Probing carbohydrate-carbohydrate interactions by photoswitchable supramolecular glycoclusters

Article abstract of DOI:10.1039/c3cc41025k

The synthesis of photo-switchable glycoclusters with distinct sugar arrangements is described and the use of these clusters to study carbohydrate-carbohydrate interactions (CCIs) is demonstrated.

Full text of DOI:10.1039/c3cc41025k

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Probing carbohydrate–carbohydrate interactions by
photoswitchable supramolecular glycoclusters†
Cite this: Chem. Commun., 2013,
4
9, 3988
Harikrishna Bavireddi,z Priya Bharatez and Raghavendra Kikkeri*
Received 6th February 2013,
Accepted 15th March 2013
DOI: 10.1039/c3cc41025k
www.rsc.org/chemcomm
6
The synthesis of photo-switchable glycoclusters with distinct sugar reported in the literature, but these research studies focused on
arrangements is described and the use of these clusters to study carbohydrate–protein interactions whereas our study explores carbo-
carbohydrate–carbohydrate interactions (CCIs) is demonstrated.
hydrate–carbohydrate interactions, a field that was unexplored before.
The synthesis of two ‘tunable’ glycoclusters based on b-cyclo-
Glycans, found on cell surfaces, interact with viruses, bacteria, dextrin (T-1 and C-1, Scheme 1) is described. The synthesis of the
toxins and other biological entities by specific carbohydrate–protein lactose substituted b-cyclodextrin 8 was started with peracetylated
interactions. Self recognition of glycans is referred to as carbo- lactose 3, followed by treatment of bromoethanol with BF ꢀEt O
3
2
1
hydrate–carbohydrate interactions (CCIs). Particular CCIs can be and thiocyanate to yield the thiocyanate–lactose derivative. The
considered on two levels, intramolecular, within glycolipids in the thiocyanate was reduced by Zn–AcOH to the corresponding thiol 5
same membrane (cis) or intermolecular, within glycolipids on and reacted with 6-hepta iodinated b-cyclodextrin 6 (ref. 7) in the
apposing membranes (trans) recognition events. These extremely presence of CsCO to yield lactose-substituted b-cyclodextrin, which
3
2
low affinity interactions serve different functions on cell surfaces.
was finally treated with base yielding compound 8. The photo-
Improved CCIs can be obtained by carbohydrate clusters.
Different carbohydrate clusters based on artificial scaffolds have benzyl bromide and 4,4 -azo-pyridine in acetonitrile for 24 hours.
been synthesised and used to study the role of the structure of the
b-Cyclodextrin complexes with 8 and 14 were prepared by mixing
chromic molecule 14 was obtained as a brown solid by reacting
0
glycans and its influence on CCIs. The groups of Penad ´e s, Basu, stoichiometric amounts of these compounds. The formation of the
1
Russell, and Kamerling have synthesised carbohydrate clusters complex was demonstrated using H-NOESY NMR spectroscopy.
attached to dendrimers, polymers, micelles and nanoparticles and Strong cross-peaks were observed in the 2D NOESY spectrum of T-1
3,4
studied weak intermolecular CCIs. In other studies, the groups of in D O that are from spacial interactions between hydrogens on the
2
Hasegawa, Sasaki, Gallagher and Schmidt have evaluated intra- phenyl group and b-CD groups. However, it was difficult to identify
molecular CCIs using calcium-mediated conformational stability specific protons of 8 involved in NOE interactions (Fig. S2, ESI†). We
of carbohydrate substituted glycopeptides and ferrocene com- have focused our attention on protons of the phenyl since we have
5
plexes. In spite of all the research in this field, the binding affinity observed a strong NOE interaction with protons from 8, confirming
and differentiation of inter and intra CCIs is still unexplored.
the encapsulation of the phenyl group in the cavity of b-CD.
In this communication we present the results of a systematic Furthermore, a weak NOE effect was found between the CH2
investigation of the CCIs of photo-switchable glycoclusters with protons of the benzyl and b-CD, referring the mode by which the
2+
8
Ca ions using the isothermal titration calorimetry (ITC) techni- benzyl group enters the b-CD cavity. The stoichiometry of the host–
que. We have demonstrated that by using photo-tunable glyco- guest complex was established by ITC titration (Fig. S3, ESI†).
clusters a straightforward and relatively fast access to different
Extensive studies, including UV-spectroscopy, imaging and photo-
sugar densities through self-assembly and tuning of intra-to inter- physical studies, were conducted in order to evaluate the effect of the
molecular CCIs can be obtained. We have used b-cyclodextrin photo-induced isomerisation of the azo-dye on the stability and
appended with lactose and maltose molecules, which formed com- orientation of the glycoclusters. T-1 has maxima of UV absorption at
plexes with 1,2-di(pyridin-4-yl) diazene derivatives, and obtained B210 nm and B280 nm. These absorptions correspond to the benzyl
glycoclusters by self-assembly. Azo–glycocluster systems were already group and p–p* transition of the azo chromophore, respectively. On
irradiation with a 120 W Xe lamp (with a cut-off filter at B350 nm) the
Indian Institute of Science Education and Research, Pune 411021, India.
intensity of the 280 nm absorption decreased (Fig. S13a) indicating a
cis–trans isomerisation process yielding the C-1 complex.
Circular dichroism (CD) spectra of C-1 and T-1 displayed an
unaltered positive Cotton effect for the benzyl–b-CD complex at
E-mail: rkikkeri@iiserpune.ac.in; Fax: +91-20-25899790; Tel: +91-20-25908207
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cc41025k
9
†
‡
Equal contribution.
3
988 Chem. Commun., 2013, 49, 3988--3990
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Fig. 1 AFM images (upper images) and the corresponding topology (lower
images) of T-1 (a) and (b) C-1.
constants (K) and thermodynamic parameters determined using
ITC are summarized in Table 1, where ‘N’ represents the number
of carbohydrate groups on the glycoclusters that are available for
binding with metal ions. In the case of 8, the best fit was obtained
ꢂ1
using an N value of 4 and a K value of 57.9 ꢁ 4.7 M (Fig. 2a). It
2+
shows that there might be 4 lactose units coordinated to Ca . Thus,
the result demonstrates that only a subset of the available ligands of
2
+
8
participated in Ca mediated CCIs and may involve intramolecular
2+
CCIs. To demonstrate the specificity of Ca and 8 binding, we
performed an ITC experiment of 8 with Na and K (Fig. S5, ESI†). As
+
+
Scheme 1 Synthesis of supramolecular complexes (T-1 and C-1); (a) bromoethanol–
expected, highly disordered enthalpy changes were observed, indi-
BF
39–46%); NaOMe–MeOH (43%); (c) benzylbromide–MeOH (31%); (d) 1 : 2 mixture
of 14 and 8 in water; (e) 350 nm Xe-lamp (14 to 15) and 430 nm lamp (15 to 14).
3 2 3
ꢀEt O (46–47%); KSCN–DMF (74–89%); Zn–AcOH (84–91%); (b) CsCO –DMF
+
+
cating that no CCIs exist in the presence of Na or K ions.
With T-1, the best fit was obtained with one set of sites model
using N = 21, indicating five-fold more lactose units involved in
CCIs compared to 8. However, the binding affinity varies by as
(
220 nm, showing configuration stability of the host–guest
complex for both isomers. However, a significant change was
observed in the 250–320 nm region. In T-1, a positive Cotton effect Table 1 Thermodynamic parameters of cis–trans isomers measured with iso-
thermal titration calorimetry; N = 1/n
at 270 nm was observed, and in C-1, a reduced Cotton effect at
2
70 nm and a new positive CD band at 298 nm appear (Fig. S13b).
ꢂ1
Binding
DH
DS (cal mol
6 3
ꢂ1
ꢂ1
ꢂ1
These spectral changes suggest that cis–trans isomerisation has a Ligand
n
N
constant (M ) (kcal mol ) 10 deg ) 10
notable influence on the overall topology of the complex. To
further support CD data on the structural rearrangement during T-1
isomerisation, we performed ITC titration with 15 and 8. The
8
0.28
0.047
4
57.9 ꢁ 4.7
ꢂ1.73 ꢁ 0.02
ꢂ3.43 ꢁ 0.81
ꢂ3.71 ꢁ 0.2
—
ꢂ5.80
ꢂ11.5
ꢂ12.4
—
21 115 ꢁ 23.0
C-1
0.0051 196 276 ꢁ 39.7
13
—
—
—
results clearly showed 1 : 1 host–guest complexation, indicating
different types of arrangement of the complex (Fig. S3b, ESI†).
Finally, AFM was employed to confirm the topologies of the cis–
trans isomers (Fig. 1a and b). A globular shape (B200 nm) was
found for T-1 which results from uniform integration of b-CD and
T-2
C-2
0.043
23 104 ꢁ 11.4
ꢂ2.18 ꢁ 1.79
ꢂ7.32
0.0052 192 205 ꢁ 87.3
ꢂ7.13 ꢁ 0.04
ꢂ23.9
14, whereas a tube shape (B200 nm width) was found for C-1,
which is most likely due to the weak host–guest complex and strong
10
p–p interactions among aromatic segments (Fig. S12, ESI†). These
results demonstrate that host–guest interactions and subsequent
isomerisation induce tuneable topologies which are crucial for
studying carbohydrate–carbohydrate interactions (CCIs).
In order to gain insight into the binding affinity and thermo-
dynamics of the interaction between glycoclusters and different
+
+
2+
cations (Na , K , Ca ) we used isothermal calorimetry (ITC). The Fig. 2 ITC profile for cis–trans isomers in the presence of CaCl
2
at 298 K in H
2
O.
ꢂ1
The top panels represent the energy (mcal s ) required to maintain isothermal
metal ions were titrated into the glycoclusters in the calorimeter
cells. Upon binding of glycoclusters to Ca heat is released,
yielding a typical titration isotherm. Based on the qualitative assess-
ment, the Ca binding isotherm was fitted to a binding model
containing one set of binding sites. The values of formation water and CaCl
2
+
conditions with respect to the reference cells and the lower panels represent the
2+
heat evolved from each injection per mole of Ca versus the molar ratio of (a)
conc. of 8 = 0.03 mM in water and CaCl solution = 20 mM in water; (b) conc. of
T-1 = 0.05 mM in water and CaCl = 20 mM in water (c) conc. of C-1 = 0.05 mM in
= 20 mM in water.
2
2
+
2
2
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sugar topology, but also exhibits specific calcium mediated CCIs.
Furthermore, we have demonstrated that sugar topology and den-
sity can tune the intra vs. intermolecular CCIs. A drawback of the
current system is the relatively small changes in the CCIs measured
for C-1 and T-1, C-2 and T-2. We believe that better results might be
X
X
obtained with more complex glycans, such as gangliosides, Le –Le ,
13
3 3
GM –Gg and sulphated b-D-GlcNAc(3S)-(1 - 3)-a-L-Fuc models.
We are currently investigating these aspects.
R.K, H.B and P.B thank IISER, Pune, Indo-German (DST-MPG)
program and DAE (grant no.2011/37C/20/BRNS) and CSIR, India,
for financial support. We thank Dr Rina Arad Yellin for critically
editing the manuscript.
Fig. 3 AFM images (upper lane) and the corresponding topology (lower lane) of
2 2
T-1 in the presence CaCl (a) and C-1 in the presence CaCl ; (b) the image scale is
in micrometers.
Notes and references
ꢂ1
much as two-fold (115 ꢁ 23 M ) from 8 to T-1 (Table 1 and
Fig. 2b). This may be attributed to the fact that the inherent sugar
density of T-1 propagates intra-to-intermolecular CCIs. A similar
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3
4
(
2
ꢂ1
increased approximately three fold more compared to the nine-
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2+
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11
models. In addition, the standard enthalpies of formation
DH) are negative in all cases indicating that the association is
exothermic in the order C-1 > T-1 > 8. These trends appear to be
general and are shown by carbohydrate–protein models.
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AFM measurements. We have found that compound 8 with Ca
did not show any aggregation, an observation that refers to the
possibility of intramolecular CCIs, whereas, T-1 in the presence of
Ca ions showed aggregates of size 200 nm and B1 mm, respec-
tively (Fig. 3a). The sizes of the small aggregates represent intra-
CCIs as their size is approximately the same as that of T-1, whereas
the large aggregations showed integration of several small aggre-
gates, representing inter-CCIs. Furthermore, Cis-1 showed random
aggregations. This may be due to the combination of intra and
intermolecular CCIs (Fig. 3b). The above approach was further
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S10, ESI†) compared to lactose analogs. These results suggest that
the glucose sugar moiety of maltose weakens the Ca ions binding
to some extent. Finally, to verify the quality and sensitivity of the
photoisomerization process, the platform was subjected to three
cycles of cis–trans isomerisation and the resultant sample was
subjected to Ca mediated CCIs. The system exhibits excellent
reproducibility (Fig. S11, ESI†).
(
12
2+
2+
6
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3
990 Chem. Commun., 2013, 49, 3988--3990
This journal is c The Royal Society of Chemistry 2013