Glycobiology Advance Access originally published online on May 4, 2005
Glycobiology 2005 15(9):861-873; doi:10.1093/glycob/cwi069
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Photoswitchable cluster glycosides as tools to probe carbohydrate–protein interactions: synthesis and lectin-binding studies of azobenzene containing multivalent sugar ligands
2 Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, Karnataka, India; and 3 Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, Karnataka, India
1 To whom correspondence should be addressed; e-mail: jayaraman{at}orgchem.iisc.ernet.in and surolia{at}mbu.iisc.ernet.in
Received on March 10, 2005; revised on April 23, 2005; accepted on April 25, 2005
 |
Abstract |
|---|
 Top Abstract IntroductionResults and discussionConclusionsSupplementary dataAcknowledgmentsReferences |
|---|
Synthetic cluster glycosides have often been used to unravel mechanisms of carbohydrate–protein interactions. Although synthetic cluster glycosides are constituted on scaffolds to achieve high avidities in lectin binding, there have been no known attempts to modulate the orientations of the sugar clusters with the aid of a functional scaffold onto which the sugar units are linked. Herein, we describe synthesis, physical, and lectin-binding studies of a series of
-D-mannopyranoside and ß-D-galactopyranosyl-(1
4)-ß-D-glucopyranoside glycoclusters that are attached to a photoswitchable azobenzenoid core. These glycoclusters were synthesized by the amidation of amine-tethered glycopyranosides with azobenzene carbonyl chlorides. From kinetic studies, the cis forms of the azobenzene-glycopyranoside derivative were found to be more stable in aqueous solutions than in organic solvents. Molecular modeling studies were performed to estimate the relative geometries of the photoswitchable glycoclusters in the trans- and cis-isomeric forms. Isothermal titration calorimetry (ITC) was employed to assess the binding of these glycoclusters to lectins peanut agglutinin (PNA) and concanavalin A (Con A). Although binding affinities were enhanced several orders higher as the valency of the sugar was increased, a biphasic-binding profile in ITC plots was observed during few glycoclusters lectin-binding processes. The biphasic-binding profile indicates a "cooperativity" in the binding process. An important outcome of this study is that in addition to inherent clustering of the sugar units as a molecular feature, an induced clustering emanates because of the isomerization of the trans form of the azobenzene scaffold to the cis-isomeric form. Key words: azobenzene / cluster glycosides / geometrical isomerism / isothermal titration calorimetry / lectins
|
Introduction |
|---|
|
TopAbstractIntroductionResults and discussionConclusionsSupplementary dataAcknowledgmentsReferences |
|---|
Carbohydrate–protein interactions are essential for the sustenance of many important biological functions (Sharon and Lis, 1989
; Varki, 1993; Dwek, 1996). A major theme in studies leading towards understanding complex carbohydrate–protein interactions is to address the stringent requirement of clustered patches of carbohydrate ligands to significantly enhance the binding strengths with the protein receptors (Drickamer, 1995; Crocker and Feizi, 1996). In a series of pioneering contributions, Lee and coworkers have established the so-called "glycoside cluster effect," wherein multivalent display of sugar residues at the binding sites of the relevant lectin led to almost a logarithmic enhancement of the binding potencies (Lee and Lee, 1995). On the basis of glycoside cluster effect, few small cluster glycosides (Fenderson et al., 1984; Kempen et al., 1984), polyvalent structures based on polymeric macromolecules (Roy and Laferrière, 1990; Spaltenstein and Whitesides, 1991; Mortell et al., 1994; Mortell et al., 1996), and structurally homogeneous dendritic macromolecules have been synthesized and studied so far (Aoi et al., 1995; Lindhorst and Kieburg, 1996; Hansen et al., 1997; Jayaraman et al., 1997; Yi et al., 1998), in an effort to identify the structural features of clustered glycosides necessary for tight binding with the receptor. An important outcome of these studies is the realization that even a small, well-defined glycoside cluster is adequate to provide a large enhancement in binding strengths upon interaction with the receptor.
With the aid of synthetic cluster glycosides and neoglycoconjugates, the concept of multivalency in carbohydrate–protein interactions is now established firmly (Lee and Lee, 1994; Mammen et al., 1998). Although multivalent or polyvalent presentation is a necessary requirement for tight binding to the binding partners, the orientation of the ligands also plays an important role in the receptor function, for example, saccharide orientation at the cell surface affects glycolipid receptor function (Stromberg et al., 1991). In the perspective of synthetic cluster glycosides, we were interested to study cluster glycosides built up on cores that in principle would allow us to modulate the orientation of the sugar ligands, and in turn, the change in orientation would allow assessing the differential receptor-binding profiles. Thus, we intended to incorporate an additional feature in the cluster glycosides, which would allow us to modify the topological properties of the clustered sugar ligands, upon an applied external stimuli. Evidently, the application of light as a source of external stimuli is attractive. In this respect, the prototypical photoresponsive moiety, namely, azobenzene has been used widely for light-induced conformational changes (Räu, 1990). Their facile and reversible trans
cis photoisomerization and cistrans thermal reversion properties have been studied in detail to follow conformation-induced effects in biological and physico-chemical systems (Whitten et al., 1971; Balasubramanian et al., 1975; Willner, 1997; Erlanger, 1980; Kunitake et al., 1981; Anzai and Osa, 1994; Delaire and Nakatani, 2000).
We have reported previously that the incorporation of photoresponsive azobenzene moiety as a component of synthetic cluster glycosides, as shown in Figure 1, allow us to study the orientation effects of the sugars, arising after the cis/trans isomerization of the azobenzene core (Srinivas et al., 2002). In these studies, we have observed from the lectin-binding studies not only variations in the binding profiles of the photoisomers, but also an anomalous "cooperativity" in the binding of these sugar ligands with a lectin. On the basis of these initial studies, we undertook to synthesize a series of multivalent cluster glycosides at different sugar densities on a photoisomerizable scaffold and to study their lectin-binding properties. Herein, we describe the synthesis of cluster glycosides built on a photoresponsive functional core, photochemical, and lectin-binding studies of individual photoisomeric states of these cluster glycosides.
|
|
Results and discussion |
|---|
|
TopAbstractIntroductionResults and discussionConclusionsSupplementary dataAcknowledgmentsReferences |
|---|
Synthesis and characterization
The features of our synthetic design were that (1) the individual sugar containing building blocks would be small and structurally well defined; (2) the central core should be able to adopt switchable conformations, so as to bring about changes in the orientation of sugar moieties with respect to each other; and (3) the branch junctures would be kept shorter and rigid, such that the structural changes of the core perturbs the molecular orientations significantly. With these features, the synthesis of cluster glycosides built upon the photoisomerizable scaffold was undertaken. The following glycopyranoside derivatives were undertaken for their covalent attachment to the photoisomerizable scaffold: (1) mono-(ß-D-galactopyranosyl-(1
4)-ß-D-glucopyranoside); (2) bis-(ß-D-galactopyranosyl-(14)-ß-D-glucopyranoside); (3) mono-(-D-mannopyranoside); and (4) bis-(-D-mannopyranoside). Photoisomerizable azobenzene cores were those that were derived from azobenzene-4,4'-dicarboxylic acid (AD) (Ameerunisha and Zacharias, 1995) and azobenzene-3,3',5,5'-tetracarboxylic acid (AT) (Wang and Advincula, 2001). The protected derivatives of amino alcohols, namely, 2-aminoethanol (1) and 2-amino-2-methyl-1,3-propane diol (2), were used as the starting materials for the synthesis of saccharide-bearing building blocks. The glycosylation of alcohol 1, using glycosyl donors, 2,3,4,6-tetra-O-benzoyl--D-mannopyranosyl bromide (3) and 2,3,4,6-tetra-O-benzoyl-ß-D-galactopyranosyl-(14)-2,3,6-tri-O-benzoyl-ß-D-glucopyranosyl bromide (4) in the presence of HgBr2/Hg(CN)2 activators in CH2Cl2, followed by hydrogenolysis afforded the -D-mannopyranoside (5) and ß-D-galactopyranosyl-(14)-ß-D-glucopyranoside (6) derivatives, respectively, in good yields (Scheme 1). Glycosylation of the diol 2 with glycosyl donors 3 and 4, followed by hydrogenolysis furnished the bis--D-mannopyranoside (7) and bis-ß-D-galactopyranosyl-(14)-ß-D-glucopyranoside (8) derivatives, respectively. Amide bond formation between amine tethered mannopyranoside derivatives 5 and 7 and azobenzene dicarbonyl chloride 9 was performed in tetrahydrofuran (THF) in the presence of Et3N to furnish the symmetrical diamides 11 and 12, respectively (Scheme 2). Similarly, azobenzene 3,3',5,5'-tetracarbonyl chloride 10 was reacted with the amine 5 (4.5 molar equivalent) in THF in the presence Et3N to obtain the tetra-amidated derivative 13. In the final step of the synthesis, the O-benzoyl-protecting groups in 11, 12, and 13 were removed (0.5 M NaOMe/MeOH) to afford the diamides bivalent Man-AD-Man (14), tetravalent Man2-AD-Man2 (15), and tetravalent Man-AT-Man (16), respectively (Scheme 3). Amidation of amine tethered lactose derivatives 6 and 8 (2.2 molar equivalent) and azobenzenedicarbonyl chloride 9 in THF in the presence of Et3N afforded the diamides 17 and 18, respectively (Scheme 4). On the other hand, amidation of 9 with amines 6 and 8 (0.8 molar equivalent) led to the isolation of monoamides 19 and 20 (Scheme 4). Removal of the O-benzoyl-protecting groups (0.5 N NaOMe/MeOH) furnished ß-D-galactopyranosyl-(14)-ß-D-glucopyranoside containing symmetrical azobenzene derivatives, abbreviated as Lac-AD-Lac (23) and Lac2-AD-Lac2 (24) and the unsymmetrical Lac-AD-COOH (25) and Lac2-AD-COOH (26) (Scheme 5). The higher generation tetravalent and octavalent azobenzene containing ß-D-galactopyranosyl-(14)-ß-D-glucopyranoside derivatives 21 and 22 were synthesized by the amidation of 3,3',5,5'-azobenzenetetracarbonyl chloride 10 (AT-Cl) with amines 6 and 8 (4.5 molar equivalent) (Scheme 4). Purification of the tetra-amide derivatives 21 and 22 was performed by using flash chromatography (SiO2). Removal of the O-benzoyl-protecting groups in derivatives 21 and 22 (NaOMe/MeOH/H2O) afforded Lac-AT-Lac (27) and Lac2-AT-Lac2 (28) (Scheme 6). All the intermediates and the final compounds were characterized by mass spectrometry (ESI-MS and MALDI-TOF), as well as by 1H and 13C nuclear magnetic resonance (NMR) spectroscopies. Electrospray ionization- mass spectrometric (ESI-MS) analysis was helpful to characterize the smaller molecular weight compounds, whereas matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was used in the characterization of higher molecular weight compounds, in both the protected and deprotected forms. MALDI-TOF mass spectrometric analysis afforded molecular ion peaks as either a Na+ or a K+ adduct ions. The 1H NMR spectra of O-benzoylated protected azobenzene-glycopyranoside derivatives were assigned by two-dimensional homonuclear and heteronuclear correlation NMR spectroscopy. The 1H NMR spectra for the fully deprotected derivatives 14–16 and 23–28 were recorded in either D2O or dimethylsulfoxide-d6 (DMSO-d6), within the concentration range of 0.5–4.0 mM. In the case of symmetrical derivatives, monosaccharide derivatives Man-AD-Man (14) and Man2-AD-Man2 (15), disaccharide derivatives Lac-AD-Lac (23) and Lac2-AD-Lac2 (24), and the chemically distinct azobenzene protons appear as a singlet in D2O. Whereas in DMSO-d6, the expected AB-type pattern was observed. We investigated the possibility of aggregation in water, because of the presence of hydrophobic azobenzene inner region and hydrophilic sugar peripheral region, by 1H NMR spectroscopy. In the case of the bivalent trans-Man-AD-Man (14) and trans-Lac-AD-Lac (23), the chemical shifts of the azobenzene protons were concentration dependent, in that the near singlet pattern of the azobenzene protons at low concentrations (up to 3.5 mM) split to a double doublet corresponding to the AB-type spin system at higher concentrations. Such changes in the 1H NMR spectra were not observed in DMSO-d6 at different concentrations, wherein the AB spin system at 8.06 and 7.97 ppm remained constant over all the concentration ranges studied. Also, in the case of tetravalent trans-Man2-AD-Man2 (15), trans-Lac2-AD-Lac2 (24), and trans-Lac-AD-COOH (25), such dependence was observed in D2O above a concentration of 12 mM. Changes in the 1H NMR resonances of the azobenzene protons were not observed with multivalent sugar clusters Man-AT-Man (16), Lac-AT-Lac (27), and Lac2-AT-Lac2 (28). The concentration-dependent chemical shifts of the trans isomers were completely absent in the cis form of all the azobenzene–sugar derivatives studied herein.
|
|
|
|
|
|
Isomerization studies
Photoisomerization studies of the azobenzene-containing glycoclusters were conducted in H2O by UV–visible spectroscopy. Photoirradiation of an aqueous solution of the sample (30–60 µM) with UV light (320–370 nm) led to isomerization of the trans isomer to the predominantly cis isomer containing photostationary state (PS) mixture (Figure 2). A decrease in absorbance (
330 nm) of the trans isomer was observed over 5–30 min and the PS was reached after 20 min. Based on the absorbance changes, 65–70% of the cis isomer was calculated for all the compounds. Thermal reversal of the cis- to the trans isomer under dark conditions was monitored at the 
max, and increase in the absorbance, which corresponds to the evolution of the trans form, was observed to be <5% even after 3 h. Thermal isomerization of the cis isomer to the trans isomer was followed by the time course measurement of the absorbance changes with aqueous solutions of Lac-AD-Lac (23), Lac2-AD-Lac2 (24), Lac-AD-COOH (25), and Lac2-AD-COOH (26) at different temperatures (50, 60, 65, 70°C). The isomerizations followed a first-order kinetics, and the corresponding rate constants for the isomerization are given in Table I. From isomerization kinetics, it was observed that the cis isomers were found to be more stable in aqueous solutions than in DMSO solutions. This increased stability of the cis isomers arise from the inherent increase in the polarity and the associated greater dipole moment of the cis isomers, and also from changes in the water of hydration around sugar units in the trans- and cis isomers. Also, it was observed that the cis isomers of the symmetrical derivatives Lac-AD-Lac (23) and Lac2-AD-Lac2 (24), bearing sugar moieties on either side, isomerized to the corresponding trans isomers relatively faster than the unsymmetrical derivatives Lac-AD-COOH (25) and Lac2-AD-COOH (26).
|
|
Molecular modeling
Molecular modeling studies were performed to visualize the energy minimized molecular conformations of the glycoside clusters in trans- and cis geometries. The minimized structures of these molecules had shown a flat and distinct 
surface for the trans geometry with the dihedral angle for the (azo)C–N=N–C(azo) around 180º and the distance of 9.1 Å between 4 and 4' carbons of the azobenzene unit. The corresponding distances for the cis isomers were found to be in the range of 6.01–7.48 Å, and the dihedral angles in the range of 5.3–6.5°. Figure 3 shows the space filled energy minimized models for the derivative Lac-AD-Lac (23) in the trans and cis geometries. For Lac-AD-Lac (23), the distance between the anomeric oxygens on either side of the trans and cis isomers was 20.8 and 8.1 Å, respectively. In the case of Man-AD-Man (18), the distance between the anomeric oxygens was found to be 18.7 Å in the trans form and 10.8 Å in the cis form. The distances between the anomeric oxygens at the reducing end of the sugar residue in octavalent Lac2-AT-Lac2 (32) on either side of azobenzene were found to be in the range of 17.1–22.6 and 6.7–14.5 Å for the trans and cis forms, respectively.
|
Lectin-binding studies
The important studies relating to the assessment of the differential binding affinities of the trans and cis isomers were performed through isothermal titration calorimetry (ITC). Evaluation by this technique offers information about the thermodynamics of the binding reaction of the lectin with the trans- and cis-isomeric forms in a quantitative manner. Two lectins, namely, peanut agglutinin (PNA) and concanavalin A (Con A), were employed for the ITC studies, as these lectins are known to be the high affinity receptors for the lactose and mannopyranoside ligands. The azobenzene–ß-D-galactopyranosyl-(1
4)-ß-D-glucopyranoside derivatives 23–28 were evaluated for their binding propensities with PNA. PNA is a homotetrameric protein with a molecular weight of 110 kDa. Crystal structure and solution state studies of the complexes of the tetrameric PNA with lactose and methyl ß-D-galactopyranoside have revealed one saccharide-binding site per monomer (Banerjee et al., 1996; Ravishankar et al., 1999; Reddy et al., 1999). On the other hand, the azobenzene–-D-mannopyranoside derivatives 14–16 were evaluated for their binding affinities with Con A. Con A is a legume lectin, it exists either as a dimer or as a tetramer depending on the pH of the solution, and it binds specifically to mannose residues (Goldstein and Poretz, 1986). Con A–mannopyranoside interactions have been studied and characterized by different techniques (Goldstein and Hayes, 1978; Bhattacharyya and Brewer, 1986; Dam et al., 2000). Crystal structures are available for Con A, both with and without bound mannopyranosides (Becker et al., 1975; Hardman and Ainsworth, 1976). In addition to a wealth of structural information, thermodynamic studies have been carried out on this particular sugar–lectin interactions (Mandal et al., 1994). ITC studies were carried out with a solution of the azobenzene–sugar derivative in which azobenzene exists either in the trans geometry or in the cis geometry predominating PS mixture. Although, we were unable to obtain completely cis-constituted PS, the overall differences in the binding between the trans isomer and the PS could be attributed to the presence of the predominantly cis form. In a typical experiment, a solution of the sugar derivative was titrated into a solution containing PNA, and the heat released or absorbed during the binding event was measured as a function of the [Sugar]/[PNA] molar ratio. The magnitude of heat release decreased progressively with each subsequent injection until complete saturation is achieved. ITC data were then fitted by using a nonlinear least square algorithm with three independent variables, namely, number of ligand-binding sites in the molecule (n), the enthalpy of the binding reaction (
H), and the binding constant (Kb) on the model based on identical and independent binding sites.
Study of the binding affinities of ß-D-galactopyranosyl-(14)-ß-D-glucopyranoside derivatives with PNA
The ITC studies were carried out separately on the trans and PS mixtures of derivatives Lac-AD-Lac (23), Lac2-AD-Lac2 (24), Lac2-AD-COOH (26), Lac-AT-Lac (27), and Lac2-AT-Lac2 (28). Owing to low solubility of Lac-AD-COOH (25) in aqueous solution, the ITC studies could not be carried out on this compound. As a comparison, binding studies were performed on ß-D-galactopyranosyl-(14)-ß-D-glucopyranose (lactose) (Reddy et al., 1999). The thermodynamic parameters obtained from the ITC studies for the binding of the above ligands to PNA is presented in Table II. In the case of the divalent Lac2-AD-COOH (26), the n value was close to 0.5 in both the trans and the PS mixture as expected, and the binding constants (per bound sugar unit) were about >10 times to that obtained for lactose alone. The H and TS values have doubled nearly, indicating the interaction involving two sugar units in Lac2-AD-COOH (26). Further, there was no noticeable difference in binding behavior of both the trans states and the PS of Lac2-AD-COOH (26). In case of the bivalent Lac-AD-Lac (23), a biphasic-binding profile was observed upon the interaction of this ligand with PNA (Srinivas et al., 2002). With tetravalent Lac2-AD-Lac2 (24), the biphasic-binding profile was observed only with the cis-predominating PS mixture. A two site binding equation was used to fit the binding profile (Figure 4) of Lac2-AD-Lac2 (24). The sum of n values in PS mixture of Lac2-AD-Lac2 was 0.27, and this value confirmed the involvement of all the sugar units in binding to the lectin. The tetravalent Lac-AT-Lac (27) showed an n value close to 0.3 upon binding to the lectin. This deviation from the expected value of 0.25 suggests that all the sugar units in this ligand did not participate in the binding reaction. The sugar residues in Lac-AT-Lac (27) are relatively more spatially dispersed when compared with Lac2-AD-Lac2 (24), and also because all the sugar residues did not participate in the binding event, the binding constants for both isomeric forms of Lac-AT-Lac (27) were found to be lesser than that of Lac2-AD-Lac2 (24). It should be mentioned that in all cases of Lac-AD-Lac (23), Lac2-AD-Lac2 (24), Lac2-AD-COOH (26), and Lac-AT-Lac (27), the binding constants per bound sugar unit increased several times when compared with constituent monomeric sugar alone. The binding profile of the octavalent Lac2-AT-Lac2 (28) to PNA (Figure 5) could not be fit satisfactorily to obtain the thermodynamic-binding parameters within expected error limits. An examination of the thermogram of 28 highlighted the pronounced cooperativity involved in the interaction. This pronounced cooperativity in the interaction precludes a reliable quantitative extraction of the binding parameters. We observe that the binding enthalpies of multivalent Lac2-AD-Lac2 (24), Lac2-AD-COOH (26), and Lac-AT-Lac (27) to the lectin do not increase linearly with increasing number of sugar residues. This is in contrast to the earlier observations that H increased linearly with the increasing number of residues in the binding of trimannopyranoside clusters to Con A and Dioclea Grandiflora lectins (Dam et al., 1998). More importantly, the observed biphasic-binding profile of the designed sugar ligand clusters Lac-AD-Lac (23) and Lac2-AD-Lac2 (24) to the lectin is anomalous. The biphasic-binding profile denotes the existence of a cooperativity, and this cooperativity during binding process is much more pronounced with the cis isomer than the trans isomer. We have also observed that contents of the calorimeter cell eventually showed some cloudiness after the ITC experiments were over. It has been observed previously that polyvalent carbohydrate ligands with two or more sugar units can form ordered cross-linked complexes, wherein the high affinity binding occurs due to simultaneous binding of the carbohydrate and cross-linked system (Bhattacharyya and Brewer, 1986). The estimation of the distances between the sugar units indicates that it is not possible for each sugar unit in a molecule to bind adjacent binding pockets of the tetrameric lectin, as the least separation between the binding sites in the lectin is about 70 Å, far above the distances between sugar units in the order of about 20–25 Å, in the trans isomers. Thus, cross-linking rather than a chelation (Fan et al., 2000; Kitov et al., 2000) is the mechanism of binding, which leads to enhancements in binding affinities. It should be noted that the ITC technique measures the thermodynamics of binding behavior of the soluble complexes during the course of the experiment and is unaffected by the competing kinetic factors of aggregation and precipitation (Dimick et al., 1999). It is to be mentioned that azobenzene di- and tetracarboxylates by themselves did not bind to the lectin (at 100 µM concentration).
|
|
|
Study of binding properties of azobenzene–-D-mannopyranoside with Con A
Isothermal titration experiments were carried out with dimeric Con A (pH 5.2). The thermodynamic parameters of the binding reaction of Con A with azobenzene–-mannopyranoside derivatives Man-AD-Man (14), Man2-AD-Man2 (15), and Man-AT-Man (16) are presented in Table III. The bivalent Man-AD-Man (14) exhibited an n value of 0.56, suggesting the involvement of both the sugar units to bind to the lectin. The corresponding PS mixture of Man-AD-Man (14) exhibited marginally better binding (Kb = 3.2 x 104 M–1), when compared with the corresponding trans form (Kb = 2.7 x 104 M–1). The n value observed for the binding of trans-Man2-AD-Man2 (15) with Con A was 0.39 and not the expected value of 0.25. This implies that all four mannose moieties present in Man2-AD-Man2 (15) did not participate in the binding event. On the other hand, the corresponding cis-enriched PS mixture exhibited a biphasic-binding profile in its binding reaction with Con A (Figure 6). The second part of the binding reaction provided a significantly enhanced binding constant (Kb2 = 6.81 x 105 M–1), when compared with the first binding reaction (Kb1 = 8.38 x 104 M–1). The binding constants observed for the binding of trans-Man-AT-Man (16) to Con A was 6.73 x 104 M–1, and the PS mixture exhibited better binding profile to Con A (Kb = 6.99 x 104 M–1). The n values, however, for the binding reaction of both the trans isomer and PS mixtures were found to be close to 0.33, which suggests that the binding of these ligands with Con A predominantly involved only three of the four mannose residues. The multivalent glycoside cluster effect in the carbohydrate–lectin interactions is caused by favorable enthalpy change based on multiple binding and entropic gain due to the preorganization of a carbohydrate unit (Mandal et al., 1994; Dam and Brewer, 2002). Azobenzene appended clustered carbohydrate ligands further afford flexibility for the modulation of geometry of the sugar ligands at the binding site of lectin. Thus, an important feature of these azobenzene–glycopyranoside derivatives studied herein is that these derivatives offer induced clustering (trans to cis isomerization), in addition to the already existing inherent clustering (the molecular feature).
|
|
Several groups have investigated the tight lectin-binding through multivalency, and many ligands with valencies ranging from 2 to 30 have been prepared for binding to a variety of lectins (Kiessling and Pohl, 1996; Mortell et al., 1996; Strenge et al., 1998; Horan et al., 1999; Frison et al., 2002; Lundquist and Toone, 2002; Thobhani et al., 2003). The true cluster effect has been observed early with the asialoglycoprotein receptor (ASGPr), primarily as a result of an exact geometric complementarity between the binding sites of the trimeric ASGPr and the triantennary oligosaccharide ligands (Lee et al., 1983; Lee, 1992). The cooperativity, which we have observed with Lac-AD-Lac (23), Lac2-AD-Lac2 (24), and Man2-AD-Man2 (15), results in the affinity enhancement of the binding of the sugar ligand with the lectin after the initial binding event. This "cooperativity" phenomenon had not been observed previously in small glycocluster–lectin-binding processes. Invariably the cooperativity was enhanced more with the cis isomer, than with the trans isomer of these azobenzene–glycocluster derivatives. Although it becomes clear now that a biphasic-binding profile, indicating a cooperative binding process, is possible for small glycoclusters–lectin interactions, the origin of the observed cooperativity itself is not clear. Qualitatively, it appears that an asymmetry with which the sugar is presented during lectin binding, as well as, a secondary structural interaction through the azobenzene core unit are likely to be the factors, leading to the cooperativity. This observation is significant and adds up as yet another facet of multivalent sugar ligand–protein interactions.
|
Conclusions |
|---|
|
TopAbstractIntroductionResults and discussionConclusionsSupplementary dataAcknowledgmentsReferences |
|---|
A general synthetic route to azobenzene–glycopyranoside derivatives with varying number of ß-D-galactopyranosyl-(1
4)-ß-D-glucopyranose and -D-mannopyranose moieties has been accomplished, and this strategy should allow the attachment of other biologically significant carbohydrates to study their binding behavior to relevant lectin receptors. Characterization by 1H, 13C NMR spectroscopic, and mass spectrometric techniques such as MALDI-TOF and ESI-MS have proved the constitution and structural integrity of the azobenzene–glycopyranoside derivatives. Molecular modeling studies allowed visualization of the overall changes in the geometry of the isomeric forms of these derivatives. The rate constants for the thermal cistrans isomerization have shown that the cis-isomeric forms of these derivatives are more stable in water than in an organic solvent. ITC investigations of the lectin-binding propensities of both the trans and cis isomer enriched PS mixtures of these derivatives have given insight into the binding of these photoswitchable clusters with the lectins. These azobenzene-appended sugar derivatives show high affinity binding with the relevant lectins, with an overall increase in binding affinity of several times relative to the monomeric sugar ligand alone. In general, isomerization of the cis form leads to better binding with the lectin than the trans form. The most significant observation during these studies is the biphasic-binding profiles of bivalent derivative Lac-AD-Lac (23), tetravalent Lac2-AD-Lac2 (24) (PS mixture) with PNA, and tetravalent Man2-AD-Man2 (15) (PS mixture) with Con A, corresponding to the existence of a cooperative nature of the binding process. Valency, spatial orientation, and geometry of the sugar ligands appear to be the prerequisites for the cooperative binding behavior to occur. |
Supplementary data |
|---|
|
TopAbstractIntroductionResults and discussionConclusionsSupplementary dataAcknowledgmentsReferences |
|---|
Supplementary data are available at Glycobiology online (http://glycob.oupjournals.org).
|
Acknowledgments |
|---|
|
TopAbstractIntroductionResults and discussionConclusionsSupplementary dataAcknowledgmentsReferences |
|---|
We are grateful to Department of Science and Technology, New Delhi, for the financial support. We thank the Sophisticated Instrumentation Facility and SERC of I.I.Sc., for the high field NMR experiments and computational facilities, respectively. O.S. thanks University Grants Commission, New Delhi, for a research fellowship.
|
Abbreviations |
|---|
AD, azobenzene-4,4'-dicarboxylic acid; AT, azobenzene-3,3',5,5'-tetracarboxylic acid; Con A, concanavalin A; DMSO, dimethylsulfoxide; ITC, isothermal titration calorimetry; NMR, nuclear magnetic resonance; PNA, peanut agglutinin; PS, photostationary state; THF, tetrahydrofuran
|
References |
|---|
|
TopAbstractIntroductionResults and discussionConclusionsSupplementary dataAcknowledgmentsReferences |
|---|
Ameerunisha, S. and Zacharias, P.S. (1995) Characterization of simple photoresponsive systems and their applications to metal ion transport. J. Chem. Soc., Perkin Trans., 2, 1679–1682.
Anzai, J.-I. and Osa, T. (1994) Photosensitive artificial membranes based on azobenzene and spirobenzopyran derivatives. Tetrahedron, 50, 4039–4070.[CrossRef]
Aoi, K., Itoh, K., and Okada, M. (1995) Globular carbohydrate macromolecules ‘sugar balls’. 1. Synthesis of novel sugar-persubstituted poly (amido amine) dendrimers. Macromolecules, 28, 5391–5393.[CrossRef]
Balasubramanian, D., Subramani, S., and Kumar, C. (1975) Modification of a model membrane structure by embedded photochrome. Nature, 254, 252–254.[CrossRef][Medline]
Banerjee, R., Das, K., Ravishankar, R., Suguna, K., Surolia, A., and Vijayan, M. (1996) Conformation, protein–carbohydrate interactions and a novel subunit association in the refined structure of peanut lectin-lactose complex. J. Mol. Biol., 259, 281–296.[CrossRef][Web of Science][Medline]
Becker, J.W., Reeke, G.N. Jr., Wang, J.L., Cunningham, B.A., and Edelman, G.M. (1975) The covalent and three-dimensional structure of concanavalin A. III. Structure of the monomer and its interactions with metals and saccharides. J. Biol. Chem., 250, 1513–1524.
Bhattacharyya, L. and Brewer, C.F. (1986) Precipitation of concanavalin A by a high mannose type glycopeptide. Biochem. Biophys. Res. Commun., 137, 670–674.[CrossRef][Web of Science][Medline]
Crocker, P.R. and Feizi, T. (1996) Carbohydrate recognition systems: functional triads in cell–cell interactions. Curr. Opin. Struct. Biol., 6, 679–691.[CrossRef][Web of Science][Medline]
Dam, T.K. and Brewer, C.F. (2002) Thermodynamic studies of lectin–carbohydrate interactions by isothermal titration calorimetry. Chem. Rev., 102, 387–430.[CrossRef][Web of Science][Medline]
Dam, T.K., Oscarson, S., and Brewer, C.F. (1998) Thermodynamics of binding of the core trimannoside of asparagine-linked carbohydrates and deoxy analogs to Dioclea grandiflora lectin. J. Biol. Chem., 273, 32812–32817.
Dam, T.K., Roy, R., Das, S.K., Oscarson, S., and Brewer, C.F. (2000) Binding of multivalent carbohydrates to concanavalin A and Dioclea grandiflora lectin. Thermodynamic analysis of the "multivalency effect." J. Biol. Chem., 275, 14223–14230.
Delaire, J.A. and Nakatani, K. (2000) Linear and nonlinear optical properties of photochromic molecules and materials. Chem. Rev., 100, 1817–1846.[CrossRef][Web of Science][Medline]
Dimick, S.M., Powell, S.C., McMahon, S.A., Moothoo, D.N., Naismith, J.H., and Toone, E.J. (1999) On the meaning of affinity: Cluster glycoside effects and concanavalin A. J. Am. Chem. Soc., 121, 10286–10296.[CrossRef]
Drickamer, K. (1995) Multiplicity of lectin–carbohydrate interactions. Nat. Struct. Biol., 2, 437–439.[CrossRef][Web of Science][Medline]
Dwek, R.A. (1996) Glycobiology: toward understanding function sugars. Chem. Rev., 96, 683–720.[CrossRef][Web of Science][Medline]
Erlanger, B.F. (1980) Models of photoregulation. Trends Biochem. Sci., 5, 110–112.
Fan, E., Zhang, Z., Minke, W.E., Hou, Z., Verlinde, C.L.M.J., and Hol, W.G.J. (2000) High-affinity pentavalent ligands of Escherichia coli heat-labile enterotoxin by modular structure-based design. J. Am. Chem. Soc., 122, 2663–2664.[CrossRef]
Fenderson, B.A., Zehavi, U., and Hakomori, S. (1984) A multivalent lacto-N-fucopentaose III-lysyllysine conjugate decompacts preimplantation mouse embryos, while the free oligosaccharide is ineffective. J. Exp. Med., 160, 1591–1596.
Frison, N., Marceau, P., Roche, A.C., Monsigny, M., and Mayer, R. (2002) Oligolysine-based saccharide clusters: synthesis and specificity. Biochem. J., 368, 111–119.[CrossRef][Web of Science][Medline]
Goldstein, I.J. and Hayes, C.E. (1978) The lectins: carbohydrate-binding proteins of plants and animals. Adv. Carbohydr. Chem. Biochem., 35, 127–340.[Medline]
Goldstein, I.J. and Poretz, R.D. (1986) The Lectins, Properties, Functions, and Applications in Biology and Medicine. Academic Press, NY.
Hansen, H.C., Haataja, S., Finne, J., and Magnusson, G. (1997) Di-, tri-, and tetravalent dendritic galabiosides that inhibit hemagglutination by streptococcus suis at nanomolar concentration. J. Am. Chem. Soc., 119, 6974–6979.[CrossRef]
Hardman, K.D. and Ainsworth, C.F. (1976) Structure of the concanavalin A-methyl alpha-D-mannopyranoside complex at 6-A resolution. Biochemistry, 15, 1120–1128.[CrossRef][Medline]
Horan, N., Yan, L., Isobe, H., Whitesides, G.M., and Kahne, D. (1999) Nonstatistical binding of a protein to clustered carbohydrates. Proc. Natl. Acad. Sci. U. S. A., 96, 11782–11786.
Jayaraman, N., Nepogodiev, S.A., and Stoddart, J.F. (1997) Synthetic carbohydrate-containing dendrimers. Chem. Eur. J., 3, 1193–1199.
Kempen, H.J., Hoes, C., van Boom, J.H., Spanjer, H.H., de Lange, J., Langendoen, A., and van Berkel, T.J. (1984) A water-soluble cholesteryl-containing tris-galactoside: synthesis, properties, and use in directing lipid-containing particles to the liver. J. Med. Chem., 27, 1306–1312.[CrossRef][Web of Science][Medline]
Kiessling, L.L. and Pohl, N.L. (1996) Strength in numbers: non-natural polyvalent carbohydrate derivatives. Chem. Biol., 3, 71–77.[CrossRef][Web of Science][Medline]
Kitov, P.I., Sadowska, J.M., Mulvey, G., Armstrong, G.D., Ling, H., Pannu, N.S., Read, R.J., and Bundle, D.R. (2000) Shiga-like toxins are neutralized by tailored multivalent carbohydrate ligands. Nature, 403, 669–672.[CrossRef][Medline]
Kunitake, T., Okahata, Y., Shimomura, M., Yasunami, S., and Takarabe, K. (1981) Formation of stable bilayer assemblies in water from single-chain amphiphiles. Relationship between the amphiphile structure and the aggregate morphology. J. Am. Chem. Soc., 103, 5401–5413.[CrossRef]
Lee, Y.C. (1992) Biochemistry of carbohydrate–protein interaction. Faseb. J., 6, 3193–3200.[Abstract]
Lee, R.T. and Lee, Y.C. (1994) Neoglycoconjugates: Preparation and Applications. Academic Press, San Diego, CA.
Lee, Y.C. and Lee, R.T. (1995) Carbohydrate–protein interactions: basis of glycobiology. Acc. Chem. Res., 28, 321–327.[CrossRef][Web of Science]
Lee, Y.C., Townsend, R.R., Hardy, M.R., Lonngren, J., Arnarp, J., Haraldsson, M., and Lonn, H. (1983) Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features. J. Biol. Chem., 258, 199–202.
Lindhorst, T.K. and Kieburg, C. (1996) Glycocoating of oligovalent amines: synthesis of thiourea-bridged cluster glycosides from glycosyl isothiocyanates. Angew. Chem. Int. Ed. Engl., 35, 1953–1956.[CrossRef]
Lundquist, J.J. and Toone, E.J. (2002) The cluster glycoside effect. Chem. Rev., 102, 555–578.[CrossRef][Web of Science][Medline]
Mammen, M., Choi, S.K., and Whitesides, G.M. (1998) Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew. Chem. Int. Ed. Engl., 37, 2754–2794.[CrossRef]
Mandal, D.K., Kishore, N., and Brewer, C.F. (1994) Thermodynamics of lectin–carbohydrate interactions. Titration microcalorimetry measurements of the binding of N-linked carbohydrates and ovalbumin to concanavalin A. Biochemistry, 33, 1149–1156.[CrossRef][Medline]
Mortell, K.H., Gingras, M., and Kiessling, L.L. (1994) Synthesis of cell agglutination inhibitors by aqueous ring-opening metathesis polymerization. J. Am. Chem. Soc., 116, 12053–12054.[CrossRef]
Mortell, K.H., Weatherman, R.V., and Kiessling, L.L. (1996) Recognition specificity of neoglycopolymers prepared by ring-opening metathesis polymerization. J. Am. Chem. Soc., 118, 2297–2298.[CrossRef]
Räu, H. (1990) Studies in Organic Chemistry: Photochromism, Molecules and Systems. Elsevier, Amsterdam, The Netherlands.
Ravishankar, R., Suguna, K., Surolia, A., and Vijayan, M. (1999) Structures of the complexes of peanut lectin with methyl-beta-galactose and N-acetyllactosamine and a comparative study of carbohydrate binding in Gal/GalNAc-specific legume lectins. Acta. Crystallogr. D Biol. Crystallogr., 55, 1375–1382.[CrossRef][Medline]
Reddy, G.B., Srinivas, V.R., Ahmad, N., and Surolia, A. (1999) Molten globule-like state of peanut lectin monomer retains its carbohydrate specificity. Implications in protein folding and legume lectin oligomerization. J. Biol. Chem., 274, 4500–4503.
Roy, R. and Laferrière, C.A. (1990) Michael addition as the key step in the syntheses of sialyloligosaccharide-protein conjugates from N-acryloylated glycopyranosylamines. J. Chem. Soc., Chem. Commun., 1709–1710.
Sharon, N. and Lis, H. (1989) Lectins as cell recognition molecules. Science, 246, 227–234.
Spaltenstein, A. and Whitesides, G.M. (1991) Polyacrylamides bearing pendant.alpha.-sialoside groups strongly inhibit agglutination of erythrocytes by influenza virus. J. Am. Chem. Soc., 113, 686–687.[CrossRef]
Srinivas, O., Mitra, N., Surolia, A., and Jayaraman, N. (2002) Photoswitchable multivalent sugar ligands: synthesis, isomerization, and lectin binding studies of azobenzene-glycopyranoside derivatives. J. Am. Chem. Soc., 124, 2124–2125.[CrossRef][Web of Science][Medline]
Strenge, K., Schauer, R., Bovin, N., Hasegawa, A., Ishida, H., Kiso, M., and Kelm, S. (1998) Glycan specificity of myelin-associated glycoprotein and sialoadhesin deduced from interactions with synthetic oligosaccharides. Eur. J. Biochem., 258, 677–685.[Web of Science][Medline]
Stromberg, N., Nyholm, P.G., Pascher, I., and Normark, S. (1991) Saccharide orientation at the cell surface affects glycolipid receptor function. Proc. Natl. Acad. Sci. U. S. A., 88, 9340–9344.
Thobhani, S., Ember, B., Siriwardena, A., and Boons, G.J. (2003) Multivalency and the mode of action of bacterial sialidases. J. Am. Chem. Soc., 125, 7154–7155.[CrossRef][Web of Science][Medline]
Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology, 3, 97–130.
Wang, S. and Advincula, R.C. (2001) Design and synthesis of photoresponsive poly (benzyl ester) dendrimers with all-azobenzene repeating units. Org. Lett., 3, 3831–3834.[CrossRef][Web of Science][Medline]
Whitten, D.G., Wildes, P.D, Pacific, J.G., and Irick, G. Jr. (1971) Solvent and substituent on the thermal isomerization of substituted azobenzenes. Flash spectroscopic study. J. Am. Chem. Soc., 93, 2004–2008.[CrossRef]
Willner, I. (1997) Photoswitchable biomaterials: en route to optobioelectronic systems. Acc. Chem. Res., 30, 347–356.
Yi, D., Lee, R.T., Longo, P., Boger, E.T., Lee, Y.C., Petri, W.A. Jr., and Schnaar, R.L. (1998) Substructural specificity and polyvalent carbohydrate recognition by the Entamoeba histolytica and rat hepatic N-acetylgalactosamine/galactose lectins. Glycobiology, 8, 1037–1043.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||