Sebastian G.
Spain
*a and
Neil R.
Cameron
*b
aSchool of Pharmacy, Boots Science Building, University of Nottingham, Nottingham, NG7 2RD, UK. E-mail: sebastian.spain@nottingham.ac.uk; Tel: +0115 8466241
bDepartment of Chemistry, University of Durham, University Science Laboratories, South Road, Durham, DH1 3LE, UK. E-mail: n.r.cameron@durham.ac.uk; Tel: +0191 3342008
First published on 4th April 2011
Mono- and polyvalent galactosides have been investigated with respect to their binding to the plant lectin Ricinus communis agglutinin 120 (RCA120). Thermodynamic parameters (Ka, ΔG, ΔH, ΔS and n) have been determined by isothermal titration calorimetry (ITC) and kinetics of binding (ka and kd) measured by surface plasmon resonance (SPR). ITC analysis using a single set of sites model found a non-statistical increase in avidity with increasing valency with the largest ligand displaying a greater than 20-fold increase in Ka compared to its monomeric precursor after correction for valency; binding was found to be enthalpically driven. SPR analysis supports the avidity increase but values of Ka observed were up to 100-fold greater than those measured by ITC. The large discrepancy between the two measurements is rationalized by the polyvalent–polyvalent interaction that is measured by SPR.
To further investigate the cluster glycoside effect, access to multivalent ligands is required. Multivalent glycoconjugates based upon dendrimers, lipids (e.g. micelles and liposomes) and nanoparticles have been investigated with regard to their lectin binding but are often, in the case of dendrimers, difficult to synthesise or, for lipids and nanoparticles, their structures are ill-defined making elucidation of structure–activity relationships, and subsequent mechanistic derivation, complex. Glycopolymers are a family of glyconjugates based upon synthetic polymer backbones that are of interest as drug-delivery vehicles and macromolecular therapeutics due to their ability to access the cluster glycoside effect using relatively facile chemistry.8 Recent developments in polymer chemistry have allowed the synthesis of complex, polyvalent glycopolymers of defined valency and architecture;9,10 consequently they have been studied extensively with respect to lectin binding.11
Herein we describe the synthesis of a series of mono- and polyvalent galactoside ligands via reversible addition–fragmentation chain transfer (RAFT) polymerisation; their interactions with the plant lectin Ricinus communis agglutinin 120 by isothermal titration calorimetry and surface plasmon resonance and the implications of these with respect to the origins of the cluster glycoside effect.
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Fig. 1 Synthesis of the mono- and polyvalent ligands studied. Reaction conditions: i. 2-hydroxyethyl methacrylate, BF3·Et2O, DCM, sonication; ii. damp MeOH, K2CO3; iii. (4-cyanopentanoic acid)-4-dithiobenzoate, 4,4′-azo(4-cyanopentanoic acid), water/EtOH, 70 °C. |
Polyvalent ligands were synthesised by RAFT polymerisation of 1 in aqueous ethanol using (4-cyanopentanoic acid)-4-dithiobenzoate and 4-4′-azobis(4-cyanopentanoic acid) as the chain transfer agent (CTA) and initiator respectively; different valencies were targeted by varying the [monomer]/[CTA] ratio. Residual 1 and other low molecular weight impurities were removed by thorough dialysis against high purity water (MWCO 3.5 kDa). Molecular weights, polydispersity indices and hydrodynamic radii were determined using aqueous size exclusion chromatography and dynamic light-scattering; the average valency of each ligand was determined from the value of Mn (Table 1).
Ligand | M n a (kDa) | M w/Mna | Valency | D h b (nm) | D h c (nm) | l d (nm) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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a Measured using triple detection aqueous size exclusion chromatography. b Determined by dynamic light-scattering in HEPES buffer. c Determined by dynamic light-scattering in PBS. d Approximate fully extended chain length assuming a C–C bond length of 1.54 Å and a dihedral angle of 109.5°. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 | n/a | n/a | 1 | n/a | n/a | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
2 | 20.1 | 1.15 | 68 | 5.4 | 6.6 | 17.2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3 | 42.3 | 1.11 | 144 | 8.0 | 7.2 | 36.2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
4 | 56.6 | 1.10 | 193 | 23.4 | 30.8 | 46.6 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
5 | 11.0 | 1.10 | 37 | 4.6 | 5.2 | 9.3 |
The previous study on poly[2-(β-D-galactosyloxy)ethyl methacrylate] binding to PNA noted that the “single set of sites” model (SSoS) within the ITC software did not produce adequate fitting to derive thermodynamic parameters; a problem attributed to precipitation of polymer–protein aggregates formed upon binding.16 Instead they used a more complex model that introduced a second enthalpic term to allow for the heat change due to the precipitation. However, it should be noted that greatest enthalpic contribution for precipitation observed was for the monomeric ligand 1 which should be incapable of causing lectin precipitation through multiple binding and questions the validity of this model. Additionally RCA120 is approximately half the size of PNA and only has 2 carbohydrate binding sites (cf. 4 on PNA) making lectin cross-linking less likely and Brewer et al. have reported successful derivation of thermodynamic parameters with multivalent ligands using the SSoS model when the concentrations of ligand and lectin were such that no precipitation was observed.20, 21 Here, although a small quantity of precipitation was observed when the largest ligand was used, the SSoS model was found to fit adequately. Fig. 2 contains example raw calorimetry data, binding isotherm and curve of best fit for the binding between ligand 3 and RCA120. Despite the relatively poor quality of the raw data (Fig. 2A), baseline correction and processing within the software resulted in an isotherm with the expected sigmoidal shape and fitting resulted in a curve in close agreement to the data. As the RCA120 tetramer is known to have two identical binding sites19n was fixed at 2 for monomeric species to simplify fitting. The ITC data were also analysed using a modified Scatchard analysis previously described by Brewer et al.20,21 This methodology introduces an additional term for the functional valency of the ligand allowing the construction of Scatchard plots for multivalent ligands (see Materials and methods for details).
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Fig. 2 Raw (A) and processed (B) isotherms for the binding of polymeric ligand 3 to RCA120. |
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Fig. 3 Thermodynamic plots for the binding between galactoside ligands and RCA120. (A) Thermodynamic parameters; (B) Compensation plot (—) Linear fit. R2 = 0.999, slope = 1.06; (C) Valency corrected Kaversus ligand valency. |
Ligand | ΔHobsa (kJ mol−1) | ΔGobsa (kJ mol−1) | TΔSobsa (kJ mol−1) | K a/M−1 | β c | n d | 1/n | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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SSoSb | Scatchard | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
a As molarities rather than activities were used the values are reported as observed. b “Single set of sites” model. c Defined as the ratio of polyvalent and monovalent interaction.22 d Stoichiometry with respect to lectin, i.e. number of ligands per RCA120 tetramer. e n was fixed during curve fitting to allow calculation of K and ΔH. f Calculated from isotherm without subtraction of heats of dilution. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 | −42.4 | −22.5 | −19.9 | 8.74 × 103 | 7.19 × 103 | 1 | 2e | 0.5 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
2 | −159 | −34.8 | −124 | 1.24 × 106 | 1.06 × 106 | 140 | 0.224 | 4.46 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3 | −245 | −40.7 | −204 | 1.01 × 107 | 9.87 × 106 | 1155 | 0.255 | 3.92 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
4 | −391 | −43.7 | −347 | 3.69 × 107 | 3.58 × 107 | 4222 | 0.130 | 7.69 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3f | −242 | −40.1 | −202 | 1.07 × 107 | n/a | 1224 | 0.260 | 3.85 |
For the polyvalent ligands, enthalpically driven binding is contrary to both modelled behaviour for multivalent binding23 and that reported by Ambrosi et al.16 The enthalpic driving force is confirmed by the compensation plot (Fig. 3B) which is linear with a slope greater than unity (1.06 ± 0.02; R2 = 0.999). The difference in thermodynamic behaviour between the two lectin bindings is probably due to a combination of effects. Ambrosi et al. partly attributed the entropic factor to displacement of water from the protein surface through aggregation. As aggregation was only observed to a slight degree here less of an entropic factor is likely. Similar behaviour has been reported by Brewer et al. for lectin binding of dendritic multivalent carbohydrates when aggregation was absent.20,21,24 Ambrosi et al. also noted that thermodynamic parameters were heavily influenced by ionic strength of the buffer used in the titration. Higher concentrations of salts resulted in an increased enthalpic contribution and a reduced entropic contribution; Ka was also reduced dramatically. This was particularly prominent when tris buffer was used, instead of citrate, with the binding becoming entropically disfavoured. It should also be noted that, due to the high heat of ionisation of tris (ΔHion ∼ 46 kJ mol−1),25 values may not have been completely accurate. Here, phosphate-buffered saline was used for all experiments due to the high solubility of RCA120 in this solution and its heats of ionisation being relatively low (– 1 ≤ ΔHion ≤ 4 kJ mol−1 depending on ionic strength).25 The minimal effect of buffer ionisation is confirmed by comparison of the thermodynamic parameters calculated with and without correction for heats of dilution/mixing for ligand 3 (entries 3 and 5 in Table 2) confirming that the lack of entropic contribution is not due to buffer choice.
Entropic favourability would also be likely if lectin binding leads to dispersion of polymer aggregates. Glycopolymers have already been shown to self-associate in aqueous solution26 with association attributed to intermolecular hydrogen bonding and hydrophobic interactions by the polymer backbone. Dynamic light-scattering at the same concentration used for ITC (see Table 1 for hydrodynamic diameters and ESI† for distributions) showed no evidence of aggregation for ligands 2 and 3. Ligand 4 is larger than expected when compared to the other ligands having a hydrodynamic radius 4-fold greater than ligand 3 despite only a <50% increase in molecular weight. It is unclear if this is due to aggregation as there is no evidence of a smaller species consistent with the unimolecular, unaggregated polymer and analysis at a 10-fold lower concentration showed no change in hydrodynamic radius. Consequently the large difference in hydrodynamic radius for ligands 3 and 4 may instead be due to a change in solution conformation with increasing molecular weight. Although dispersion of possible aggregates during binding is still a possibility the apparent solution stability of 4 suggests that these effects may be negligible. Overall binding would be expected to be entropically disfavoured as a bound polymer will have fewer possible solution conformations and thus a reduced number of degrees of freedom. This is particularly true where ligands are binding to multiple lectin moieties which both restricts the degrees of freedom and reduces the number of discrete species in solution.
Interestingly, as molecular weight increases, the ratio ΔH/TΔS decreases and tends towards 1 (black squares in Fig. 3A). Similar behaviour has been noted in dendritic systems where ΔH was seen to scale approximately with valency, i.e. for a tetravalent system ΔH was approximately 4-fold that of the monovalent, but −TΔS became more positive disproportionately.27 This behaviour was attributed to the inability of a relatively rigid ligand to fill both binding sites on an individual lectin, instead binding one lectin for each ligand presented, resulting in a greater entropic cost. The same explanation is possible here as, although the ligands in question are relatively flexible molecules, the two binding sites on RCA120 are approximately 11 nm apart (measured with Jmol28,29 using the Protein Data Bank file 1RZO30). The hydrodynamic diameter and fully extended chain lengths of ligands 2–4 are given in Table 1. From these it may be possible for 3 and 4 to bridge both sites but to do so would come at great entropic cost due to the restriction of the degrees of freedom on the polymer's conformation; particularly ligand 3 which would be very extended. Consequently it may be more entropically favourable to bind a single site of multiple RCA120 molecules which is supported by the values of 1/n (the number of lectins per ligand). Unsurprisingly the stoichiometry of the interaction also varies considerably with valency. More interestingly there is a decrease in stoichiometry between ligands 2 and 3. This may be an artifact of imperfect fitting of the SSoS model and will be discussed in more detail (vide infra).
Ligand 4 (average valency 193) has an overall avidity over 4000-fold greater than monomeric ligand 1; on a per sugar basis this 22-fold increase is no less striking. The non-linear behaviour observed is similar to that demonstrated by Kiessling et al. who also found a limiting molecular weight after which avidity no longer increased; this may also be the case here but would require larger ligands to be synthesised and analysed.6,31 Kiessling et al. attributed the large increase to the larger ligands' ability to bridge binding sites on a single lectin.
Sensorgrams for the two ligands at various concentrations are shown in Fig. 4. The data from these were fitted within the BIAcore software using a single sites (1:
1 Langmuir Binding) model. For each polymer five consecutive concentrations were chosen from those analysed; 5 nM–1 μM for ligand 5 and 500 pM–50 nM for ligand 3. The highest concentrations were removed as the binding was too rapid to enable accurate fitting. For ligand 5, the lowest two concentrations were removed as the response was too weak to allow accurate fitting. Thermodynamic and kinetic parameters for these polymers are shown in Table 3. As was seen with the ITC experiment, there is a large increase in Ka with increased molecular weight, here a ∼4-fold increase in valency leads to a near 8-fold increase in avidity. The larger polymer also displays more than a 100-fold increase in Ka compared to the value measured by ITC (1.39 × 109 M−1cf. 1.01 × 107 M−1); an increase attributable to the now multivalent–multivalent interaction. The kinetic parameters for the two ligands are more interesting. Despite the large difference in Ka there is less than a 2-fold difference in the association rate constant, ka. Looking at this value alone it would be expected that these ligands would have similar avidities. However, ligand 5 has a value of kd 5-fold higher than that of ligand 3 and, consequently, has a far lower avidity (Ka = ka/kd).
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Fig. 5 Schematic of the possible binding mechanism between RCA120 and polyvalent galacotsides. |
The SPR data also supports a “bind and slide” type mechanism. Ligand 5 is too small to be able to bind both sites on RCA120 as its fully extended chain length is ≤10 nm. It is also unlikely that it could bind two neighbouring lectins on the surface as the modification density was relatively low. Consequently its high avidity for the lectin-coated surface can only arise from rapid association following a dissociation event resulting in a low overall dissocation rate. 3 may be able to bind two sites simultaneously but to do so would require a conformational change compared to its solution structure and the SPR “two state–conformational change” model was found to fit poorly to the data. Additionally, the increased avidity of 3cf.5 is dominated by a decreased off rate which is in agreement with a “bind and slide” type mechanism and an increased probability of rebinding. If the ligands are approximated to a deformable sphere then they can be considered to have a footprint that will be proportional to their radius. In the same manner, a tyre of a larger diameter will maintain a larger contact area with the road than a smaller one; assuming all other factors to be the same. Deformable spheres have been modelled to roll along ‘sticky’, i.e. interacting, surfaces,37,38 a phenomenon that has also been modelled and observed experimentally in leukocytes.39,40 An increased contact area during rolling will result in a greater probability of secondary binding events occuring, resulting in increased retention on the surface. Thus smaller polymers, with their reduced footprints will be shed more readily, increasing kd values.
Found C, 51.88; H, 6.18; C20H28O12 requires C, 52.17; H, 6.13. FT-IRν/cm−1: 1751 (CO of acetate groups) 1719 (C
O of methacrylate ester) 1637, 1320, 1298 (C
C). δH: (400 MHz, CDCl3) 1.98 (3H, s, 3 × H-3), 2.01, 2.04, 2.05, 2.15, (3H × 4, 4s, Ac × 4), 3.84 (1H, ddd, J6a, 6b 11.5 Hz, J5a, 6a 7.5 Hz, J5b, 6a 4.0 Hz, H-6a), 3.92 (1H, td, Jt = J5′, 6a′ = J5′, 6b′ 6.6 Hz, Jd = J4′, 5′ 1.1 Hz, H-5′), 4.06 (1H, ddd, J6a, 6b 11.6 Hz, J5b, 6b 5.9 Hz, J5a, 6b 3.7 Hz, H-6b), 4.10 (1H, dd, J6′a, 6′b 10.3 Hz, J5′, 6′a 6.7 Hz, H-6′a), 4.18 (1H, dd, J6′a, 6′b 10.9 Hz, J5′, 6′b 6.5 Hz, H-6′b), 4.29 (2H, m, H-5a,b), 4.55 (1H, d, J1′, 2′ 8.05 Hz, H-1′), 5.01 (1H, dd, J2′, 3′ 10.4 Hz, J3′, 4′ 3.6 Hz, H-3′), 5.23 (1H, dd, J2′, 3′ 10.4 Hz, J1′, 2′ 8.0 Hz, H-2′), 5.39 (1H, dd, J3′, 4′ 4.5 Hz, J4′, 5′ 1.2 Hz H-4′), 5.59–5.61 (1H, m, H-1 Z to Me–C
C), 6.13–6.14 (1H, m, H-1 E to Me–C
C). δC: (100.26 MHz, decoupled, 1H 400 MHz; CDCl3) 18.25 (C-3), 20.6, 20.8, 21.0 (4 × H3CCO2, 2 resonances overlap), 61.3 (C-6′), 63.5 (C-5), 66.8 (C-4′), 67.4 (C-6), 68.7 (C-2′), 70.7 (C-3′), 70.9 (C-5′), 101.3 (C-1′), 125.9 (C-1), 136.1 (C-2), 167.1 (C-4), 169.4, 170.2, 170.3 (MeCO2). LR-MS (ES+) m/z requires 483.4, found 483.2 (M + Na+).
Found C, 49.12; H, 6.94; C12H20O8 requires C, 49.31; H, 6.90. FT-IR (NaCl Plates) ν/cm−1: 3360 (br, OH), 1708 (CO of methacrylate ester) 1636, 1320, 1298 (C
C). δH: (500 MHz; D2O) 1.94 (3H, m, 3 × H-3), 3.48 (1H, dd, J2′, 3′ 9.7 Hz, J3′, 4′ 3.3 Hz, H-3′), 3.53 (2H, dt, Jt 5.09 Hz, Jd 5.7 Hz, H-5′), 3.74 (2H, m, H-6′) 3.84 (1H, m, H-4′) 3.86 (1H, ddd, J6a, 6b 11.8, J5b, 6b 6.0, J5b, 6a 3.8 Hz, H-6a) 4.12 (1H, ddd,J6a, 6b 11.6, J5b, 6b 5.9, J5a, 6b 3.7 Hz, H-6b) 4.29 (1H, d, 7.5, H-1′) 4.35 (2H, m, H-5) 5.64 (1H, m, H-1 Z to Me–C
C) 6.14 (1H, m, H-1 E to Me–C
C); δC: (100.62 MHz; decoupled 1H 500 MHz; D2O) 18.4 (C-3), 62.5 (C-6′), 65.3 (C-5), 68.5 (C-6), 70.3 (C-4′), 72.4 (C-2′), 74.9 (C-3′), 76.7 (C-5′), 103.2 (C-1′), 126.4 (C-1), 137.7 (C-2), 168.8 (C-4). LR-MS (ES+) m/z requires 315.3, found 315.1 (M + Na+).
δ H (500 MHz, D2O) 0.61–1.50 (3H, br, m CH3–C), 1.53–2.41 (2H, br, backbone CH2), 3.37–3.62, 3.62–3.72, 3.72–3.86, 3.86–4.02, 4.02–4.16, 4.16–4.36 (10H, protons of carbohydrate and methylene side chain), 4.44 (1H, br, anomeric proton), 7.52 (Hmeta, arom.), 7.70 (Hpara, arom.), 7.99 (Hortho, arom). Aromatic resonances from terminal dithiobenzoate moiety.
Xb(i) = [Q(i)/ΔH·V] + Xb(i − 1) | (1) |
Xf(i) = Xt(i) − Xb(i) | (2) |
In a normal Scatchard analysis, the average number of ligands bound per lectin (r(i)) is the ratio Xb(i)/Mt(i). The modified Scatchard analysis introduces an additional term to allow for multivalent ligands and thus:
![]() | (3) |
Polymer solutions (500 pM–10 mM) were prepared in the same HEPES buffer. Sensorgrams for each polymer concentration were recorded with a 180 s injection of polymer solution (on period) followed by 240 s of buffer alone (off period). After this time the chip was regenerated by a 30 s injection of methylβ-D-galactoside (1 mg mL−1) and 60 s of buffer alone. After regeneration was complete the next sample was injected. For each polymer 5 consecutive concentrations were used for kinetic analysis using a single set of sites (1:
1 Langmuir Binding) model in the BIAevalulation 4.1 software.
Footnote |
† Electronic Supplementary Information (ESI) available: Solution size distributions of polymeric ligands by dynamic light-scattering. See DOI: 10.1039/c1py00030f/ |
This journal is © The Royal Society of Chemistry 2011 |