 (16 mg, 0.026 mmol,
100%). [a]D =+16.1 (c=0.25 in MeOH); 1H NMR (D2O, 400 MHz): d=
7.73 (s, 1H; H-triaz), 3.26–3.80 (m, 12H; H-4Neu, H-5Neu, H-6Neu, H-
7Neu, H-8Neu, 2ꢅH-9Neu, H-3Gal, H-4Gal, H-5Gal, 2ꢅH-6Gal), 3.17
(t, J=7.5 Hz, 1H; H-2Gal), 2.97–3.10 (m, 3H; CH2NHC(O), H-3eqNeu),
2.93 (dt, J1 =7.5, J2 =2.3 Hz, 1H; H-1Gal), 2.78 (t, J=7.2 Hz,1H;
CH2CH2C(O)NH), 2.78 (t, J=7.2 Hz, 1H; CH2CH2C(O)NH), 1.99 (t, J=
11.2 Hz, 1H; H-3axNeu), 1.82 (s, 3H; NHC(O)CH3), 1.72–1.76 (m, 1H;
CH2bCH2NH), 1.30 ppm (m, 1H; CH2aCH2NH); 13C NMR (D2O,
400 MHz): d=173.7, 169.5 (C=O), 145.0 (C-triaz), 119.7 (CH-triaz), 89.6
(C-2Neu), 77.3, 73.0, 72.8, 69.5, 68.0, 66.9 (C-3Gal, C-4Gal, C-5Gal, C-
4Neu, C-6Neu, C-7Neu, C-8Neu), 76.6 (C-1Gal), 69.9 (C-2Gal), 61.5, 60.2
(C-6Gal, C-9Neu), 50.4 (C-5Neu), 38.4 (C-3Neu), 34.8 (CH2NHC(O)),
34.2 (CH2CH2C(O)NH), 29.3 (CH2CH2NHC(O)), 20.9 (NHCOCH3),
20.2 ppm (CH2CH2C(O)NH); HRMS (FT-ICR, ESI): m/z calcd for
C24H39N5O14: 620.241530 [MÀH]À; found: 620.24065.
Ligand 42: Deprotection of 31 (16 mg, 0.016 mmol) following the general
procedure described above afforded ligand 42 (10 mg, 0.016 mmol, 97%).
[a]D =+80.8 (c=0.06 in MeOH); 1H NMR (D2O, 400 MHz): d=8.07 (s,
1H; H-triaz), 4.10–4.14 (m, 1H; H-1Gal), 3.60–4.06 (m, 13H; H-4Neu,
H-5Neu, H-6Neu, H-7Neu, H-8Neu, 2ꢅH-9Neu, H-2Gal, H-3Gal, H-
4Gal, H-5Gal, 2ꢅH-6Gal), 3.22–3.35 (m, 3H; H-3eqNeu, CH2NHC(O)),
2.79 (t, J=7.2 Hz, 2H; CH2ACHTNUTRGNE(UNG CH2)2C(O)NH), 2.22–2.37 (m, 3H;
CH2CH2C(O)NH, H-3axNeu), 2.08 (s,1H; NHC(O)CH3), 1.75–2.05 ppm
(m, 4H; CH2CH2CH2, CH2CH2NHC(O)); 13C NMR (D2O, 400 MHz):
d=177.3, 175.0 (C=O), 119.9 (CH-triaz), 72.1 (C-1Gal), 72.9, 71.0, 70.4,
70.7, 68.8, 68.5, 67.1 (C-2Gal, C-3Gal, C-4Gal, C-5Gal, C-4Neu, C-5Neu,
C-6Neu, C-7Neu, C-8Neu), 61.6, 60.03 (C-6Gal, C-9Neu), 51.2 (C-5Neu),
38.4 (C-3Neu), 35.0 (CH2NHC(O)), 33.8 (CH2CH2C(O)NH), 23.6 (CH2-
Ligand 45: Deprotection of 34 (20 mg, 0.020 mmol) following the general
procedure described above afforded ligand 45 (12 mg, 0.019 mmol, 93%).
[a]D =+8.5 (c=0.64 in MeOH); 1H NMR (D2O, 400 MHz): d=7.89 (s,
1H; H-triaz), 3.37–3.90 (m, 12H; H-4Neu, H-5Neu, H-6Neu, H-7Neu, H-
8Neu, 2ꢅH-9Neu, H-3Gal, H-4Gal, H-5Gal, 2ꢅH-6Gal), 3.28 (t, J=
9.5 Hz, 1H; H-2Gal), 2.99–3.23 (m, 4H; CH2NHC(O), H-3eqNeu, H-
1Gal), 2.60 (t, J=9.2 Hz,1H; CH2CH2C(O)NH), 2.01–2.17 (m, 3H;
CH2CH2C(O)NH, H-3axNeu), 1.75–19.5 (m, 6H; CH2CH2CH2,
NHC(O)CH3, CH2bCH2NH), 1.42–1.52 ppm (m, 1H; CH2aCH2NH);
13C NMR (D2O, 400 MHz): d=173.7 (C=O), 120.27 (CH-triaz), 89.6 (C-
2Neu), 77.3, 72.9, 72.8, 70.1, 68.4, 68.0, 66.9, 66.8 (C-3Gal, C-4Gal, C-
5Gal, C-4Neu, C-6Neu, C-7Neu, C-8Neu), 76.6 (C-1Gal), 69.6 (C-2Gal),
61.6, 60.2 (C-6Gal, C-9Neu), 50.3 (C-5Neu), 38.4 (C-3Neu), 34.8
ACHTUNGTRENNUNG(CH2)2C(O)NH), 20.9 (NHCOCH3), 21.0 (CH2CH2C(O)NH), 20.7 ppm
1964
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 1951 – 1967

Bidentate Cholera Toxin Ligands
FULL PAPER
(CH2NHC(O)), 33.7 (CH2CH2C(O)NH), 29.3 (CH2CH2NHC(O)), 23.8
(CH2CH2CH2), 22.7 (CH2CH2C(O)NH), 20.9 ppm (NHCOCH3); HRMS
(FT-ICR, ESI): m/z calcd for C25H41N5O14: 634.257180 [MÀH]À; found:
634.25636.
Ligand 48: Deprotection of 37 (9.2 mg, 0.0096 mmol) following the gen-
eral procedure described above afforded ligand 48 (4.0 mg, 0.0453 mmol,
69%). 1H NMR (400 MHz, D2O): d=7.87 (s, 1H; CH-Triaz), 3.65–3.89
(m, 6H; H-2Gal, H-3Gal, H-4Neu, H-7Neu, H-8Neu, H-9aNeu), 3.40–
3.54 (m, 6H; H-4Gal, H-6Gal, H-5Neu, H-6Neu, H-9aNeu), 3.31–3.39
(m, 3H; H-1Gal, NHCH2CH2Triaz), 3.27 (t, J=10 Hz, 1H; H-5Gal), 3.09
(dd, J1 =4, J2 =12 Hz, 1H; H-3eqNeu), 2.78 (t, J=6 Hz, 2H;
NHCH2CH2Triaz), 2.58 (dd, J1 =3, J2 =15 Hz, 1H; CH-2aGal), 2.21 (dd,
J1 =9, J2 =15 Hz, 1H; CH-2bGal), 2.10 (dd, J1 =11, J2 =12 Hz, 1H; H-
3axNeu), 1.90 ppm (s, 3H; CH3CONH); 13C NMR (400 MHz, D2O): d=
173.9, 172.5, 169.5 (CH2NHCO, C-1Neu, CH3CONHNeu), 77.3 75.62,
7.95, 72.6, 70.2, 69.5, 67.9, 66.93, 66.87 (C-1Gal, C-2Gal, C-3Gal, C-4Gal,
C-5Gal, C-4Neu, C-6Neu, C-7Neu, C-8Neu), 61.6 (C-9Neu), 60.0 (C-
6Gal), 50.4 (C-5Neu), 38.4 (C-3Neu), 37.6 (NHCH2CH2Triaz), 37.5
(GalCH2), 23.5 (NHCH2CH2Triaz), 20.9 ppm (NHCOCH3); HRMS (FT-
ICR, ESI): m/z calcd for C23H36N5O14
:
606.22642 [MÀH]À; found:
606.22665.
Ligand 46: Deprotection of 35 (13 mg, 0.013 mmol) following the general
procedure described above afforded ligand 46 (8 mg, 0.012 mmol, 95%).
[a]D =+27.4 (c=0.11 in MeOH); 1H NMR (D2O, 400 MHz): d=7.81 (s,
1H; H-triaz), 3.31–3.90 (m, 12H; H-4Neu, H-5Neu, H-6Neu, H-7Neu, H-
8Neu, 2ꢅH-9Neu, H-3Gal, H-4Gal, H-5Gal, 2ꢅH-6Gal), 3.27 (t, J=
9.4 Hz, 1H; H-2Gal), 3.00–3.23 (m, 4H; CH2NHC(O), H-3eqNeu, H-
1Gal), 2.57 (t, J=9.4 Hz,1H; CH2CH2C(O)NH), 2.03–2.15 (m, 3H;
CH2CH2C(O)NH, H-3axNeu), 1.83–1.96 (m, 4H; NHC(O)CH3,
CH2bCH2NH), 1.37–1.57 ppm (m, 5H; (CH2)2, CH2aCH2NH); 13C NMR
(D2O, 400 MHz): d=175.5, 173.8, 169.7 (C=O), 146.8 (C-triaz), 119.5
(CH-triaz), 89.5 (C-2Neu), 77.4, 72.9, 72.8, 70.2, 68.0, 66.9, 66.8 (C-3Gal,
C-4Gal, C-5Gal, C-4Neu, C-6Neu, C-7Neu, C-8Neu), 76.2 (C-1Gal), 69.6
(C-2Gal), 61.6, 60.2 (C-6Gal, C-9Neu), 50.4 (C-5Neu), 38.4 (C-3Neu),
34.7 (CH2NHC(O)), 34.4 (CH2CH2C(O)NH), 29.3, 26.7, 23.6
Ligand 52: Aqueous sodium ascorbate (0.1 mL, 1m) and an aqueous so-
lution of CuSO4 (0.1 mL, 0.3m) were added to a solution of alkyne 51
(20 mg, 0.039 mmol) and 26[9] (24 mg, 0.047 mmol) in methanol (1 mL).
The reaction vessel was protected from light and the reaction mixture
was vigorously stirred overnight. The solvent then was evaporated and
the solid residue was extracted with CH2Cl2 (15 mL). The extracts were
concentrated and purified by flash chromatography to give the crude ace-
tylated coupling product (27 mg, 0.026 mmol), which was not further pu-
rified, but subsequently deprotected, as described above for compounds
38–48, to provide ligand 52 (17 mg, 0.025 mmol) as a mixture of stereo-
(CH2CH2NHC(O), (CH2)2), 23.0
(CH2CH2C(O)NH),
20.9 ppm
(NHCOCH3); HRMS (FT-ICR, ESI): m/z calcd for C26H43N5O14
648.272830 [MÀH]À; found: 648.27212.
:
1
isomers, in 64% overall yield. H NMR (400 MHz, D2O): d=7.96 (s, 1H;
H-triaz), 4.42–4.50 (m, 1H; CHACHTNUTRGENNG(U NHAc)CH2), 3.41–4.05 (m, 12H; H-
4Neu, H-5Neu, H-6Neu, H-7Neu, H-8Neu, 2ꢅH-9Neu, H-3Gal, H-4Gal,
H-5Gal, 2ꢅH-6Gal), 3.34 (t, J=10 Hz, 1H; H-2Gal), 2.95–3.28 (m, 5H;
CHACTHNUTGRNE(UGN NHAc)CH2, CH2CH2NHC(O), H-3axNeu), 2.15 (m, 1H; H-3eqNeu),
1.84–2.00 (m, 4H; CH3CO, CH2bCH2NHC(O)), 1.42–1.52 ppm (m, 1H;
CH2aCH2NHC(O)). 13C NMR (400 MHz, D2O): d=122.0 (C-triaz), 79.0,
77.3, 74.0, 71.2, 70.0, 68.9, 67.6 (C-3Gal, C-4Gal, C-5Gal, C-4Neu, C-
6Neu, C-7Neu, C-8Neu), 77.6 (C-1Gal), 70.3 (C-2Gal), 62.2, 61.0 (C-
Ligand 47: Deprotection of 36 (136.1 mg, 0.144 mmol) following the gen-
eral procedure described above afforded ligand 47 (26.9 mg,
0.0453 mmol, 31.4%). 1H NMR (400 MHz, D2O): d=8.14 (s, 1H; CH-
Triaz.), 3.54(s, 2H; CH2-Triaz), 3.86–4.08 (m, 6H; H-2Gal, H-3Gal, H-
4Neu, H-7Neu, H-8Neu, H-9aNeu), 3.59–3.74 (m, 7H; H-1Gal, H-4Gal,
H-6Gal, H-5Neu, H-6Neu, H-9aNeu), 3.50 (t, J=10 Hz, 1H; H-5Gal),
3.28 (dd, J1 =4, J2 =13 Hz, 1H; H-3eqNeu), 2.86 (dd, J1 =3, J2 =15 Hz,
1H; CH-2aGal), 2.50 (dd, J1 =9, J2 =15 Hz, 1H; CH-2bGal), 2.28 (dd,
J1 =11, J2 =13 Hz, 1H; H-3axNeu), 2.10 ppm (s, 3H; CH3CONH);
13C NMR (400 MHz, D2O): d=173.83, 172.44, 169.43 (CH2NHCO, C-
1Neu, CH3CONHNeu), 143.2 (CqTriaz.), 120.4 (CH-Triaz.), 89.7 (C-
2Neu), 77.3, 75.5, 72.9, 72.6, 70.1, 69.4, 67.9, 66.9, 66.8 (C-1Gal, C-2Gal,
C-3Gal, C-4Gal, C-5Gal, C-4Neu, C-6Neu, C-7Neu, C-8Neu), 61.6 (C-
9Neu), 60.0 (C-6Gal), 50.4 (C-5Neu), 38.5 (C-3Neu), 37.5 (GalCH2), 33.3
(NHCH2Triaz), 20.9 ppm (NHCOCH3); HRMS (FT-ICR, ESI): m/z calcd
for C22H34N5O14: 592.21077 [MÀH]À; found: 592.21028.
6Gal, C-9Neu), 54.3 (CHACHTNUTRGNE(NUG NHAc)CH2), 52.1 (C-5Neu), 39.4 (C-3Neu),
30.7 (CH2CH2NHC(O)), 26.8 (CH2CH2NHC(O)), 21.9 ppm (CH3CO);
HRMS (FT-ICR, ESI): m/z calcd for C26H42N6O15: 677.262994 [MÀH]À;
found: 677.26189.
Ligand 56: CF3COOH (900 mL) was added to a solution of 55 (15.2 mg,
0.014 mmol) in MeOH (2 mL). The mixture was stirred for ꢀ2 h until de-
protection of the amino group was complete. Then the solvent was
evaporated and the acid was eliminated by co-evaporation with toluene.
The resulting amine was dissolved in anhydrous THF (1.5 mL), and trie-
thylamine (21 mL, 0.15 mmol) and benzoyl chloride (7 mL, 0.06 mmol)
were added. The mixture was stirred at RT for 4 h then the solvent was
Chem. Eur. J. 2010, 16, 1951 – 1967
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1965

A. Bernardi et al.
evaporated, and the crude product was purified by flash chromatography
(CH2Cl2/acetone gradient). Deprotection of the product (9 mg,
by co-evaporation with toluene. The amine was dissolved in anhydrous
pyridine (1.5 mL), then anhydrous Et3N (79 mL, 0.561 mmol) and
PhNCO (20 mL, 0.168 mmol) were added. The mixture was stirred over-
night at RT, then the solvent was evaporated in vacuum and the mixture
was purified by flash chromatography (CH2Cl2/acetone gradient). The
product was deprotected as described above to afford 58 (31.8 mg,
0.0421 mmol, 75%). 1H NMR (400 MHz, D2O): d=8.08 (s, 1H; CH-
Triaz), 7.39 (t, J=8 Hz, 2H; H-Phmeta), 7.30–7.35 (m, 2H; H-Phortho), 7.17
(t, J=7 Hz, 1H; H-Phpara), 4.52 (t, J=6 Hz, 1H; CHNH), 3.13–4.04 (m,
19H; H-1Gal, H-2Gal, H-3Gal, H-4Gal, H-5Gal, H-6Gal, H-1Neu, H-
2Neu, H-3eqNeu, H-4Neu, H-5Neu, H-6Neu, H-7Neu, H-8Neu, H-9Neu,
GalCH2CH2NH, CHCH2-Triaz), 2.20–2.30 (m, 1H; H-3axNeu), 2.02–2.12
(m, 1H; GalCH2a), 1.94 (s, 3H; CH3CONH), 1.55–1.65 ppm (m, 1H;
GalCH2b); 13C NMR (400 MHz, D2O): d=173.9, 172.1, 168.9, 155.9
(CH2NHCO, C-1Neu, CH3CONHNeu, NHCONH), 136.54 (CqTriaz),
128.1 (C-Phmeta), 123.0 (C-Phpara), 120.0 (CH-Triaz), 119.2 (C-Phortho), 77.3,
76.5, 76.1, 73.0, 72.8, 70.2, 69.6, 68.0, 66.9 (C-1Gal, C-2Gal, C-3Gal, C-
4Gal, C-5Gal, C-4Neu, C-6Neu, C-7Neu, C-8Neu), 61.5 (C-9Neu), 60.3
(C-6Gal), 52.9 (CHNH), 50.4 (C-5Neu), 38.6 (C-3Neu), 35.1
(GalCH2CH2NH), 29.1 (GalCH2), 26.3 (CH2Triaz), 20.9 ppm
0.0082 mmol) afforded ligand 56 as
a 1:1 mixture of diastereomers
(6.0 mg, 0.0081 mmol, 58%). 1H NMR (400 MHz, D2O): d=8.13 (s, 1H;
H-triaz), 7.77 (dd, J=4.3, 5.9 Hz, 2H; Ph), 7.67–7.73 (m, 1H; Ph), 7.60 (t,
J=7.5 Hz, 2H; Ph), 4.85> (brt, J=6.5 Hz, 1H; CHACTHNUTRGENUG(N NHBz)CH2), 3.48–
4.00 (m, 12H; H-4Neu, H-5Neu, H-6Neu, H-7Neu, H-8Neu, 2ꢅH-9Neu,
H-3Gal, H-4Gal, H-5Gal, 2ꢅH-6Gal), 3.43 (m, 1H; H-2Gal), 3.17–3.38
(m, 5H; CHACHTUNGTRENNUNG(NHBz)CH2, CH2CH2NHC(O), H-3axNeu), 2.20–2.30 (m,
1H; H-3eqNeu), 1.85–2.07 (m, 4H; CH3CO, CH2bCH2NHC(O)), 1.50–
1.64 ppm (m, 1H; CH2aCH2NHC(O)); 13C NMR (400 MHz, D2O): d=
132.6, 128.8, 127.8 (Ph), 122.1 (C-triaz), 78.7, 77.5, 74.1, 71.2, 69.7, 68.7,
67.7 (C-3Gal, C-4Gal, C-5Gal, C-4Neu, C-6Neu, C-7Neu, C-8Neu), 77.5
(C-1Gal), 70.7 (C-2Gal), 62.4, 61.2 (C-6Gal, C-9Neu), 54.1 (CH-
ACHTUNGTRENNUNG(NHAc)CH2), 51.3 (C-5Neu), 39.5 (C-3Neu), 30.1 (CH2CH2NHC(O)),
27.0 (CH2CH2NHC(O)), 21.8 ppm (CH3CO); HRMS (FT-ICR, ESI): m/z
calcd for C31H43N6O15: 739.27919 [MÀH]À; found: 739.27851.
(NHCOCH3); HRMS (FT-ICR, ESI): m/z calcd for C31H44N7O15
754.29009 [MÀH]À; found: 754.29121.
:
Ligand 57: CF3COOH (250 mL) was added to a solution of 55 (20.2 mg,
0.0186 mmol) in CH2Cl2 (1 mL). The mixture was stirred for ꢀ2 h until
deprotection of the amino group was complete. Then the solvent was
evaporated and the acid was removed by co-evaporation with toluene.
The amine was dissolved in anhydrous THF (0.5 mL) and treated with
the pentafluorophenyl ester of phenylacetic acid (8.4 mg, 0.0279 mmol)
and triethylamine (13 mL, 0.0929 mmol) for 4 h. The solvent was evapo-
rated in vacuum and the product was purified by flash chromatography
(CH2Cl2/acetone gradient). The product was deprotected as described
above to afford ligand 57 (15.9 mg, 0.0144 mmol, 71%). 1H NMR
Ligand 59: CF3COOH (300 mL) was added to a solution of 55 (24.8 mg,
0.0228 mmol) in CH2Cl2 (1.2 mL). The mixture was stirred for ꢀ2 h until
deprotection of the amino group was complete, then the solvent was
evaporated, and CF3COOH was removed by co-evaporation with tolu-
ene. The amine was dissolved in anhydrous pyridine (0.6 mL), then anhy-
drous Et3N (32 mL, 0.228 mmol) and BnNCO (6 mL, 0.0456 mmol) were
added. The mixture was stirred overnight at RT then the solvent was
evaporated in vacuum and the mixture was purified by flash chromatog-
raphy (CH2Cl2/acetone gradient). The product was deprotected as de-
scribed above to afford 59 (0.013 mmol, 56%). 1H NMR (400 MHz,
D2O): d=8.07 (s, 1H; CH-Triaz), 7.00–7.43 (m, 5H; Ph), 4.43–4.53 (m,
1H; CHNH), 4.29 (qd, J1 =5, J2 =16 Hz, NHCH2Ph), 3.07–4.16 (m, 19H;
H-1Gal, H-2Gal, H-3Gal, H-4Gal, H-5Gal, H-6Gal, H-1Neu, H-2Neu,
H-3eqNeu, H-4Neu, H-5Neu, H-6Neu, H-7Neu, H-8Neu, H-9Neu,
GalCH2CH2NH, CHCH2-Triaz), 2.00–2.25 (m, 1H; H-3axNeu), 2.08 (s,
3H; CH3CONH), 1.98–2.07 (m, 1H; GalCH2a), 1.55–1.65 ppm (m, 1H;
GalCH2b); 13C NMR (400 MHz, D2O): d=173.9, 172.3, 169.1
(CH2NHCO, C-1Neu, CH3CONHNeu), 158.1 (NHCONH), 138.2
(CqTriaz), 127.6 (C-Phmeta), 125.4 (C-Phortho), 125.5 (C-Phortho), 77.4, 76.5,
76.2, 73.0, 72.8, 70.1, 69.2, 68.0, 67.1, 66.9 (C-1Gal, C-2Gal, C-3Gal, C-
4Gal, C-5Gal, C-4Neu, C-6Neu, C-7Neu, C-8Neu), 61.5 (C-9Neu), 60.2
(C-6Gal), 53.0 (CHNH), 50.4 (C-5Neu), 42.1 (CH2Ph), 38.5 (C-3Neu),
34.7 (GalCH2CH2NH), 29.2 (GalCH2), 25.9 (CH2Triaz), 20.9 ppm
(400 MHz, D2O): d=8.04 (d, 1H; CH-Triaz
ACHTUNGTNER(NUNG R/S)), 7.34–7.47 (m, 3H; H-
Phmeta H-Phpara), 7.30–7.25 (m, 2H; H-Phortho), 4.45–4.65 (m, 1H;
,
CHNH), 3.11–4.09 (m, 21H; H-1Gal, H-2Gal, H-3Gal, H-4Gal, H-5Gal,
H-6Gal, H-1Neu, H-2Neu, H-3eqNeu, H-4Neu, H-5Neu, H-6Neu, H-
7Neu, H-8Neu, H-9Neu, GalCH2CH2NH, CHCH2-Triaz, COCH2Ph),
2.19 (dd, J1 =11, J2 =18 Hz, 1H; H-3axNeu), 2.00–2.10 (m, 1H; GalCH2a),
2.10 (s, 3H; CH3CONH), 1.50–1.64 ppm (m, 1H; GalCH2b); 13C NMR
(400 MHz, D2O): d=173.9, 173.2, 171.0, 169.3 (CH2NHCO, C-1Neu,
CH3CONHNeu, NHCOCH2Ph), 133.5 (CqTriaz), 127.7–127.9 (C-Phortho
,
C-Phmeta), 126.2 (C-Phpara), 77.3, 76.3, 76.0, 72.9, 72.8, 70.2, 69.6, 68.0, 66.9
(C-1Gal, C-2Gal, C-3Gal, C-4Gal, C-5Gal, C-4Neu, C-6Neu, C-7Neu, C-
8Neu), 61.5 (C-9Neu), 60.3 (C-6Gal), 52.6 (CHNH), 50.4 (C-5Neu), 40.8
(CH2Ph), 38.5 (C-3Neu), 34.9, 34.6 (GalCH2CH2NH), 29.2 (GalCH2),
26.0 (CH2Triaz), 20.9 ppm (NHCOCH3); HRMS (FT-ICR, ESI): m/z
calcd for C32H45N6O15: 753.29484 [MÀH]À; found: 753.29366.
Ligand 58: CF3COOH (750 mL) was added to a solution of Boc-protect-
ed acetylated ligand 55 (61.0 mg, 0.0561 mmol) in CH2Cl2 (3 mL). The
mixture was stirred for ꢀ2 h until deprotection of the amino group was
complete, then the solvent was evaporated and CF3COOH was removed
1966
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ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 1951 – 1967

Bidentate Cholera Toxin Ligands
FULL PAPER
(NHCOCH3); HRMS (FT-ICR, ESI): m/z calcd for C32H46N7O15
768.30574 [MÀH]À; found: 768.30668.
:
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Acknowledgements
Support by COST-D34 and Consorzio Interuniversitario Nazionale Meto-
dologie e Processi Innovativi di Sintesi (CINMPIS) is gratefully acknowl-
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Received: September 7, 2009
Published online: December 28, 2009
Chem. Eur. J. 2010, 16, 1951 – 1967
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1967