Comparative studies on lectin-carbohydrate interactions in low and high density homo- and heteroglycoclusters

Article abstract of DOI:10.1039/b920048g

A versatile synthetic procedure to construct series of high- and low-density homo- and heteroglycoclusters is reported. The binding properties of these synthetic multivalent glycoconjugates to concanavalin A (Con A), a model lectin, have been assessed by

Full text of DOI:10.1039/b920048g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Comparative studies on lectin–carbohydrate interactions in low and high
density homo- and heteroglycoclusters†
a
b
c
d
a
Marta G o´ mez-Garc ´ı a, Juan M. Benito, Ricardo Guti e´ rrez-Gallego, Alfredo Maestre, Carmen Ortiz Mellet,
b
a
Jos e´ M. Garc ´ı a Fern a´ ndez and Jos e´ L. Jim e´ nez Blanco*
Received 25th September 2009, Accepted 26th January 2010
First published as an Advance Article on the web 24th February 2010
DOI: 10.1039/b920048g
A versatile synthetic procedure to construct series of high- and low-density homo- and
heteroglycoclusters is reported. The binding properties of these synthetic multivalent glycoconjugates to
concanavalin A (Con A), a model lectin, have been assessed by using a range of competitive and
non-competitive binding assays including enzyme-linked lectin assays (ELLA), isothermal titration
microcalorimetry (ITC) and surface plasmon resonance (SPR). In all cases, highly dense glycoclusters
showed a substantial amplification of the lectin-binding strength in comparison with low-density
counterparts. Interestingly, highly-dense glycoligand presentations, regardless of their homo- or
heteroglycoligand pattern, furnished similar Con A binding properties, supporting the existence of a
synergic effect (heterocluster effect) due to secondary interactions of “non-active” structural motifs in
the presence of a certain density of “active” glycoligands.
Introduction
Multivalent ligands can interact with receptors via many
possible mechanisms such as the chelate effect, subsite binding,
steric stabilization, statistical rebinding, or receptor clustering
and, therefore, thorough understanding of biological systems
is a complex task. Synthetic multivalent glycoconjugates with
well-defined structures have become powerful tools for the
Molecular recognition of carbohydrates represents a research area
with strong potential in drug discovery and biotech applications.
Thus, protein–carbohydrate interactions play central roles in
many biological and pathological processes, such as cell de-
velopment, differentiation, morphogenesis, fertilization, immune
response, implantation, cell migration, and cancer metastasis.
Carbohydrate-binding proteins (lectins) are extensively involved
in processes associated with cell proliferation and cell death,
just to mention a few examples.
events with selective small molecular inhibitors/effectors offers
1
10
elucidation of the basis ruling carbohydrate recognition events
2
3,8,11
and the design of novel glycodrugs.
Typically, these systems
incorporate several copies of single sugar structural motifs on
an appropriate scaffold (molecular, dendritic, polymeric). How-
ever, despite efficiently imitating functional ligand multivalent
presentation in terms of valency and density, these models barely
reflect the inherent heterogeneity of biological systems, therefore
systematically underestimating its potential contribution. In fact,
it has been observed that variations in the expression levels of
a particular sugar motif in a heterogeneous environment may
affect not only the individual binding affinity of a given protein
1
,3,4
The ability to control these
5
enormous potential in glycobiology. Because these binding events
are linked to well-defined cellular responses, one might expect
them to be highly potent and specific. Notwithstanding, most
saccharide ligands bind to their protein receptors only weakly,
with association constants (K ) typically in the mM range. Nature
a
has circumvented this problem through a multivalent presentation
of the binding motifs, thus leading to affinities exceeding those
expected from the simple addition of individual interactions. This
phenomenon, first noted by Lee and co-workers, and referred to
as the “cluster” or “multivalent” glycoside effect, has found a
12
receptor but also its selectivity. However, only a few procedures
13
that allow the construction of well-defined glycoclusters and
14
glycopolymers from different sugar moieties have been described.
6
Additionally, the generation and deconvolution of combinatorial
libraries of di-, tri- and tetra-valent heteroglycoclusters have also
7
3
,5,8,9
wide range of applications in biology and medicine.
15,16
been reported.
Model systems to address binding events involving hetero-
geneous carbohydrate displays should comply with the need
for polyvalency and high density and, simultaneously, allow a
total control of the relative proportions and presentation of
the different structural motifs. In this context, we and others
have recently reported an efficient synthetic procedure for the
construction of multi-antennary homo- and heteroglycoclusters
a
Departamento de Qu ´ı mica Org a´ nica, Facultad de Qu ´ı mica, Universidad
de Sevilla, Apartado 1203, E-41012 Sevilla, Spain. E-mail: jljb@us.es;
Fax: +34 954624960; Tel: +34 954559998
b
Instituto de Investigaciones Qu ´ı micas, CSIC, Am e´ rico Vespucio 49, Isla de
la Cartuja, E-41092 Sevilla, Spain
c
IMIM Hospital del MAR & Universidad Pompeu Fabra. Barcelona Biomed-
17,18
ical Research Park (PRBB), C/Doctor Aiguader 88, 08003 Barcelona,
Spain
based on a b-cyclodextrin (bCD) core (Fig. 1).
Preliminary
binding affinity studies with the a-D-mannopyranose specific plant
d
Departamento de Qu ´ı mica F ´ı sica, Facultad de Qu ´ı mica, Universidad de
lectin concanavalin A (Con A) as model receptor by enzyme-
Sevilla, Apartado 1203, E-41012 Sevilla, Spain
19
linked lectin assay (ELLA) showed that the mixed-type a-Man–
b-Glc heteroglycoclusters, in spite of displaying a lower number
†
Electronic supplementary information (ESI) available: Experimental
details and copies of the NMR spectra. See DOI: 10.1039/b920048g
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The homology between both core molecules and spacers was pur-
posely chosen to detect any possible influence of the scaffold in the
binding affinities. A modular strategy that takes advantage of the
27–29
radical addition of thiols to double bonds
has been developed
for the sequential incorporation of the desired monosaccharide
motifs. A critical advantage of the methodology is that it
allows sampling compounds with varied, yet perfectly defined
densities of the constitutive sugars in an overall architecture that
favours a highly compact packing by orientational bias. Dendrons
themselves constitute interesting models to assess lectin binding
but, more importantly, they can be assembled on the appropriate
scaffold to attain a controlled ligand density.
Synthesis of homo- and heteroglycodendrons
To test the potential of the radical addition of thiosugars to double
bonds for the construction of molecularly diverse structures,
the stepwise addition of per-O-acetyl-1-thio-a-D-mannopyranose
Fig. 1 Representative bCD-centered a-Man–b-Glc heteroglycocluster.
of putative selective binding motifs, exhibited Con A-binding
affinities similar or even higher as compared to homogeneous
30
31
(2) and per-O-acetyl-1-thio-b-D-glucopyranose (3) to the tri-
17
32
a-mannosylated conjugates. Such unexpected behaviour could
allylated pentaerythritol derivative 1 was first explored. The
not be explained in terms of a difference in effective binding motif
choice of these glycoligands is motivated by the fact that a-D-
mannopyranosyl residues are specifically recognised by Con A
whereas b-D-glucopyranosyl derivatives are not. Previous studies
had shown that a-D-thiomannopyranosyl ligands bind to Con A
lectin with similar affinities to a-D-mannopyranosides and with
20
concentration and pointed to the existence of a “heterocluster
effect”, a term that refers to the increase in the intrinsic binding
affinity of a carbohydrate ligand towards a lectin in the presence
of a second sugar that itself is not a ligand for this lectin
28b
(
intrinsic affinity means on a per-ligand basis, keeping the same
higher affinities than N-linked analogues.
overall topology and preventing contributions to binding from
aggregation phenomena).
Homoglycodendrons 4 and 7 were prepared by UV-promoted
radical addition of 2 and 3, respectively, to the triallyl derivative
1. Alternatively, azo-bis(isobutyronitrile) (AIBN) was used as
radical starter. Anti-Markonikov addition led to adducts 4 and
7 with complete regioselectivity. Conventional deacetylation of
these compounds yielded the corresponding trivalent unprotected
derivatives TriMan-OH and TriGlc-OH. Triflyl activation of the
primary hydroxyl group of 4 and 7, followed by azide displacement
(→ 5 and 8) and isothiocyanation using the triphenylphosphine–
The nature of this effect remains uncertain and seems to be
strongly dependent on the overall architecture. To get a deeper
insight into this matter, we have now prepared a series of molec-
ularly well-defined homo- and heteromultivalent glycoclusters.
Low- and high-density central cores have been used to discrim-
inate the influence of a pointed heterogeneity and a repetitive
multiheterogeneity in a space-oriented platform. A comparative
analysis of the performance of the different compounds towards
33
carbon disulfide (TPP–CS ) system led to the corresponding
2
21
Con A, an extensively studied mannose-binding lectin, by a
isothiocyanate-armed dendrons 6 and 9, respectively (Scheme 1).
1
9,22
set of complementary techniques, namely ELLA,
isothermal
Interestingly, the radical addition reaction of thiosugars 2 and 3
to 1 could be experimentally controlled by modulating the number
of equivalents of the thiols in order to preferentially obtain the
diaddition products 10 and 13 in good yields (Scheme 1). The
diaddition products were further used to prepare the heteroge-
neous glycodendrons 14 and 17 by addition of the diastereomeric
thiosugars. A reaction sequence analogous to that above discussed
for the preparation of 6 and 9 allowed the synthesis of the mixed
a-Man–b-Glc isothiocyanate-armed glycodendrons 12, 16 and
19 (Scheme 1). Conventional deacetylation of 10, 14 and 17
yielded the corresponding unprotected pentaerythritol-centered
heteroglycoligands DiMan-OH, DiManGlc-OH, and ManDiGlc-
OH.
As a monovalent control compound for binding studies, the
mono-S-mannopyranosyl ligand (Man-OH) bearing a linear
spacer similar to that present in the pentaerythritol deriva-
tives TriMan-, TriGlc-, DiMan-, DiManGlc- and ManDiGlc-
OH was elaborated (Scheme 2). The synthesis of Man-OH was
achieved by radical addition of the per-O-acetyl-1-thiomannose
2 to allyl 2-hydroxypropyl ether 20, obtained by selective al-
lylation of propane-1,3-diol with allyl bromide in THF (40%
yield), followed by conventional deacetylation. Additionally, the
21,23
titration calorimetry (ITC),
and surface plasmon resonance
24
(
SPR) is reported.
Results and discussion
To evaluate how heterogeneity affects specific recognition of
multivalent a-mannosides by Con A, the design of a set of suitable
heteroglycoclusters featuring structurally diverse yet well-defined
molecular architectures is mandatory. For such purpose, we have
envisioned the synthesis of a series of multivalent displays ranging
from fully homogeneous in the putative a-D-mannopyranoside
ligand to fully homogeneous in b-D-glucopyranoside residue, with
all intermediate combinations among them. b-Glucopyranosides
have previously been shown to be unrecognized by Con A
25
in control experiments. Two different scaffolds were selected
to prepare low-density and high-density multivalent systems,
namely C-6-substituted methyl a-D-glucopyranoside and per-
(
C-6)-substituted-bCD, respectively. The contribution of these
15,26
scaffolds to Con A recognition was shown to be negligible.
In both cases, a cysteaminyl spacer was incorporated to warrant
accessibility of the coating binding motifs to recognition events.
1
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2 2 2 2
Scheme 1 Synthesis of homo- and heterogeneous glycodendrons. Reagents and conditions: a, hn (250 nm), MeOH, Ar, RT; b, i) Tf O, CH Cl , Py, N ,
◦
+
-
3 2
25 C, 20 min; ii) NaN , DMF, RT, 3 h; c, TPP, CS , dioxane, Ar, RT, 24 h; d, NaMeO, MeOH, RT, 1 h, then Amberlite IR 120 (H ).
(
→ 22), and TPP–CS
2
-mediated isothiocyanation in good overall
yield (Scheme 2).
Synthesis of methyl a-D-glucopyranoside and b-CD scaffolded
homo- and heteroglyconjugates
Previous attempts to access hyperbranched cyclodextrin-centered
conjugates by direct nucleophilic addition of per-(6-amino-6-
17
deoxy)-bCD to isothiocyanates proved unsatisfactory. Probably,
the proximity of the nucleophilic centres in the cyclooligosac-
charide structure results in strong steric hindrance and poor
accessibility. To avoid this problem, we have used instead the
Scheme 2 Synthesis of mono-S-mannosyl ligand. Reagents and condi-
◦
tions: a, AIBN, dioxane, Ar, 75 C, 45 min; b, i) TsCl, CH
2
Cl
2
, DMAP,
◦
RT, 24 h; ii) NaN
3
, DMF, 80 C, 4 h; c, TPP, CS
2
, dioxane, RT, 16 h; d,
17
+
cysteaminyl derivatives 24 (see Supporting Information) and 25
NaMeO, MeOH, RT, 1 h, then Amberlite IR 120 (H ).
as amine-armed cores to construct our low- and high-density
glycoclusters, respectively. Compounds 24 and 25 were readily
obtained by displacement of the corresponding 6-bromo-6-deoxy
monomannosylated isothiocyanate-functionalized building block
23 was prepared by tosylation of compound 21, azide displacement
This journal is © The Royal Society of Chemistry 2010
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derivatives by cysteamine. Coupling reaction with isothiocyanate-
armed homo- and hetero-glycodendrons by exploiting the thiourea
in comparison with the carbons of the external glycoligands. Nev-
1
3
1
ertheless, both the C NMR and H NMR spectra were consistent
with the expected C symmetry for homogeneously C-6 substituted
34
forming reaction let us obtain a collection of homogeneous and
heterogeneous glycoclusters. Scheme 3 summarizes the synthesis
of the glycoconjugate library, based on a two-step strategy involv-
ing the base-catalyzed amine–isothiocyanate coupling followed
by acetyl group cleavage (mixed Zemplen trans-esterification–
saponification). TLC- and NMR-monitoring of the reaction
mixtures evidenced outstanding chemical yields for the amine–
isothiocyanate coupling both in the aMe and bCD series,
though chromatographic purification of the high molecular weight
hemiacetylated intermediates in the bCD series handicapped the
final yields in certain cases. Final acetyl cleavage quantitatively
furnished the target multivalent glycoconjugates split into two sep-
arate batches: the low density glucopyranosyl adducts (aMe) and
the high density CD-centered (bCD) constructs. The homogeneity
and purity of all structures were confirmed by mass spectrometry
7
bCD-centered clusters. In the case of the homoglycoconjugates,
proton spectra showed two spin systems at the characteristic
sugar spectral region, one corresponding to the cyclodextrin and
another to the coating monosaccharide. For heteroglycoclusters,
three different spin systems were distinguished (see e.g., Fig. 2).
The location of the trivalent dendrons at the primary face
of the truncated-cone bCD platform provides a well defined
topology in which binding motifs are oriented towards the same
space region, providing a high-density glycoside surface. A three-
dimensional view of compound DiManGlc-bCD is presented in
Fig. 3 (MACROMODEL 6.0, MM2*). It reveals an external coat
composed by a-Man and b-Glc monomers that mimics highly
dense and heterogeneous regions of the cell surface glycocalix.
˚
The glycosidic crown spans to a maximum of 47 A, shorter
1
13
(
MS), NMR spectroscopy and microanalysis. H and C NMR
spectra of the final fully unprotected conjugates in D O showed
the typical line broadening associated with restricted rotation
than the distance between two adjacent recognition sites in the
Con A tetramer, therefore preventing any possible contribution
2
10
associated to the chelate effect.
35
at the pseudoamide NH–C(=S) bonds, which remained ev-
ident in the hyperbranched bCD derivatives also at elevated
temperatures (333–353 K). The drastic decrease of motion at the
central core region of the macromolecular conjugates provokes an
increase in the relaxation time for the corresponding carbon atoms
Evaluation of concanavalin A binding capabilities
The binding affinity of the library of multivalent glycoligands
synthesized against the tetrameric plant lectin concanavalin A
(Con A) was extensively evaluated by enzyme-linked lectin assay
1
3
that translates into much lower intensities in the C NMR spectra
Scheme 3 Synthesis of a-D-glucopyranoside and b-CD scaffolded glycoconjugates. Reagents and conditions: a, i) Py, Et
3
N, RT, 16 h; ii) NaMeO, MeOH,
+
+
+
then Amberlite IR 120 (H ); b, i) H
2
O, acetone pH 8 (NaHCO
3
), RT; ii) NaMeO, MeOH, H
2
O, then Amberlite IR 120 (H ), Duolite MB 6113 (H ,
-
OH ).
1
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Fig. 3 3D view of heteromultivalent conjugate DiManGlc-bCD.
concentration needed to achieve 50% inhibition (IC50) is then
assumed to be inversely proportional to the lectin-saccharide free
energy of binding. The experimental ELLA data are summarized
in Table 1. In general, results are in agreement with the known
Con A specificity and the expected increase in affinity with
a-Man ligand valency. In fact, a substantial amplification of
the binding affinity was observed for homogeneous a-Man
clusters when monosaccharide units were presented in triads, in
15,24
agreement with previous results.
Conversely, trivalent b-Glc
conjugates TriGlc-OH and TriGlc-aMe were not recognised by
Con A in the concentration range explored (up to ca. 4 mM).
Additionally, comparative analysis of the IC50 data discarded
significant contributions to the binding energy ascribable to the
scaffolds. Furthermore, the presence of b-Glc monomers in low
density heteroclusters is irrelevant regarding Con A binding to the
a-Man ligands.
1
Fig. 2 H NMR (500 MHz, D
2
O, 353 K) of compound ManDiGlc-bCD.
The 1D TOCSY spectra of bCD and b-Glc units are included.
Highly dense glycoclusters featured a completely different sce-
nario. The presence of b-Glc residues significantly contributes to
the enhancement of lectin affinity in this highly-dense architecture.
Though TriMan-bCD still features two and three fold better
affinity than heterovalent DiManGlc-bCD and ManDiGlc-bCD,
respectively, on a relative potency (mannose molar) basis this
difference tends to level (7.51, 4.75 and 6.86, respectively, Table 1).
The fact that the IC50 value for DiMan-bCD (14 a-Man residues
and 7 allyl groups) is higher than that of TriMan-bCD indicates
that the presence of allyl groups does not contribute to any
significant level to the recognition process and, more importantly,
(
ELLA). In addition, isothermal titration microcalorimetry (ITC)
and surface plasmon resonance (SPR) measurements were carried
out on the hyperbranched glycoclusters TriMan-bCD, DiMan-
bCD, DiManGlc-bCD and ManDiGlc-bCD.
Enzyme-linked lectin assays (ELLA)
ELLA measures the ability of a soluble ligand to inhibit the
association between a labelled lectin and a polymeric ligand
attached to the microtiter well (horseradish peroxidase-labelled
Con A and yeast mannan, respectively, in the present case). The
Table 1 Inhibition of yeast mannan–Con A binding measured by ELLA
a
Compound
Man units
Glc units
IC50 (mM)
Relative potency
Relative potency (molar)
Man-OH
ManDiGlc-OH
DiMan-OH
DiManGlc-OH
TriMan-OH
ManDiGlc-aMe
DiManGlc-aMe
TriMan-aMe
Man-bCD
ManDiGlc-bCD
DiMan-bCD
DiManGlc-bCD
TriMan-bCD
1
1
2
2
3
1
2
3
7
0
2
0
1
0
2
1
0
0
800
605
319
275
46
790
69
53
67
18
76
1
1
1.32
2.51
2.91
17.3
1.10
12.5
16.3
12.9
48.1
11.4
66.5
157.8
1.32
1.25
1.45
5.77
1.10
6.30
5.43
1.84
6.86
0.81
4.75
7.51
7
14
0
7
14
14
21
13
5.5
0
a
36
Relative values are compared to methyl-a-D-Man (IC50 865 mM). The IC50 are mean values obtained from at least three estimations. Errors are estimated
in the range ±15%.
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Table 2 Thermodynamic parameters of CD-centered glycocluster–Con A binding determined by ITC (n.d.: not determined)
◦
◦
◦
Compound
Man units
Glc units
-DG kJ/mol
-DH kJ/mol
TDS kJ/mol
TriGlc-bCD
ManDiGlc-bCD
DiMan-bCD
DiManGlc-bCD
TriMan-bCD
0
7
14
14
21
21
14
0
7
0
no binding detected
a
32.4
21.1
32.3
8.3
n.d.
24.1
n.d.
b
c
71.4
-39.1
d
33.3
152.8
-119.1
a
5
b
3
c
5
d
5
Association constant values, calculated according to the Gibbs equation, are 4.8 ¥ 10 , 5.0 ¥ 10 , 4.6 ¥ 10 , 6.9 ¥ 10 .
supports that the glycosidic nature of the secondary ligand is
necessary to boost the affinity towards Con A.
hand, the gain in enthalpy of binding when the a-Man valency
increases, keeping unchanged the total sugar content, is balanced
by the unfavourable entropy that characterises specific association
processes in aqueous solutions. The active role of b-Glc was
confirmed by the dramatic decrease in the free energy of binding
of compound DiMan-bCD as compared with that of DiManGlc-
bCD, having identical a-Man valency. The heterocluster effect
implies a synergy between the primary (a-Man) and the secondary
ligand (b-Glc) since the homogeneous derivative TriGlc-bCD did
not bind to the lectin.
Since the presence of the horseradish peroxidase (HPR) label
used in ELLA is supposed to prevent cross-linking processes,
19a
7,17
unless very long spacer arms are used,
these hyperbranched
bCD probably interact with a single binding site in the lectin.
Consequently, the observed affinity enhancement for heterovalent
derivatives cannot be ascribed to aggregation. It is possible that
11,37
weak secondary interactions,
either in the recognition site of
the primary ligand or proximal to this region, operate when the
a-Man and b-Glc motifs are closely packed together in a highly
dense surface.
Surface plasmon resonance (SPR) experiments
SPR detection offers several unique advantages in comparison to
other techniques. Both kinetic and thermodynamic parameters
can be monitored in real time using minor amounts of materials
Isothermal titration microcalorimetry assays (ITC)
To get thermodynamic information that could reliably support the
implication of b-Glc residues in the reinforcement of the a-Man–
Con A interaction, ITC measurements were carried out. In these
experiments soluble glycodendrons were titrated against a solution
containing Con A, and the heat evolved or absorbed during
binding was measured as a function of ligand concentration.
HRP-labelled Con A was also used in these experiments in order
to fully imitate the ELLA binding process. Deconvolution of
these data yielded the binding constant, enthalpy of binding,
and the stoichiometry of the interaction. The binding constant
is easily related to the free energy of association, and the entropy
is calculated through subtraction. The results confirmed the 1 : 1
stoichiometry, in agreement with the absence of precipitation.
The obtained thermodynamic data are collected in Table 2.
Paralleling ELLA assays, heterogeneous a-Man–b-Glc clusters
featured free energies of binding 50% higher than that found
for the homo-14-valent conjugate DiMan-bCD. The 21-valent
TriMan-bCD, DiManGlc-bCD and ManDiGlc-bCD displayed
38,39
without requiring specific labelling of the interacting partners.
To determine association constants (K ) of our ligands with
Con A, SPR measurements were carried out in which the
a
38
lectin was immobilized on the chip surface. While the opposite
configuration, i.e. using immobilized glycoligands, has obvious
advantages in terms of sensitivity, in our case that would have
required the modification of the glycoclusters without altering
the overall architecture, which implies a non-evident synthetic
strategy.
The poor lectin affinity of low-density glycoclusters was trans-
lated into very weak K (data not shown). For this reason, no
a
reliable data could be obtained in these cases, except for the
compounds displaying the trivalent homomannosides (TriMan-
OH and TriManaMe), confirming the benefits of this type of
presentation in low-density ligands. No synergic effect due to the
presence of additional b-glucopyranosyl motifs was detected for
low-density heterovalent derivatives, in full agreement with ELLA
data.
In the case of the CD-scaffolded glycocluster series, SPR
experiments were carried out using low and high density Con
A-functionalized chips. To evaluate lectin density and stability of
the lectin anchoring after a set of SPR experiments, ribonuclease
B was used as standard ligand. Channel responses upon injection
of 400 nM ribonuclease B (30 mL for 3 min) were in the range
of 200 RU for the low-density channel and over 900 RU for the
high-density chip, respectively. The results, summarized in Table 3,
showed a divergence of approximately one order of magnitude in
◦
◦
rather similar DG values. However the enthalpic and entropic con-
tributions to DG were significantly different. Data are indicative
of partial enthalpy–entropy compensation between glycocluster
analogues having different mannose valency. Thus, glycocluster
ManDiGlc-bCD compensates a lower enthalpic contribution with
higher entropy of binding, suggesting that a sliding mechanism,
promoted by the presence of the b-Glc ligand, is operating. The
bind and slide (internal diffusion) model allows, for instance, a
small fraction of bound lectin molecules to dynamically move
from carbohydrate to carbohydrate epitope in globular and linear
glycoproteins, and has been suggested to be a general mechanism
the K
a
values calculated using the different chips, probably due to
23b
38,40
of binding of ligands to biopolymers. In our system, partial
entropy–enthalpy compensation might be at the origin of the
observed heterocluster effect. Actually, ManDiGlc-bCD binding
to Con A is, basically, an entropically driven process. On the other
mass transport effects.
As a general trend, the homo- and heteromultivalent CD-
conjugates showed a much higher Con A affinity (K ) than their
a
low-density counterparts. In contrast to ELLA and ITC data,
1
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a
Table 3 Con A binding K calculated from SPR-sensorgrams
a
K )
(M-
1
a
Compound
Man units
Glc units
4
5
4
4
4
5
4
5
3
3
b
c
b
c
b
c
b
c
b
c
TriMan-bCD
DiManGlc-bCD
DiMan-bCD
ManDiGlc-bCD
Man-bCD
21
14
14
7
0
7
2.03 ¥ 10
.70 ¥ 10
5.33 ¥ 10
.48 ¥ 10
7.57 ¥ 10
.98 ¥ 10
5.53 ¥ 10
.78 ¥ 10
2.92 ¥ 10
4
6
0
5
14
0
2
7
6
.37 ¥ 10
TriGlc-bCD
0
21
no binding detected
a
b
Average values from at least four experiments (S.D. = ±10%). Low
c
Con A-surface density sensorchip flow cell. High Con A-surface density
sensorchip flow cell.
valency-dependent Con A affinity increases for the highly-dense
systems and so did not require the presence of the mannosyl
ligands in triads to maximize it. The different nature of the
phenomena measured in SPR and ELLA or ITC probably
accounts for these contrasting observations. The surface-ligand
interaction taking place in SPR is probably easier to saturate by
densely-decorated clusters than those taking place in the bulk
solution (ELLA and ITC).
In any case, SPR experiments were consistent with the hete-
rocluster effect observed by ELLA and ITC. While Man-bCD
3
-1
Fig. 4 SPR-Sensorgrams of TriMan-bCD (A), DiManGlc-bCD (B), and
ManDiGlc-bCD (C) at 12.5, 25, 50, 100, 200, and 400 mM in phosphate
buffer.
featured moderate Con A binding affinity (K 6.37 ¥ 10 M ),
a
insertion of additional b-Glc ligands (ManDiGlc-bCD) increased
Con A affinity to a similar level as the homogeneously decorated
5
5
-1
2
1-valent TriMan-bCD (2.78 ¥ 10 and 4.70 ¥ 10 M , respectively).
Sensorgrams of the homo- and heteroclusters are rather similar
Fig. 4 and Supporting Information), supporting the reliability of
mixed constructs with identical a-Man valency evidenced affinity
increases due to the presence of b-Glc residues, which however
are not ligands for the lectin themselves. Our results point to
entropy–enthalpy compensation as the basis of this effect. We
hypothesize that, once a-Man residues have been recognized by
Con A, a sliding mechanism promoted by the presence of the b-Glc
monomers might operate that results in improved binding. Further
investigations to assess the scope and its possible biological
relevance of the heterocluster effect are being developed in our
laboratories.
(
the measurements and, therefore, the relevance of secondary inter-
actions at high saccharide-surface densities also for immobilized
biological receptors.
The homotetradecavalent derivative DiMan-bCD, featuring
5
-1
seven allyl groups, afforded the highest K value (5.98 ¥ 10 M ),
a
as determined by SPR, in this series. However, in this case the
observed affinity increase is probably motivated on non-specific
hydrophobic interactions of the clusterized allyl groups with the
surface. A similar observation has been recently reported for
16
a series of cyclopeptide-templated glycoclusters. Furthermore,
hydrophobic residues have been shown to beneficially contribute to
Experimental section
41
enhancing lectin–glycoconjugate binding in particular systems.
Horseradish peroxidase-labelled concanavalin A (HRP-Con A;
Sigma), mannan from Saccharomyces cerevisiae (Sigma) and
all other common reagents and materials were purchased from
commercial sources. Optical rotations were measured at room
temperature in 1 cm or 1 dm tubes on a Perkin-Elmer 141 MC
polarimeter. Infrared (IR) spectra were recorded on a Bomem
Conclusions
In this study, an efficient methodology to access monodisperse,
well-defined homo- and heteromultivalent glycoclusters has been
implemented. This diversity-oriented synthetic strategy allows an
exquisite control of the molecular architecture of mixed-type
glycoclusters and is very well suited to explore the effects of
glycoligand density and nature on lectin binding. The binding
affinity towards Con A was evaluated by a number of techniques
including ELLA, ITC and SPR. The combined data showed that,
while low density glycoclusters behaved according to the classical
cluster effect, with a gradual increase in affinity towards Con
A associated with putative ligand (a-Man) valency, highly dense
1
13
Michelson MB-120 FTIR spectrophotometer. H (and C NMR)
1
3
13
spectra were recorded at 300 (75.5 for C) and 500 (125.7 for C)
1
MHz with Bruker 300 and 500 DRX instruments. 1D H TOCSY,
1
1
13
2D H TOCSY, COSY, H– C HMQC and HSQC experiments
were used to assist NMR assignments. Thin-layer chromatography
(TLC) was carried out on aluminium sheets coated with Kieselgel
60 F254 (E. Merck), with visualization by UV light and by charring
with 10% H
2
SO or 0.2% ninhydrin. Column chromatography
4
was carried out on Silica Gel 60 (E. Merck, 230–400 mesh).
This journal is © The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 1849–1860 | 1855

View Online
Gel permeation chromatography (GPC) of the fully unprotected
bCD adducts was carried out on a Sephadex G-25 (eluent H O)
binding studies were obtained by gel permeation chromatography
2
(Sephadex G-25, H O). For the notation of atoms in NMR
2
column (Pharmacia Amersham) attached to a Gradifrac system
using a UV detector set at 248 nm. FAB mass spectra were
obtained using a Kratos MS-80 RFA instrument. The operating
conditions were the following: the primary beam consisted of
Xe atoms with a maximum energy of 8 keV; the samples were
dissolved in thioglycerol, and the positive ions were separated
and accelerated over a potential of 7 keV; NaI was added as
cationising agent. MALDI-TOF mass spectra were acquired on
a GSG System spectrometer operating in the positive-ion mode
with an accelerating voltage of 28 keV. Samples were dissolved
assignments, see Fig. 5 and the diagrams hereinafter.
in H O at millimolar concentration and mixed with a standard
2
-
1
solution of 2,5-dihydroxybenzoic acid (DHB; 10 mg mL in 10%
aq EtOH, 2 mL) in 1 : 1 v/v relative proportions; 1 mL of the
mixture was loaded onto the target plate, then allowed to air-dry
at room temperature. Elemental analyses were performed at the
Instituto de Investigaciones Qu ´ı micas (Sevilla, Spain).
Fig. 5 Structure of compound Man-bCD with labels indicating the
different spin systems and proton numbers.
Heptakis[6-[2-[N¢-[2,2,2-tris[5-(2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosylthio)-2-oxapentyl]ethyl]thioureido]ethylthio]]cyclo-
maltoheptaose (32). Reaction time: 48 h. Yield: 47 mg (37%);
Triphenylphosphine, trifluoromethanesulfonic anhydride and
N,N-dimethylformamide are indicated by the acronyms TPP,
Tf
2
O
and DMF, respectively. 2,3,4,6-Tetra-O-acetyl-1-thio-
1
[
a]
D
= -11.4 (c 1.0, MeOH); H NMR (500 MHz, CD
3
OD, 323
a-D-mannopyranose (2) and 2,3,4,6-tetra-O-acetyl-1-thio-b-D-
glucopyranose (3) were prepared from the corresponding per-O-
acetates in three steps by transformation into the corresponding
K): d = 5.27 (t, 21 H, J2,3 = J3,4 = 9.5 Hz, H-3Glc), 5.04 (t, 21 H,
J
1
1
4,5 = 10.0 Hz, H-4Glc), 5.03 (s, 7 H, H-1), 4.96 (t, 21 H, J1,2
=
=
0.0 Hz, H-2Glc), 4.76 (d, 21 H, H-1Glc), 4.26 (dd, 21 H, J6a,6b
42
glycosyl halides, treatment with thiourea and subsequent hydrol-
ysis of the resulting isothiouronium salt with potassium metabisul-
2.0 Hz, J5,6a = 5.0 Hz, H-6aGlc), 4.16 (dd, 21 H, J5,6b = 2.5 Hz,
H-6bGlc), 3.97 (m, 7 H, H-5), 3.92 (ddd, 21 H, H-5Glc), 3.83 (t, 7
H, J2,3 = J3,4 = 9.5 Hz, H-3), 3.72 (m, 14 H, CH Cyst), 3.55 (m,
4 H, H-2, H-4), 3.52 (m, 42 H, H-3Pent), 3.42 (s, 56 H, CH
H-1Pent), 3.21 (m, 7 H, H-6a), 3.02 (m, 7 H, H-6b), 2.91 (bt, 14 H,
2
9,43
1
fite (K
2
S
2
O
5
).
Experimental procedures and fully assigned H
2
N
1
3
and C NMR data for pentaerythritol glycoclusters (TriMan-
OH, TriGlc-OH, DiMan-OH, ManDiGlc-OH), 4-oxahept-6-en-1-
ol derivatives (21 and Man-OH) and monoconjugate derivatives
of methyl 6-(2-aminoethylthio)-a-D-glucopyranoside (3, TriGlc-
aMe, 4, TriMan-aMe, 10, DiMan-aMe, 14, DiManGlc-aMe,
1
2
N
Branch
,
44
2
3
CH
2
S
Cyst), 2.82, 2.78 (2 dt, 42 H, JH,H = 13.0 Hz, JH,H = 6.0 Hz,
45
H-5Pent), 2.07, 2.05, 2.01, 1.95 (4 s, 252 H, MeCO), 1.89 (m, 42
13
H, H-4Pent); C NMR (125.7 MHz, CD
3
OD, 323 K): d = 185.4
17, ManDiGlc-aMe, 21, and Man-aMe) are described in the
(
(
CS), 172.3, 171.7, 171.3, 171.2 (CO), 103.8 (C-1), 86.1 (C-4), 84.9
C-1Glc), 76.9 (C-5Glc), 75.5 (C-3Glc), 74.6 (C-3), 74.4 (C-2, C-5),
Supporting Information.
7
6
3
2.1 (C-1Pent), 71.8 (C-2Glc), 71.2 (C-3Pent), 70.2 (C-4Glc), 63.7 (C-
Glc), 45.9 (C , CH Cyst), 35.1 (C-6), 34.2 (CH
1.4 (C-4Pent), 28.5 (C-5Pent), 21.1-20.7 (MeCO). Anal. Calcd. for
35: C 48.43, H 6.14, N 1.74. Found: C 48.08, H
N
Branch, CH
2
N
2
SCyst),
General procedure for the preparation of heptaconjugates of
heptakis[6-(2-aminoethylthio)]cyclomaltoheptaose (TriGlc-bCD,
TriMan-bCD, DiMan-bCD, DiManGlc-bCD, ManDiGlc-bCD
and Man-bCD)
q
2
C
455
H
686
N
14
238
O S
6.02, N 1.69.
1
7
A solution of heptacysteaminyl bCD 25 (20 mg, 11.08 mmol)
in H O (1 mL) was adjusted to pH 8–9 with solid NaHCO
Heptakis[6-[2-[N¢-[2,2,2-tris[5-(b-D-glucopyranosylthio)-2-
oxapentyl]ethyl]thioureido]ethylthio]]cyclomaltoheptaose (TriGlc-
2
3
1
and stirred for 16 h at room temperature. A solution of the
corresponding isothiocyanate (115 mmol, 1.5 eq) in acetone
1 mL) was then added and the reaction mixture was stirred
bCD). Yield: 32 mg (99%); [a]
(500 MHz, D
D
= -16.2 (c 1.4 in H
2
O); H NMR
O, 343 K): d = 5.46 (bd, 7 H, J1,2 = 3.0 Hz, H-1),
2
(
4.86 (d, 21 H, J1,2 = 9.5 Hz, H-1Glc), 4.29 (m, 7 H, H-5), 4.28 (m,
7 H, H-3), 4.27 (d, 21 H, J6a,6b = 12.0 Hz, H-6aGlc), 4.13 (d, 21 H,
at room temperature until total consumption of 25. Acetone
was evaporated under reduced pressure, the remaining aqueous
suspension was freeze-dried and the solid residue was purified by
column chromatography, using MeCN → 10 : 1 MeCN–H
eluent, to give the corresponding hemiacetylated C -symmetric
adducts (32–37). Deacetylation was effected by treatment with
N NaOMe in MeOH (0.1 eq per mol of acetates) at room
temperature. After 5 min a white precipitate appeared, which
was redissolved by addition of H O. The solution was stirred
for 15 min, neutralised using Amberlite IR-120 (H ) ion ex-
H-6bGlc), 4.09 (m, 14 H, CH
2
N
Cyst), 4.07 (m, 7 H, H-2), 3.98 (t, 42
3
H, JH,H = 6.5 Hz, H-3Pent), 3.97 (m, 7 H, H-4), 3.89 (t, 21 H, J2,3
=
2
O as
J
3,4 = 9.3 Hz, H-3Glc), 3.84 (m, 98 H, H-4Glc, H-5Glc, CH
2
N
Branch
,
7
H-1Pent), 3.73 (t, 21 H, H-2Glc), 3.65 (m, 7 H, H-6a), 3.38 (m, 7 H,
3
H-6b), 3.32 (bt, 14 H, JH,H = 6.5 Hz, CH
2
SCyst), 3.22, 3.18 (2 dt,
2
3
1
42 H, JH,H = 13.0 Hz, JH,H = 5.5 Hz, H-5Pent), 2.33 (m, 42 H, H-
1
3
4Pent); C NMR (125.7 MHz, 343 K, D
2
O): d = 181.4 (CS), 102.6
2
(C-1), 86.3 (C-1Glc), 85.1 (C-4), 80.5 (C-5Glc), 78.0 (C-3Glc), 73.4
(C-3), 73.2 (C-2Glc), 72.8 (C-2, C-5), 71.2 (C-1Pent), 70.4 (C-3Pent),
+
+
-
change resin, demineralised with Duolite MB-6113 (H , OH )
ion exchange resin and freeze-dried to give the fully unprotected
conjugates (TriGlc-bCD, TriMan-bCD, DiMan-bCD, DiManGlc-
bCD, ManDiGlc-bCD, Man-bCD). Analytical samples for lectin-
70.3 (C-4Glc), 61.8 (C-6Glc), 46.5 (C
q
), 45.0 (CH
2
N
Branch, CH NCyst),
2
34.2 (C-6), 33.1 (CH
TOFMS: m/z 7745 [M + H] . Anal. Calcd. for C287
2
S
Cyst), 30.0 (C-4Pent), 27.4 (C-5Pent); MALDI-
+
H
518
N
14
O
154
35
S :
C 44.46, H 6.75, N 2.53. Found: C 44.32, H 6.67, N 2.34.
1
856 | Org. Biomol. Chem., 2010, 8, 1849–1860
This journal is © The Royal Society of Chemistry 2010

View Online
Heptakis[6-[2-[N¢-[2,2,2-tris[5-(2,3,4,6-tetra-O-acetyl-a-D-man-
nopyranosylthio)-2-oxapentyl]ethyl]thioureido]ethylthio]]cyclo-
maltoheptaose (33). Reaction time: 48 h. Yield: 75 mg (60%);
1
Man), 74.4 (C-3), 73.7 (CH
2
CH=), 73.5 (C-2, C-5), 72.5 (C-2Man),
72.2 (C-1Pent), 71.8 (CH OAll), 71.2 (C-3Man), 71.1 (C-3Pent), 70.6
2
(C-5Man), 67.8 (C-4Man), 63.8 (C-6Man), 45.8 (C
CH Cyst), 35.0 (CH Cyst), 34.0 (C-6), 30.9 (C-4Pent), 29.6 (C-5Pent),
21.1-20.6 (MeCO). Anal. Calcd for C357 28: C 49.10, H
6.30, N 2.25. Found: C 49.59, H 6.51, N 2.24.
q
), 45.6 (CH
2
N
Branch
,
[
a]
D
= +78.1 (c 1.0, MeOH); R
f
0.48 (10 : 1 : 1 MeCN–H
2
O–
2
N
2
S
1
NH
4
OH); H NMR (500 MHz, CD
3
OD, 313 K): d = 5.36 (s,
H
546
N
14
175
O S
2
1
7
4
3
3
3
2
2
1 H, H-1Man), 5.32 (bs, 21 H, H-2Man), 5.26 (t, 21 H, J3,4 = J4,5 =
0.0 Hz, H-4Man), 5.19 (dd, 21 H, J2,3 = 2.5 Hz, H-3Man), 5.01 (bs,
H, H-1), 4.37 (ddd, 21 H, J5,6a = 5.0 Hz, J5,6b = 2.0 Hz, H-5Man),
.28 (dd, 21 H, J6a,6b = 12.5 Hz, H-6aMan), 4.12 (dd, 21 H, H-6bMan),
Heptakis[6-[2-[N¢-[2,2-bis[5-(a-D-mannopyranosylthio)-2-oxa-
pentyl]-2-(2-oxapent-4-enyl)ethyl]thioureido]ethylthio]]cyclomalt-
.97 (m, 7 H, H-5), 3.82 (m, 7 H, H-3), 3.73 (m, 14 H, CH
2
N
Cist),
Branch, H-1Pent),
Cyst),
oheptaose (DiMan-bCD). Yield: 59 mg (99%); [a]
1.0 in H O); R 0.45 (5 : 3 : 5 MeCN–H O–NH
(500 MHz, D
D
= +89.0 (c
1
.56 (m, 56 H, H-2, H-4, H-3Pent), 3.43 (s, 56 H, CH
.23 (m, 7 H, H-6a), 3.00 (m, 7 H, H-6b), 2.91 (m, 14 H, CH
.82, 2.80 (2 dt, 42 H, JH,H = 13.0 Hz, JH,H = 6.5 Hz, H-5Pent),
.15–1.92 (4 s, 252 H, MeCO), 1.93 (m, 42 H, H-4Pent); C NMR
125.7 MHz, CD
2
N
2
f
2
4
OH); H NMR
2
S
2
O, 333 K): d = 6.26 (m, 7 H, OCH
2
CH=), 5.64 (m,
2
3
3
7 H, JH,H = 17.5 Hz, =CH
a
), 5.59 (d, 14 H, J1,2 = 1.7 Hz, H-1Man),
1
3
3
5.57 (bd, 7 H, JH,H = 10.5 Hz, =CH
b
), 5.39 (bs, 7 H, H-1), 4.34
(
3
OD, 313 K): d = 183.5 (CS), 172.6–171.5 (CO),
(m, 28 H, H-2Man, OCH
14 H, H-5, H-3), 4.13 (m, 14 H, H-6aMan), 4.06 (m, 28 H, H-3Man
H-6bMan), 4.04 (m, 14 H, H-4Man), 4.00 (m, 2 H, CH
(bd, 7 H, J2,3 = 9.5 Hz, H-2), 3.87 (m, 35 H, H-3Pent, H-4), 3.74
(m, 44 H, H-1Pent, CH OAll), 3.58 (m, 7 H, H-6a), 3.29
(m, 7 H, H-6b), 3.23 (m, 2 H, CH
2
CH=), 4.21 (m, 14 H, H-5Man), 4.19 (m,
1
7
6
3
03.9 (C-1), 86.0 (C-4), 84.0 (C-1Man), 74.4 (C-3), 73.7 (C-2, C-5),
2.5 (C-2Man), 72.1 (C-1Pent), 71.2 (C-3Man, C-3Pent), 70.5 (C-5Man),
,
2
NCyst), 3.97
7.7 (C-4Man), 63.8 (C-6Man), 45.9 (C
5.0 (C-6), 34.0 (CH Cyst), 30.9 (C-4Pent), 29.6 (C-5Pent), 21.1-20.6
35: C 48.43, H 6.14, N
q
), 45.7 (CH
2
N
Ram, CH
2
NCyst),
2
S
2
N
Branch, CH
2
(
MeCO). Anal. Calcd. for C455
H
686
N
14
O
238
S
2
S
Cyst), 3.03 (m, 28 H, H-5Pent),
2.23 (m, 28 H, H-4Pent); C NMR (125.7 MHz, D
81.5 (CS), 131.3 (CH ), 103.0 (C-1), 85.6
CH=), 118.0 (=CH
C-1Man), 83.6 (C-4), 73.8 (C-5Man), 73.2 (C-3), 72.8 (CH
CH=),
2.6 (C-2, C-5), 72.5 (C-2Man), 71.9 (C-3Man), 70.9 (C-1Pent), 70.6
C-3Pent, CH OAll), 67.5 (C-4Man), 61.4 (C-6Man), 44.8, 44.2 (C
Branch), 33.2 (C-6), 31.8 (CH
1
3
1
.74. Found: C 48.30, H 6.04, N 1.55.
2
O, 333 K): d =
1
(
7
(
2
2
Heptakis[6-[2-[N¢-[2,2,2-tris[5-(a-D-mannopyranosylthio)-2-
oxapentyl]ethyl]thioureido]ethylthio]]cyclomaltoheptaose (TriMan-
2
1
bCD). Yield: 52 mg (99%); [a]
D
= +104.0 (c 1.0 in H
O, 353 K): d = 5.82 (s, 21 H, H-1Man), 5.61
bs, 7 H, H-1), 4.57 (m, 21 H, H-2Man), 4.46 (m, 7 H, H-5), 4.43 (m,
2
O); H
2
q
,
NMR (500 MHz, D
2
CH
2
N
Cyst, CH
2
N
2
S
Cyst), 29.6 (C-4Pent),
(
+
2
8.4 (C-5Pent); MALDI-TOFMS: m/z 6394.53 [M + Na] . Anal.
Calcd for C245 28: C 46.14, H 6.86, N 3.07. Found: C
6.62, H 6.83, N 2.87.
2
8 H, H-3, H-5Man), 4.36 (m, 63 H, H-3Man, H-6aMan, H-6bMan), 4.29
Cyst), 4.19 (m, 7 H, H-2),
.09 (m, 49 H, H-4, H-3Pent), 3.96 (m, 56 H, CH
.82 (m, 7 H, H-6a), 3.50 (m, 7 H, H-6b), 3.47 (m, 14 H, CH
H
434
N
14
119
O S
(
m, 21 H, H-4Man), 4.23 (m, 14 H, CH N
2
4
4
3
3
2
NBranch, H-1Pent),
2
S
Cyst),
13
Heptakis[6-[2-[N¢-[2-[5-(2,3,4,6-tetra-O-acetyl-b-D-glucopyran-
2
.26 (bt, 42 H, JH,H = 11.5 Hz, H-5Pent), 2.46 (m, 42 H, H-4Pent); C
osylthio)-2-oxapentyl]-2,2-bis[5-(2,3,4,6-tetra-O-acetyl-a-D-man-
nopyranosylthio)-2-oxapentyl]ethyl]thioureido]ethylthio]]cyclo-
maltoheptaose (35). Reaction time: 48 h. Yield: 24 mg (26%);
NMR (125.7 MHz, D
2
O, 343 K): d = 181.9 (CS), 102.7 (C-1), 85.3
C-1Man), 85.1 (C-4), 73.9 (C-5Man), 73.4 (C-3), 72.9 (C-2, C-5), 72.6
C-2Man), 72.1 (C-3Man), 71.1 (C-1Pent), 70.9 (C-3Pent), 67.7 (C-4Man),
(
(
[
a]
D
= +76.8 (c 1.0, MeOH); R
f
0.67 (10 : 1 : 1 MeCN–H
2
O–
6
1.6 (C-6Man), 45.1 (CH
2
N
Branch, CH
2
N
Cyst), 45.0 (C ), 34.1 (C-6),
q
1
NH
4
OH); H NMR (500 MHz, CD
3
OD, 323 K): d = 5.36 (bs,
3
3.2 (CH
2
S
Cyst), 29.8 (C-4Pent), 28.5 (C-5Pent); MALDI-TOFMS:
1
5
J
4 H, H-1Man), 5.33 (14 H, dd, J2,3 = 3.0 Hz, J1,2 = 1.5 Hz, H-2Man),
+
m/z 7767.50 [M + Na] . Anal. Calcd for C287
H
518
N
14
154
O S35: C
.27 (t, 14 H, J3,4 = J4,5 = 10.0 Hz, H-4Man), 5.26 (t, 7 H, J2,3
3,4 = 9.0 Hz, H-3Glc), 5.21 (dd, 14 H, H-3Man), 5.03 (t, 7 H, J4,5
=
=
4
4.46, H 6.75, N 2.53. Found: C 44.19, H 6.36, N 2.43.
Heptakis[6-[2-[N¢-[2,2-bis[5-(2,3,4,6-tetra-O-acetyl-a-D-manno-
pyranosylthio)-2-oxapentyl]-2-(2-oxapent-4-enyl)ethyl]thioureido]-
ethylthio]]cyclomaltoheptaose (34). Reaction time: 24 h. Yield:
10.0 Hz, H-4Glc), 5.02 (m, 7 H, H-1), 4.95 (t, 7 H, J1,2 = 10.0 Hz,
H-2Glc), 4.74 (d, 7 H, H-1Glc), 4.39 (ddd, 14 H, J5,6a = 5.0 Hz, J5,6b =
2.0 Hz, H-5Man), 4.28 (dd, 14 H, J6a,6b = 12.5 Hz, H-6aMan), 4.24
(dd, 7 H, J6a,6b = 12.5 Hz, J5,6a = 4.0 Hz, H-6aGlc), 4.15 (dd, 7 H,
8
1.2 mg (84%); [a]
D
= +7.0 (c 1.0, MeOH); R
f
0.54 (10 : 1 : 1
OD, 323 K):
1
MeCN–H O–NH
2
4
OH); H NMR (500 MHz, CD
3
J
5,6b = 1.5 Hz, H-6bGlc), 4.13 (dd, 14 H, H-6bMan), 3.96 (m, 7 H,
3
3
3
d = 5.93 (ddt, 7 H, JH,H = 17.0 Hz, JH,H = 10.5 Hz, JH,H
.0 Hz, OCH
CH=), 5.35 (bd, 14 H, J1,2 = 3.3 Hz, H-1Man), 5.32
dd, 14 H, J2,3 = 3.0 Hz, H-2Man), 5.26 (t, 14 H, J3,4 = J4,5 = 10.0 Hz,
=
H-5), 3.90 (ddd, 7 H, H-5Glc), 3.83 (t, 7 H, J2,3 = J3,4 = 8.5 Hz, H-3),
3.72 (m, 14 H, CH Cyst), 3.55 (m, 42 H, H-3Pent), 3.54 (m, 14 H,
H-2, H-4), 3.43 (s, 56 H, CH Branch, H-1Pent), 3.22 (m, 7 H, H-6a),
3.01 (m, 7 H, H-6b), 2.91 (14 H, m, CH Cyst), 2.81, 2.78 (2 dt, 42
H, JH,H = 12.5 Hz, JH,H = 5.5 Hz, H-5Pent), 2.14–1.96 (s, 252 H,
MeCO), 1.99 (42 H, m, H-4Pent); C NMR (125.7 MHz, CD OD,
323 K): d = 183.6 (CS), 172.7-169.9 (CO), 103.8 (C-1), 85.9 (C-4),
84.9 (C-1Glc), 84.0 (C-1Man), 76.9 (C-5Glc), 75.5 (C-3Glc), 74.6 (C-3),
74.3 (C-2, C-5), 72.5 (C-2Man), 72.1 (C-1Pent), 71.8 (C-2Glc), 71.2
(C-3Man), 71.1 (C-3Pent), 70.6 (C-5Man), 70.1 (C-4Glc), 67.8 (C-4Man),
5
(
2
2
N
2
N
H-4Man), 5.29 (bd, 7H, =CHa), 5.20 (dd, 14 H, H-3Man), 5.18 (da,
2
S
2
3
7
J
4
7
H, =CHb), 5.01 (bs, 7 H, H-1), 4.37 (ddd, 14 H, J5,6a = 5.0 Hz,
1
3
5,6b = 2.0 Hz, H-5Man), 4.27 (dd, 14 H, J6a,6b = 12.5 Hz, H-6aMan),
3
.12 (dd, 14 H, H-6bMan), 4.00 (bd, 14 H, OCH
2
CH=), 3.96 (m,
H, H-5), 3.82 (t, 7 H, J2,3 = J3,4 = 9.5 Hz, H-3), 3.71 (m, 14
3
H, CH
2
N
Cyst), 3.55 (t, 28 H, JH,H = 5.5 Hz, H-3Pent), 3.51 (m, 14
H, H-2, H-4), 3.44 (m, 56 H, H-1Pent, CH
2
OAll, CH
2
N
Branch), 3.20
Cyst),
(
m, 7 H, H-6a), 2.96 (m, 7 H, H-6b), 2.91 (m, 14 H, CH
2
S
63.8 (C-6Man), 63.6 (C-6Glc), 45.9 (C
35.1 (C-6), 34.0 (CH Cyst), 31.4 (C-4PentGlc), 31.0 (C-4PentMan), 29.6
(C-5PentMan), 28.5 (C-5PentGlc), 21.1-20.7 (MeCO). Anal. Calcd. for
q
), 45.7 (CH
2
N
Branch, CH
2
NCyst),
2
3
2
2
.80, 2.77 (2 dt, 28 H, JH,H = 13.0 Hz, JH,H = 6.5 Hz, H-5Pent),
.13–1.95 (4 s, 168 H, MeCO), 1.92 (m, 28 H, H-4Pent); C NMR
OD, 323 K): d = 183.6 (CS), 172.3–171.5 (CO),
CH=), 117.6 (=CH ), 103.8 (C-1), 85.9 (C-4), 84.0 (C-
2
S
1
3
(
125.7 MHz, CD
3
C
455
H
686
N
14
O
238
S
35: C 48.43, H 6.14, N 1.74. Found: C 48.31, H
136.4 (CH
2
2
5.88, N 1.70.
This journal is © The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 1849–1860 | 1857

View Online
Heptakis[6-[2-[N¢-[2-[5-(b-D-glucopyranosylthio)-2-oxapentyl]-
,2-bis[5-(a-D-mannopyranosylthio)-2-oxapentyl]ethyl]thioureido]-
ethylthio]]cyclomaltoheptaose (DiManGlc-bCD). Yield: 17 mg
H, H-2Man), 4.42 (m, 7 H, H-5Man), 4.41 (m, 7 H, H-5), 4.39 (d,
14 H, J6a,6b = 12.0 Hz, H-6aGlc), 4.38 (m, 7 H, H-3), 4.36 (m, 21
H, H-3Man, H-6aMan, H-6bMan), 4.29 (m, 7 H, H-4Man), 4.25 (dd,
2
1
(
3
99%); [a]
D
= +66.0 (c 1.0 in H
2
O); H NMR (500 MHz, D
2
O,
14 H, J5,6b = 4.5 Hz, H-6bGlc), 4.22 (m, 14 H, CH
2
NCyst), 4.17 (d,
3
53 K): d = 5.67 (s, 14 H, H-1Man), 5.47 (bs, 7 H, H-1), 4.86 (d, 7
7 H, H-2), 4.09 (t, 42 H, JH,H = 5.5 Hz, H-3Pent), 4.08 (m, 7 H,
H-4), 4.02 (t, 14 H, J2,3 = J3,4 = 9.4 Hz, H-3Glc), 3.96 (m, 84 H,
H, J1,2 = 10.0 Hz, H-1Glc), 4.43 (m, 14 H, H-2Man), 4.29 (m, 21 H,
H-5Man, H-5), 4.26 (m, 7 H, H-3), 4.25 (d, 7 H, J6a,6b = 12.0 Hz,
H-6aGlc), 4.14 (m, 42 H, H-3Man, H-6aMan, H-6bMan), 4.14 (m, 14 H,
H-4Glc, H-5Glc, CH
2
N
Branch, H-1Pent), 3.85 (t, 14 H, H-2Glc), 3.78 (m,
3
7 H, H-6a), 3.51 (m, 7 H, H-6b), 3.46 (bt, 14 H, JH,H = 6.5 Hz,
2
3
H-4Man), 4.09 (d, 7 H, H-6bGlc), 4.08 (m, 14 H, CH
2
N
Cyst), 4.04 (m,
H, H-2), 3.95 (m, 49 H, H-3Pent, H-4), 3.85 (t, 7 H, J2,3 = J3,4
.9 Hz, H-3Glc), 3.80 (m, 70 H, H-4Glc, H-5Glc, CH
CH
2
S
Cyst), 3.34, 3.32 (2 dt, 42 H, JH,H = 13.0 Hz, JH,H = 6.0 Hz,
1
3
7
8
3
=
H-5Pent), 2.45 (m, 42 H, H-4Pent); C NMR (125.7 MHz, D
2
O,
2
N
Branch, H-1Pent),
353 K): d = 181.9 (CS), 102.8 (C-1), 86.3 (C-1Glc), 86.0 (C-1Man),
85.3 (C-4), 80.7 (C-5Glc), 78.3 (C-3Glc), 74.1 (C-5Man), 73.6 (C-3),
73.4 (C-2Glc), 73.0 (C-2, C-5), 72.8 (C-2Man), 72.3 (C-3Man), 71.4
(C-1Pent), 71.0 (C-3Pent), 70.6 (C-4Glc), 67.9 (C-4Man), 62.0 (C-6Glc),
.71 (t, 7 H, H-2Glc), 3.61 (m, 7 H, H-6a), 3.37 (m, 7 H, H-6b),
2
3
.32 (m, 14 H, CH
J
NMR (125.7 MHz, D
2
S
Cyst), 3.17, 3.12 (2 dt, 42 H, JH,H = 13.5 Hz,
3
3
13
H,H = 6.5 Hz, H-5Pent), 2.30 (t, 42 H, JH,H = 6.5 Hz, H-4Pent);
C
2
O, 353 K): d = 182.0 (CS), 102.7 (C-1), 86.1
C-1Glc), 85.7 (C-1Man), 85.2 (C-4), 80.5 (C-5Glc), 78.0 (C-3Glc), 73.9
C-5Man), 73.1 (C-3), 73.0 (C-2Glc), 72.8 (C-2, C-5), 72.6 (C-2Man),
61.7 (C-6Man), 47.0 (C
q
), 45.0 (CH
2
N
Branch, CH NCyst), 34.5 (C-6),
2
(
(
33.3 (CH
2
S
Cyst), 30.4 (C-4PentGlc), 30.0 (C-4PentMan), 28.7 (C-5PentMan),
+
27.6 (C-5PentGlc); MALDI-TOFMS: m/z 7768 [M + Na] . Anal.
7
4
3
2.0 (C-3Man), 71.1 (C-1Pent), 70.8 (C-3 Pent), 70.3 (C-4Glc), 67.7 (C-
Man), 61.8 (C-6Glc), 61.5 (C-6Man), 45.1 (CH ),
4.2 (C-6), 33.1 (CH Cyst), 30.2 (C-4PentGlc), 29.3 (C-4PentMan), 28.5
Calcd. for C287
44.29, H 6.63, N 2.34.
H
518
N
14
O S35: C 44.46, H 6.75, N 2.53. Found: C
154
2
N
Branch, CH
2
N
Cyst, C
q
2
S
Heptakis[6-[2-[N¢-[7-(a-D-mannopyranosylthio)-4-oxaheptyl]-
thioureido]ethylthio]]cyclomaltoheptaose (Man-bCD). Yield:
O–NH OH); [a]
O); H NMR (500 MHz, D
O, 343 K): d =
(
C-5PentMan), 27.5 (C-5PentGlc); MALDI-TOFMS: m/z 7768 [M +
+
Na] . Anal. Calcd. for C287
Found: C 44.33, H 6.67, N 2.44.
H
518
N
14
154
O S35: C 44.46, H 6.75, N 2.53.
2
0.4 mg (46%); R
f
0.51 (5 : 3 : 5 MeCN–H
2
4
D
=
1
+
10.8 (c 1.0 in H
2
2
Heptakis[6-[2-[N¢-[2,2-bis[5-(2,3,4,6-tetra-O-acetyl-b-D-gluco-
pyranosylthio)-2-oxapentyl]-2-[5-(2,3,4,6-tetra-O-acetyl-a-D-man-
nopyranosylthio)-2-oxapentyl]ethyl]thioureido]ethylthio]]cyclo-
maltoheptaose (36). Reaction time: 5 days. Yield: 30 mg (30%);
5.69 (s, 7 H, H-1Man), 5.49 (d, 7 H, J1,2 = 3.0 Hz, H-1), 4.44 (m,
7 H, H-2Man), 4.32 (m, 14 H, H-5Man, H-5), 4.27 (t, 7 H, J2,3
=
J
3,4 = 9.0 Hz, H-3), 4.23 (m, 14 H, H-6aMan, H-6bMan), 4.17 (dd, 7
H, J3,4 = 9.3 Hz, J2,3 = 2.9 Hz, H-3Man), 4.12 (m, 21 H, H-4Man
,
3
[a]
D
= +32.3 (c 1.0, MeOH); R
f
0.50 (10 : 1 : 1 MeCN–H
2
O–
CH
2
N
Cyst), 4.06 (dd, 7 H, H-2), 3.98 (t, 14 H, JH,H = 6.2 Hz,
1
3
NH
7
J
4
OH); H NMR (500 MHz, CD
3
OD, 313 K): d = 5.39 (s,
H-5Hept), 3.96 (t, 14 H, JH,H = 6.0 Hz, H-3Hept), 3.94 (m, 7 H,
H, H-1Man), 5.34 (bd, 7 H, J2,3 = 5.0 Hz, H-2Man), 5.28 (t, 14 H,
2,3 = J3,4 = 9.5 Hz, H-3Glc), 5.27 (t, 7 H, J3,4 = J4,5 = 10.5 Hz,
H-4), 3.89 (m, 14 H, H-1Hept), 3.67 (m, 7 H, H-6a), 3.40 (m, 7 H,
3
H-6b), 3.34 (bt, 14 H, JH,H = 6.5 Hz, CH
2
SCyst), 3.15, 3.11 (2 dt,
2
3
H-4Man), 5.20 (dd, 7 H, H-3Man), 5.15 (m, 7 H, H-1), 5.04 (t, 14 H,
J
14 H, JH,H = 13.2 Hz, JH,H = 6.4 Hz, H-7Hept), 2.32 (m, 14 H,
1
3
4,5 = 10.0 Hz, H-4Glc), 4.96 (t, 14 H, J1,2 = 10.0 Hz, H-2Glc), 4.76
H-6Hept), 2.27 (m, 14 H, H-2Hept); C NMR (125.7 MHz, D O,
2
(
d, 14 H, H-1Glc), 4.39 (ddd, 7 H, J5,6a = 2.5 Hz, J5,6b = 1.5 Hz,
343 K): d = 181.0 (CS), 102.7 (C-1), 85.7 (C-1Man), 85.0 (C-4),
73.9 (C-5Man), 73.5 (C-3), 72.8 (C-2, C-5), 72.6 (C-2Man), 72.0
(C-3Man), 69.8 (C-5Hept), 68.9 (C-3Hept), 67.7 (C-4Man), 61.6 (C-6Man),
H-5Man), 4.29 (dd, 7 H, J6a,6b = 10.5 Hz, H-6aMan), 4.26 (dd, 14 H,
J
6a,6b = 12.5 Hz, J5,6a = 4.5 Hz, H-6aGlc), 4.15 (dd, 14 H, J5,6b =
2.5 Hz, H-6bGlc), 4.13 (dd, 7 H, H-6bMan), 3.96 (m, 7 H, H-5), 3.92
44.5 (CH
2
N
Cyst), 42.0 (C-1Hept), 34.1 (C-6), 33.1 (CH SCyst), 29.8
2
(
(
2
3
ddd, 14 H, H-5Glc), 3.82 (t, 7 H, J2,3 = J3,4 = 10.0 Hz, H-3), 3.73
m, 14 H, CH Cyst), 3.54 (m, 42 H, H-3Pent), 3.52 (m, 14 H, H-
, H-4), 3.42 (s, 56 H, CH Branch, H-1Pent), 3.22 (m, 7 H, H-6a),
.00 (m, 7 H, H-6b), 2.92 (m, 14 H, CH Cyst), 2.83, 2.79 (2 dt, 42
(C-2Hept), 29.3 (C-6Hept), 28.4 (C-7Hept); MALDI-TOFMS: m/z
+
2
N
4036.74 [M + H] . Anal. Calcd for C147
H
266
N
14
70
O S21: C 43.89, H
2
N
6.66, N 4.87. Found: C 44.13, H 6.51, N 4.76.
2
S
2
3
Enzyme-linked lectin assay (ELLA)
H, JH,H = 11.5 Hz, JH,H = 6.5 Hz, H-5Pent), 2.07–1.97 (s, 252 H,
1
3
MeCO), 1.92 (m, 42 H, H-4Pent); C NMR (125.7 MHz, CD OD,
3
TM
Nunc-Inmuno(tm) plates (MaxiSorp ) were coated overnight
3
8
7
(
6
3
(
C
13 K): d = 181.9 (CS), 172.2–171.1 (CO), 103.7 (C-1), 85.9 (C-4),
with yeast mannan at 100 mL/well diluted from a stock solution
4.9 (C-1Glc), 84.0 (C-1Man), 76.9 (C-5Glc), 75.4 (C-3Glc), 74.5 (C-3),
4.3 (C-2, C-5), 72.5 (C-2Man), 72.0 (C-1Pent), 71.7 (C-2Glc), 71.2
-
1
of 10 mg mL in 0.01 M phosphate buffer saline (PBS, pH 7.3
2
+
2+
containing 0.1 mM Ca and 0.1 mM Mn ) at room temperature.
The wells were then washed three times with 300 mL of washing
buffer (containing 0.05% (v/v) Tween 20) (PBST). The washing
procedure was repeated after each of the incubations throughout
C-3Man), 71.1 (C-3Pent), 70.5 (C-5Man), 70.1 (C-4Glc), 67.7 (C-4Man),
3.8 (C-6Man), 63.6 (C-6Glc), 45.9 (C
q
), 45.7 (CH
2
N
Branch, CH
2
NCyst),
5.0 (C-6), 34.0 (CH Cyst), 31.4 (C-4PentGlc), 30.9 (C-4PentMan), 29.6
2
S
C-5PentMan), 28.1 (C-5PentGlc), 21.2-20.9 (MeCO). Anal. Calcd for
35: C 48.43, H 6.14, N 1.74. Found: C 48.30, H
the assay. The wells were then blocked with 150 mL/well of
455
H
686
N
14
238
O S
◦
1
% BSA/PBS for 1 h at 37 C. After washing, the wells were
5
.98, N 1.63.
Heptakis[6-[2-[N¢-[2,2-bis[5-(b-D-glucopyranosylthio)-2-oxa-
pentyl]-2-[5-(a-D-mannopyranosylthio)-2-oxapentyl]ethyl]thiour-
eido]ethylthio]]cyclomaltoheptaose (ManDiGlc-bCD). Yield:
1 mg (99%); [a]
= +25.0 (c 1.0 in H
filled with 100 mL of serial dilutions of horseradish peroxidase
-
1
-5
-1
labelled concanavalin A (Con A-HRP) from 10 to 10 mg mL
◦
in PBS, and incubated at 37 C for 1 h. The plates were
washed and 50 mL/well of 2,2¢-azinobis-(3-ethylbenzothiazoline-
1
-1
2
D
3
D
2
O); H NMR (500 MHz,
6-sulfonic acid) diammonium salt (ABTS) (0.25 mg mL ) in
2
O, 353 K): d = 5.87 (s, 7 H, H-1Man), 5.60 (bd, 7 H, J1,2
=
citrate buffer (0.2 M, pH 4.0 with 0.015% H
2
2
O ) was added. The
.5 Hz, H-1), 5.00 (d, 14 H, J1,2 = 10.0 Hz, H-1Glc), 4.57 (m, 7
reaction was stopped after 20 min by adding 50 mL/well of 1 M
1
858 | Org. Biomol. Chem., 2010, 8, 1849–1860
This journal is © The Royal Society of Chemistry 2010

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H
2
SO
4
and the absorbances were measured at 415 nm. Blank wells
dialysis buffer as the protein. A separate experiment was run for
each titration in order to determine the heat of dilution of the
ligand in the dialysis buffer.
contained citrate-phosphate buffer. The concentration of lectin–
enzyme conjugate that displayed an absorbance between 0.8 and
1
.0 was used for inhibition experiments.
In order to carry out the inhibition experiments, each inhibitor
was added in a serial of 2-fold dilutions (60 mL/well) in PBS with
0 mL of the desired Con A–peroxidase conjugate concentration
Surface plasmon resonance experiments (SPR)
6
Con A and ribonuclease B (RNase B, EC 3.1.27.5) were purchased
from Sigma. Red cellulose filters were from Agilent technologies
(0.22 mm ¥ 13 mm) or from Millipore (0.22 mm ¥ 47 mm). All
on Nunclon(tm) (Delta) microtiter plates and incubated for 1 h
◦
at 37 C. The above solutions (100 mL) were then transferred to
◦
the mannan-coated microplates, which were incubated for 1 h at
SPR experiments were performed at 25 C using a BIAcore 3000
◦
3
(
7 C. The plates were washed and the ABTS substrate was added
system (BIAcore International AB, Uppsala, Sweden), using a
PBS buffer (pH 7.3) consisting of 10 mM sodium phosphate,
50 mL/well). Color development was stopped after 20 min and
the absorbances were measured. The percent of inhibition was
10 mM NaCl, 0.1 mM CaCl
2
, and 0.1 mM MgCl
2
as running
calculated as follows:
buffer. To account for the overestimation of K
a
associated to
37
%
Inhibition = (A(no inhibitor) - A(with inhibitor))/A(no inhibitor) ¥ 100.
mass transport limitation effects at the surface of the chip,
high and low density channels were used. Due to the high
molecular weight of CD-glycoconjugates, mass transport effects
in the flow channel on the sensor chip could not be discarded
completely, even at the low density surfaces. Low and high-density
chips were prepared by immobilizing Con A as follows. For the
experiments a CM5 sensorchip (Pharmacia) was activated and
Con A (in 10 mM NaOAc, pH 4.0) was immobilised to three flow
channels (FC2, FC3 and FC4, ~11600 RU). As a reference surface
Results in triplicate were used for plotting the inhibition
curves for each individual ELLA experiment. Typically, the IC50
values (concentration required for 50% inhibition of the Con A
coating mannan association) obtained from several independently
performed tests were in the range of ±15%. Nevertheless, the
relative inhibition values calculated from independent series of
data were highly reproducible.
49
(
FC1) an activated-deactivated carboxymethylated dextran layer
Isothermal titration calorimetry experiments (ITC)
was employed. Furthermore, the lectin bound to channel 3 was
denatured by flowing 6 M guanidinium chloride (pH 1.0) and 0.5%
SDS solution across the flow cell. The denatured lectin was used to
measure the level of nonspecific binding and to serve as reference
The lectin solutions to be used for ITC were prepared in 0.1 M
2
+
sodium phosphate buffer (pH 7.4) containing 0.1 mM Ca and
2
+
0
.1 mM Mn , dialysed against a large volume of the same buffer
40
and centrifuged to remove any insoluble material. The protein
concentrations were determined colorimetrically following the
channel for analysis of the data. Experiments were performed at
-
1
a flow rate of 10 mL min . For interaction experiments, typically,
a solution of carbohydrate containing material (15–30 mL) was
flowed across the sensor-chip for the association phase. Then,
spontaneous dissociation was allowed to occur for another 3 min
and subsequently the surface was regenerated, and stabilised for
the next round of binding.
46
Bradford’s method, measuring the absorbance at 595 nm of a
solution (1 mL) containing 200 mL of the commercial reagent
(
BIO-RAD protein assay), distilled H O (780 mL) and 20 mL
2
of protein solution containing 2–10 mg of protein. Calibration
of the commercial reagent was carried out using a 10% (w/v)
solution of BSA. The extinction coefficient determined for the
For calculation of the kinetics of the binding of the different
compounds, samples were appropriately diluted in phosphate
buffer, to cover the concentration range from 0.5 mM to 4 mM
for alcohol and model glycodendrimers, and from 12.5 mM to
400 mM for bCD glycoclusters. Sample solutions at different
concentrations were flown across the surfaces for 3 min, followed
by a dissociation period of 6 min prior to regeneration. After a
dissociation period (~ 3-6 min) remaining material was eluted
from the surface using two consecutive injections of 5 mM
sodium hydroxide (15 mL) and two consecutive injections of
100 mM (15 mL) methyl a-D-mannopyranoside in running buffer
(regeneration A) or using two consecutive injections of 5 mM
sodium hydroxide (5 mL) and two consecutive injections of
200 mM (10 mL) methyl a-D-mannopyranoside in running buffer
(regeneration B). Occasionally (in between different compounds),
surface regeneration was performed using 10 mM sodium acetate
(pH 4.2) followed by a re-equilibration injection employing a
running buffer.
-
1
-1
colorimetric complex was e595 = 45 mL mg cm . A multichannel
thermal activity monitor (TAM) isothermal heat conduction
microcalorimeter (Thermometric AB 2277/201, J a¨ rf a¨ lla, Sweden)
was used. This instrument has been described in detail by
47
Suurkuusk and Wads o¨ . The microcalorimeter was connected
to an external H O circulator (Hato) and the whole system was
2
placed in a room in which the temperature was kept constant
◦
within ±0.5 C. A 1.1 mL titration vessel was equipped with a
stirring system, loaded with 0.8 mL of protein solution using a
◦
microsyringe, thermostated at 25 C and continuously stirred at
6
0 rpm. The ligand was injected through a stainless steel cannula
connected to a 250 mL microsyringe, which was positioned in
a computer controlled syringe pump (Hamilton Microlab M).
Injections were made over a period of 10 s with intervals of
6
4
min. The experiment was computer controlled using Digitam
.1 software (Thermometric); the same program was used for data
analysis. The microcalorimeter was electrically calibrated before
each experiment. Nevertheless, in order to check the performance,
Association and dissociation rate constants were calculated
by nonlinear fitting of the primary sensorgram data using the
BIAevaluation 3.0 software. The precision of the fit for each
estimated parameter is described by the statistical c . The
association constant (K
2
+
the binding of Ba (aq) to 18-crown-6 was used. The results
-
1
-1
obtained, K
c
= 5667 ± 680 M and DH = -(31.46 ± 0.70) kJ mol
m
48
2
are in agreement with those reported by Briggner and Wads o¨ :
-
1
-1
K
c
= 5900 ± 200 M and DH
m
= -(31.42 ± 0.20) kJ mol . To
a
) was calculated both experimentally
minimize dilution artefacts, the ligand was dissolved in the same
from the association (k
a
) and dissociation (k ) rate constants,
d
This journal is © The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 1849–1860 | 1859

View Online
and by Scatchard analyses based on the equilibrium steady-state
kinetics.
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Acknowledgements
We thank the Spanish Ministerio de Educaci o´ n y Ciencia
3
1366.
(
6
contracts number CTQ2006-15515-C02-01/BQU and CTQ2007-
1180/PPQ) and the Junta de Andaluc ´ı a for financial support.
2
2
2
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860 | Org. Biomol. Chem., 2010, 8, 1849–1860
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