Photoswitchable multivalent sugar ligands: Synthesis, isomerization, and lectin binding studies of azobenzene-glycopyranoside derivatives

Article abstract of DOI:10.1021/ja0173066

Coating of azobenzene chromophore with multivalent sugar ligands has been accomplished. Such sugar coating allows the study of the isomerization properties of this chromophore in aqueous solutions. The predominantly cis-isomer-containing photostationary s

Full text of DOI:10.1021/ja0173066

Published on Web 02/16/2002
Photoswitchable Multivalent Sugar Ligands: Synthesis, Isomerization, and
Lectin Binding Studies of Azobenzene-Glycopyranoside Derivatives
Oruganti Srinivas,‡ Nivedita Mitra,§ Avadhesha Surolia,*,§ and Narayanaswamy Jayaraman*,‡
Department of Organic Chemistry and Molecular Biophysics Unit, Indian Institute of Science,
Bangalore 560 012, India
Received October 16, 2001
Scheme 1 a
Among biological recognition processes, carbohydrate-protein
recognitions are known to be rather weak (Ka < 103 M-1).1 A
significant carbohydrate-protein interaction ensues in natural
systems when one or both of the interacting partners is multiply
presented at the binding sites.2 In a series of pioneering contribu-
tions, Lee and co-workers have established3 the so-called “glycoside
cluster effect”, wherein multivalent display of sugar residues at the
binding sites of relevant lectin led to almost a logarithmic
enhancement of binding potency. A number of small cluster
glycosides,4 polyvalent structures based on polymeric macromol-
ecules,5 and structurally homogeneous dendritic macromolecules6
have been synthesized that have shown enhanced binding affinity
to relevant lectins. In our desire toward synthesis of glycoclusters
a Conditions: (i) Et3N, THF, 0 °C to room temperature, 24 h. (ii) 0.5 M
NaOMe/MeOH, room temperature, 12 h.
carrying a photoresponsive moiety, which will allow probing the
biological recognition event, we have investigated the reversible
photoisomerization properties of azobenzene-containing glycocon-
jugates at the interface of carbohydrate-lectin recognition pro-
cesses. The facile and reversible trans T cis isomerization properties
of azobenzene chromophore have been studied in detail,7 including
in many biological8 and physicochemical systems.9 In this com-
munication, we report the synthesis, photoisomerization, and lectin
binding studies of new types of azobenzene-glycopyranoside
derivatives. Most importantly, the preliminary studies described
herein illustrate the existence of a cooperativity in the binding of
a lactose bearing bivalent azobenzene derivative toward its high-
affinity lectin, namely, peanut agglutinin (PNA), as judged from
isothermal titration calorimetric (ITC) measurements.
The synthesis of azobenzene-glycopyranoside derivatives was
accomplished by forming amide bonds between amine tethered
glycopyranoside derivatives, namely, 2-aminoethyl-(2,3,4,6-tetra-
O-benzoyl)-â-D-galactopyranoside (1), 2-aminoethyl-(2,3,4,6-tetra-
O-benzoyl)-â-D-galactopyranosyl-(1 f 4)-(2,3,6-tri-O- benzoyl)-
â-D-glucopyranoside (2), and 1,1′-bis-(2,3,4,6- tetra-O-benzoyl-â-
D-galactopyranosyloxymethyl)ethylamine (3) and azobenzene-
4,4′-dicarbonyl chloride 4,10 followed by removal of the O-benzoyl
protecting groups to afford free sugar containing diamides 5-7 and
monoamides 8-10 (Scheme 1), respectively.
Table 1. Rate Constants (k) and Activation Energies (Ea)a for
Thermal Cis-Trans Isomerization of 6 and 9
70 °C
65°C
60°C
50°C
E
a
6
9
k (×104 s-1
)
)
1.40
1.41
1.14
1.20
0.56
0.34
0.21
0.18
20.54
22.57
k (×104 s-1
a Ea (kcal mol-1) values were obtained from the Arrhenius plots.
determined the cis:trans ratio at the PS state by NMR spectroscopy
in D2O (1-2 mM). In the case of symmetrical diamide derivatives
5-7, the protons of the azobenzene unit having an AB type spin
system in the trans isomer appear as a sharp singlet at ∼7.85 ppm.
After irradiation for 2 h, a set of new peaks in the form of doublets
at ∼7.50 and ∼7.00 ppm (J ) 7.8 Hz) was observed, which upon
integration, afforded ∼60-65% cis isomer at the PS state in these
samples. Thermal isomerization from the cis isomer to the trans
isomer was followed by the time course measurement of the
absorbance changes (330 nm) for the photoirradiated solutions of
6 and 9 at different temperatures. The isomerizations followed first-
order kinetics and the corresponding rate constants and the
activation energies for 6 and 9 are given in Table 1.
Despite several reports on the interactions of cluster glycosides
with multivalent lectins, the true cluster effect has been observed
only with the asialoglycoprotein receptor (ASGPr),3 primarily
because of an exact geometric complementarity between the binding
sites of the trimeric ASGPr and the triantennary oligosaccharide
ligands. In most other cases, the interaction of the ligand occurs
with independent, unconnected multivalent lectin molecules.12
Consequently a true cluster effect is not seen. Also, in none of
these cases has cooperativity during these interactions been
observed. We explored the nature of binding by photoisomerisable
bivalent lactoside derivative 6, in which the azobenzene unit acts
as the switchable scaffold. Our ITC studies pertain largely to the
interaction of bivalent 6 with PNA, as lactose13a is known to have
Photoisomerization studies11 on compounds 5-10 were con-
ducted in H2O (40-60 µM) by UV-vis spectroscopy. A decrease
in absorbance (∼330 nm) of the trans-isomer of 5-10 was observed
over a period of 5 to 30 min and the so-called photostationary state
(PS) reached after ∼20 min. On the basis of the absorbance changes,
∼55-60% of cis isomer formation was calculated for all the
compounds. Thermal reversal of the cis to trans isomer in dark
conditions was monitored at ∼330 nm and the increase in
absorbance, which corresponds to the evolution of the trans form,
of the PS state was less than 5% even after 3 h. We have also
‡ Department of Organic Chemistry.
§ Molecular Biophysics Unit.
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2124 VOL. 124, NO. 10, 2002 J. AM. CHEM. SOC.
10.1021/ja0173066 CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
In conclusion, the newly synthesized azobenzene-lacto- and
galactopyranoside derivatives exhibit rather slow cis f trans
thermal relaxation processes in aqueous solution. Lectin binding
studies of bivalent derivative 6 with PNA, using ITC, have shown
a biphasic binding profile, corresponding to a cooperative nature
of the binding process. Although a number of multivalent glycosides
have been tested previously, no report concerning the existence of
cooperativity is reported so far. To the best of our knowledge, the
present observation is the first report that such phenomena can exist
in glycoside-lectin interactions.
Acknowledgment. This paper is dedicated to the memory of
Dr. Darshan Ranganathan. We are grateful to CSIR (New Delhi)
for financial support. O.S. thanks UGC for a research fellowship.
Supporting Information Available: Experimental procedures of
5-10 and method of ITC studies (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
Figure 1. Binding profiles of the lactose derivative 6 in the trans and the
PS (inset) isomeric states. The cooperativity is seen to be more in the case
of the PS mixture than in the trans isomer.
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PS mixture
Kb1 (×10-4
)
5.1 ( 0.2
4.9 ( 0.3
∆H1
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-2.1 ( 0.1
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a Values of Kb and ∆H, ∆G, and Τ∆S are expressed in M-1 and kcal
mol-1, respectively. The studies were performed in 20 mM phosphate buffer,
pH 7.4, and 150 mM NaCl. The titrations were done at 298 K.
higher affinity for PNA than galactose.13b As shown in Figure 1,
the recognition of bivalent 6 by PNA exhibits pronounced coop-
erativity. The experimentally obtained thermodynamic parameters
are given in Table 2. The distance between 4,4′-positions of the
azobenzene unit is about 9 Å, while that between the anomeric
oxygen at the reducing end is not expected to be greater than 17
Å. Consequently, a true cluster effect for its binding to the PNA
molecule is not observed, as the least separation between these
binding sites is 69 Å.14 Even in the absence of a cluster effect, we
still observe a much higher binding constant (Kb1 ) 5.1 × 104
M-1) for the low-affinity part of the binding reaction, when
compared to 7, 8, and 10 (Kb ) 3.5 × 103, 1.7 × 103, and 1.9 ×
103 M-1, respectively).15
Although the binding invokes interaction of the ligand with the
separate unconnected subunits of tetrameric PNA, the geometry of
the trans-azobenzene derivative 6 is such that a mere separation of
17 Å between its two lactose units promotes a cooperativity during
the reaction. Further, the orientation of 6 increases the affinity of
the second site in a striking manner. This can be explained on the
basis that the binding of the first lactose moiety to a subunit of the
first PNA tetramer aligns the second moiety in an orientation that
increases the affinity for the recognition of the latter by another
PNA molecule. Irrespective of the origin of the cooperativity, the
affinity becomes even more pronounced in the cis-isomer (Kb2),
where the distance between the two lactosides is close to 10 Å.16
(15) 7, 8, and 10 had shown that the binding profiles were monophasic in
nature, indicating the absence of any cooperativity, probably due to the
fact that galactose itself exhibits low affinity for PNA. Low aqueous
solubilities precluded ITC studies of 5 and 9.
(16) It should be mentioned here that the contributions of the cis and trans
forms were not taken into account separately during the ITC analysis of
the PS mixture of bivalent derivative 6 because of practical difficulties.
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