Divalent cation interactions with a carboxylated derivative of scleroglucan

Macromol. Chem. Phys. 196,3979-3989 (1995) 3979

Divalent cation interactions with a carboxylated derivative of scleroglucan

Dedicated to the memory of Prof. Mario Farina

Marc0 Bosco a), Fabiana Sussich b), Amelia Gaminib), Edoardo Reisenhoferc), Giampiero Adamic), Roberto Rizzoa, b)*

a) Centro Ricerche POLY-bios, Area della Ricerca di Trieste, Padriciano 99, 34012 Tkieste, Italy

b, Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Universita di Trieste, Via Licio Giorgieri 1, 34127 Trieste, Italy

c, Dipartimento di Scienze Chimiche, UniversitA di Trieste, Via Licio Giorgieri 1 , 34127 Tkieste, Italy

(Received: March 20, 1995; revised manuscript of May 24, 1995)

SUMMARY Divalent cation binding was investigated on a semisynthetic carboxylate derivative

obtained from the fungal polysaccharide scleroglucan. Polarography, circular dichroism and isothermal microcalorimetry were used to study both the binding ability of Cu(II), Pb(II), and Zn(I1) ions and the macromolecular properties of the systems under investigation. A theoretical model, proposed by Schwarz for the cooperative binding, was used to evaluate binding constants and the parameter Q related to the degree of cooperativity. All findings are discussed in terms of the differences in the binding ability of the different cations investigated. Pb(I1) ions exhibit the higher affinity for oxidised scleroglucan and, in addition to this, its binding process is characterised by a cooperative behavior due to the onset of a disorder-to-order conformational transition of the polysaccharidic backbone. On the other hand, Cu(I1) ions are less effective in the binding and unable to induce the conformational transition.

Introduction

Investigation in polysaccharide chemistry has received growing attention in the last decades both for the well-established exploitation of polysaccharides in industry and for the advances in the understanding of their role in various biological processes 1,2).

In the past, the polysaccharides were mainly investigated because of their technological interest. In particular, besides the most exploited cellulose and starch, other plant polysaccharides (alginate, pectate, and carrageenans) were actively studied for their ion-induced gelling ability (for a comprehensive bibliography see ref. I)). Nowadays, an impressive number of papers on animal and bacterial polysaccharides is currently published for the relevance they could have in interesting applications, even in sectors where synthetic polymers still have a major role. The fact that such polymers are extracted from natural renewable sources makes them most suitable for the up-to-date policy of resources utilisation. In addition to this, it is worth noting that polysaccharides are generally considered as environmentally-friendly molecules.

0 1995, Huthig & Wepf Verlag, Zug CCC 1022-1 352/95/$10.00

3980 M. Bosco, F. Sussich, A. Gamini, E. Reisenhofer, G. Adami, R. Rizzo

Besides native polymers, semisynthetic polysaccharide derivatives are increasingly investigated with the aim of obtaining new polymers tailored for specific applications'). Until recently the chemistry carried out on the polysaccharidic backbones was able to give almost exclusively randomly derivatised polymers. Today much effort is going to be devoted to the tuning of reaction strategies which are able to give specific regioselective derivatisation and also mild enough to preserve the macromolecular nature of the modified molecules.

In two previous papers4p5) we described both the synthesis and the properties of a carboxylated derivative of agar, a galactan produced by some species of marine red macroalgae. The derivative was an 0-carboxymethylagar which exhibited a degree of substitution (DS) equal to 1.2 carboxymethyl groups per agar repeating unit (the maximum theoretical being DS = 4). The carboxylated compound obtained was tested for divalent cation complexation and it turned out to exhibit a certain degree of selectivity between different cations.

One problem connected with carboxymethylagar arose from the fact that the substitution of carboxymethyl groups was not regioselective so that substituents were almost randomly distributed on all possible substitution sites. This feature led to the formation of different binding sites for cation complexation, thus rendering the analysis of the structure-function relationships difficult to be performed.

To overcome this problem, we studied a carboxylated derivative of the fungal polysaccharide scleroglucan6*", already synthesized by Crescenzi et al. '3 9), which exhibits a regular distribution of carboxylic groups. In fact, the (I -+ 3)-8-~-Glcp main chain of scleroglucan was kept unchanged, whereas the glucose residue, present as lateral chain, was conveniently and regioselectively split in order to obtain two new carboxylic groups regularly distributed along the polysaccharidic backbone.

Experimental part

Scleroglucan was a kind gift from SANOFI BIO-INDUSTRIES, Paris, France (Actigum CS11, lot no. 662,6/92). The polymer was purified by means of precipitation with acetone, redissolution in water and dialysis. Pb(II), Cu(II), and Zn(I1) ions were always used in the form of perchlorate salts.

Oxidised scleroglucan was obtained according to Crescenzi et al. ') Oxidation reaction was carried out by means of an excess of sodium periodate on a 0.6% (w/v) scleroglucan solution (24 h, 4 "C, in the dark). After dialysis against water, the weak gel obtained was then treated with 0.15 M sodium chlorite in the presence of acetic acid for 24 h at room temperature under nitrogen atmosphere, in order to transform the dialdehyde into the carboxylic derivative.

NMR experiments were performed on a Bruker AC 200 spectrometer using 1s aquisition time and 16 kHz spectral width. Chemical shifts were referred to the 31.07 ppm resonance relative to methyl groups of aceton. Samples were dissolved in D,O and spectra were recorded at 80 "C. Molecular weight averages were determined by means of flow injection analysis using an LDC-Chromatix CMS-100 low angle laser light scattering photometer, equipped with a He-Ne laser ( A = 632.8 nm). For this purpose, measurements were performed in 0.15 M NaCI; the starting polymer concentration was 5.36 - lo-' g/L. All solutions were filtered on a 0.2 wm pore size membrane (Millipore, Millex FG5) before injection into the pump.

Divalent cation interactions with a carboxylated derivative of scleroglucan 3981

The concentrations of divalent cation standard solutions were always checked by means of complexometric titrations.

Polarographic experiments were carried out using a polarographic cell equipped with three electrodes: a dropping mercury electrode, a platinum wire auxiliary electrode and a saturated calomel reference electrode. The calomel electrode was connected to the measurement cell by means of a KNO, salt bridge in order to avoid contamination by chloride. In all measurements a 0.05 M NaCIO, buffer solution was used and all solutions, were used under a nitrogen flux. The experimental apparatus was purchased from Princeton Applied Research Corp. (Electrochemical System Mod. 170). A differential pulsed technique (modulation amplitude 50 mV * s- ' , scan rate 2 mV * s -', drop time 2 s) was used to'gain suitable accuracy on data obtained at very low concentration of free ions ([M"'] <

Circular dichroism measurements were carried out on a JASCO-600 spectropolarimeter using a quartz cell of 0.5 cm optical pathway. Polymer concentration was 1.35 * lo-, eq/L.

Isothermal microcalorimetry measurements were carried out at 25 "C by means of a batch LKB 10700 instrumentation equipped with gold cells. Polymer concentration was 1.30. eq/L.

In both circular dichroism and microcalorimetric measurements a 0.05 M NaCIO, buffer solution was used to dissolve both polymer and divalent cation salts.

F). Polymer concentration was 3.95 lo-, eq/L.

Results

The oxidation reaction conditions were aimed at the obtainment of the 100% oxidation. For this purpose, an excess of both sodium periodate and sodium chlorite was used. In order to avoid extensive polymer degradation during periodate oxidation, the reactor was kept at 4°C and in the dark. For the same purpose, the chlorite treatment was carried out under nitrogen atmosphere.

After the synthesis, the oxidised polysaccharide obtained was analysed to asses the chemical structure. Both lH and 13C NMR spectra were in very good agreement with structure 1, which was schematically proposed in ref.*) The 13C NMR spectrum of oxidised scleroglucan is shown in Fig. 1.

OH C

OH

1

Potentiometric titration were performed in order to figure out the degree of oxidation which turned out to be equal to 2, i. e. two carboxylic groups per scleroglucan repeating structure. Flow injection analysis coupled with low angle laser light scattering detection was used to establish the weight average molecular weight of the ionic derivative


C2(A*B*C1 CL1A.B.C) C2lDl I ,

CI(A.B)

. .

110 105 100 95 90 85 80 75 70 65 60 6 in ppm

Fig. 1. I3C NMR spectrum of oxidised scleroglucan with peak assignment. Symbols refer to labels in formula 1. The spectrum was obtained at 50.32 MHz in D,O and 80 "C

obtained. This analysis was performed in 0.15 M NaCl and the value obtained was MW = 260000 g * mol-'. As a comparison, the MW of the parent scleroglucan polysaccharide, measured in water where this polymer is known to assume a triple-strand conformation, is about 6 - lo6 "*"), whereas the value obtained in dimethyl sulfoxide (DMSO), where the same polymer assumes a random coil single-strand conformation, is 1.64 - lo6 lo). From the above findings, it follows that the oxidation reactions produce a certain degree of polymer degradation, but the macromolecular nature of the compound obtained is maintained, provided that we reasonably suppose that, even in water, the semisynthetic ionic compound is able to assume a single-strand conformation due to negative charge repulsions.

Polarographic, circular dichroism and isothermal microcalorimetric experiments were carried out in order to test the binding ability of the oxidised scleroglucan (hereafter referred as SCLOX). For this purpose, Cu(II), Pb(II), Zn(I1) ions were selected on the basis of preliminary polarographic experiments carried out on a larger number of ionic species.

Polarograpic data are shown in Fig. 2 where the amount of free cations (Mf& in the presence of a given amount of SCLOX, is reported as a function of the increasing amount of the cations added (MtOt). In this figure, the solid straight line represents the free ion concentration in the absence of SCLOX. As can be seen, all ionic species selected interact with the polymer, but lead ions show the highest degree of affinity. In fact, the amount of free Pb(I1) ions is very low up to a ratio ( R ) between the moles of the cations added and the equivalents of polymer equal to 0.5.

Cation interaction with SCLOX was further investigated by means of circular dichroism (CD). CD spectra can bring to evidence the perturbations induced by the bound cations on the electronic transitions of the carboxylate chromophores. Alternatively, as in the case of Pb(I1) ions which show a charge-transfer dichroic band at 230 nm, the circularly dichroic differential absorption is due to the perturbations


Fig. 2. Polarographic data reported as free [M2+] as a function of the total [M2+] added, without SCLOX (solid line) and in the presence of 3.95 *

polymer equiv./L: ( 0 )

Zn(II), ( 0 ) CW), Pb(I1)

0 1 2 3 L 5 6 7 8 9 10'. IMtotl/lmol-L?

induced by the chiral polymeric matrix on the electronic transitions of the bound cation.

CD data obtained on SCLOX in the presence of Cu(II), Pb(II), and Zn(I1) ions are shown in Fig. 3. In this figure the molar ellipticity, 0, at a given wavelength (220 nm for copper and zinc, 230 nm for lead ions, respectively) is reported as a function of the increasing amount of divalent cations (Mtot). As can be seen easily the binding curve relative to the copper ion addition exhibits a regular increasing behaviour which finally reaches a plateau. On the other hand, CD experiments carried out in the presence of

Fig. 3. Molar ellipticity variation for copper (V), zinc (M) and lead ( 0 ) addition in the presence of

polymer equiv./L). R represents the ratio of [Mz+] added (moles) to the polymer concentration (equivalents)

scmx (1.26.10-3

- - E -0 \ N

E

2500

2000

1500

1000

500

0

-500

-1000

-1500

-2000 0 0.25 0.50 3.00

R=IMtOtl/lpoll


Pb(I1) and Zn(I1) ions show a rather different trend. In fact, in both cases, binding curves exhibit a sigmoidal behaviour indicating a cooperative effect related to cation interaction. Unfortunately, a plateau cannot be reached for the Pb(I1) addition because of the occurrence of precipitation at an R ratio as high as 0.4. The cooperative effect observed can be safely traced back to the macromolecular nature of the binding matrix. As a matter of fact, it is known'2) that SCLOX can give rise to a disorder-to-order conformational transition as a function of either pH or ionic strength. Quite surprisingly, Cu(I1) ions seem to be unable to induce the conformational transition. This point will be thoroughly discussed later on in this paper. In this context, it has to be stressed that, according to Crescenzi et al.") the 0.05 M NaClO, ionic strength present in the buffer solution is unable to induce any conformational transition on SCLOX. Monovalent cations are effective only at an ionic strength higher than 0.5 M ' 2 ) .

Isothermal microcalorimetry data obtained for the mixing of Cu(II), Pb(II), and Zn(I1) ions with SCLOX are shown in Fig. 4. The heat of mixing, reported as a function of the increasing amount of divalent cations (M,,J resulted to be endothermic for both copper and zinc ions and exothermic for lead ions addition. On the one hand, the endothermic behaviour clearly suggests that the binding process is an entropy-driven one and, in the case of Zn ion addition, the rather large heat effect can disguise any other contribution, such as that due to the concomitant conformational transition occurrence as revealed by means of circular dichroism studies. On the other hand, calorimetric data obtained in the presence of lead ions exhibit the typical exothermic behaviour due to the conformational rearrangement, in good agreement with CD data''). In fact, at low Pb(I1) concentration the cation binding occurs with a slightly endothermic effect, but at higher lead ion concentration a major exothermic effect superimposes to the previous one. This effect is mainly due to those intramolecular

2000

g 1750

2 1500

- 1250

1000

750

500

250

0

-250

-500

-750

0

3

1 1 1 1 1 1 1 1 1 1 1 1

\* * . '+-I

Fig. 4. Mixing enthalpy of ( 0 ) Zn(II), (V Cu(II), and (W Pb(I1) ions with SCLoX(Cp = 5.2 equiv./L). R represents the ratio of [M2+] added (moles) to the polymer concentration (equivalents)

0 0.25 0.50 0.75 1.30 R=lM"1/Ipoll


energetic contributions which stabilise the ordered conformation (e. g. hydrogen- bonding formation).

Discussion

In order to extract more information from the above shown experimental results, the data were processed to evaluate the concentrations of both bound and free cations. Having this information, it is possible to use the mathematical model of cation binding accompanied by a conformational transition which was formerly introduced by Schwarz 14). This leads to the evaluation of both the binding constant and a parameter bound to the cooperativity of the investigated system. The model defines an equilibrium constant, K, for the binding to an isolated site (nucleation process) and a second constant, KI, for the binding of sites in the immediate neighbourhood of an already bound one (propagation process). The ratio between these two constants is defined as a = K/K* and represents the degree of cooperativity. According to this definition, the co-operative binding, where the propagation constant is much higher than the nucleation one, is characterised by a much lower than unity. Under these definitions, the application of a matrix algorithm leads to a general expression (Eq. 1) for the number of the occupied sites per mole of carboxylate groups as a function of the free metal cations.

(1) 1 (1 - K* * [Md) o n . 1 -

[ 1 / (I - K*’[MJ)2 + 4.a.K**[Mf] tMb1 - [poll 2

Since the stoichiometry of the complexation is not known “a prior?’, the concentration of binding sites has to be defined as the product of the concentration of carboxylate groups and a stoichiometric coefficient n, so that the fitting parameters are: n, K* and a. The best fitting procedure needs the knowledge of both the bound- and free-cation concentrations which can be obtained from the experiments.

Concerning the polarographic data, the concentration of free ions can be obtained directly from a calibration curve using a standard solution of known cation concentration, in the absence of the polymer or other complexing agents. In these experiments, the height of the polarographic peak is linearly proportional to the concentration of the cations which are free in solution. On the contrary, polarographic measurements carried out in the presence of the complexing polymer show peaks lower than those obtained in the calibration experiments since part of the added cations is no longer able to reduce onto the dropping electrode. The concentration of the bound cations is then easily obtained as the difference between the free cations concentration and the analytical one.

According to the model, polarographic experimental data are reported in Fig. 5 as the concentration o i bound cations (Mbound) as a function of the concentration of free cations @if&. As expected, the three ions behave differently. Lead ions interact pretty efficiently and cooperatively with SCLOX so that in the first steps of the binding process the amount of free ions is very low. Free ion concentration starts to be significantly higher only when a plateau is reached and the bound-ion concentration

3986 M. Bosco, F. Sussich, A. Gamini, E. Reisenhofer, G. Adami, R. Rivo

0.3

0.2

0.1

0

Fig. 5 . Bound ions vs. free ions plot obtained from the polarographic data. An enlargement of the plot for Pb(I1) ion addition is shown in the insert to better underline the cooperative behaviour. (0) Zn(I1); (m) Pb(I1); (V) Cu(11). Solid lines represent the results of the best fitting procedure

0 1 2 3 L 13 14 1O4-IM,,,J/1mol- L-')

remains constant. The value of the plateau corresponds to 0.5 moles of bound ions per mole of carboxylate groups, i. e. each Pb(I1) ion is able to bind two carboxylate groups. Very reasonably, both carboxylate groups belong to the same oxidised lateral chain and hydroxyl groups belonging either to the polysaccharidic chain or to the solvent are filling the remaining binding sites of the co-ordination sphere.

The same binding stoichiometry was found for Zn(I1) ions. However, in this case, the binding was less efficient than that observed in the presence of lead ions; as a matter of fact, the amount of free Zn(I1) cations was rather large even at low total cation concentration, and the plateau was reached much more slowly. In fact, the value of the plateau was obtained only after the best-fitting procedure using the above mentioned mathematical model.

A different situation is obtained in the presence of copper ions. Here, the binding is again less efficient than that obtained with lead ions, and, in addition to this, the plateau, as estimated from the plot, is characterised by a value near to 0.25 moles of bound copper ions per mole of carboxylate groups, i.e. each Cu(I1) ion binds 4 carboxylate groups. It is evident that such stoichiometry implies that two lateral chains, belonging either to the same polysaccharidic chain or to different chains, are complexed with copper ions. It has to be said, however, that the value of the plateau obtained after the best-fitting procedure is 0.32, which implies a stoichiometry between 1 : 2 and I : 4 cation-to-carboxylate groups. However, by performing very simple calculations, it is possible to evaluate that, even when using this plateau value, more than 70% of the bound copper ions assume a 1 : 4 stoichiometry.

The difference in the complexation stoichiometry, as obtained by polarographic data, suggests a possible explanation of the finding that, contrary to Pb(I1) and Zn(II), copper ions are unable to induce the SCLOX conformational transition. As a matter of fact, if we assume that two not-contiguous lateral chains are involved in the copper


complexation, the topolocigal constraints imposed on the chain stretches between the two complexation sites can inhibit the disorder-to-order conformational transition. It can be easily seen that such considerations equally hold if the two lateral chains belong either to the same polysaccharidic backbone or to different chains. In the latter case, one could expect that the interchain ion-bridges would eventually lead to polymer precipitation. This does not occur in the presence of Cu(I1) ions probably because the extent of ion complexation is not high enough to prevent polyanion negative charges self-repulsion and, in addition to this, the persistence of a disordered structure opens the system to solvent permeation so that the small aggregates still remain in the solution phase.

A plot of the bound ion as a function of free ion concentrations can also be obtained from the experimental data collected by means of circular dichroism. However, contrary to polarography, a direct estimation of either bound or free ion concentrations is not feasible. The situation gets even more complicated when, like in the case of copper and zinc ions addition, the observed changes in the CD spectra do not refer to the cation electronic transitions, but to those pertaining to the carboxylate chromophores. In this case, CD spectra are sensitive to the conformational changes of the polysaccharidic backbone and any correlation between the observed molar ellipticity, 0, and the concentration of the bound cations might be affected by errors. In addition to this, the cation binding process and the conformational transition might not proceed in a parallel way. For instance, binding can still occur when the conformational transition has already reached its end.

The discrepancies between polarographic and CD data are evidenced in Fig. 6, where both experimental results are reported for each divalent cation. On the vertical axis the fraction of the completeness of the process detected is reported independently from its nature, i. e. being them “cation binding” (as detected by means of polarography) or “polymeric chromophore perturbation” (as detected by means of circular dichroism) obtained upon divalent cation addition. The solid lines reported in the plots of Fig. 6 are the result of the best fitting procedure. It is clearly seen from the figure that polarographic and CD results superimpose pretty well in the case of Pb(I1) addition, where both techniques look at the “cation state”. On the contrary, concerning Zn(I1) addition, the differences are very large since polarographic response is proportional to the free-ion concentration whereas CD effects depend mainly on the conformational state of the polymeric chromophores.

As far as calorimetric data are concerned, the situation is even more critical because side effects can produce additive contributions to the enthalpy of mixing which prevent the use of the Schwarz model.

The fitting parameters n, K * , and 6, as obtained from the polarographic data, are reported in lhb. 1. The figures reported quantitatively confirm the experimental findings. As expected, the interaction of SCLOX with different divalent cations exhibits a character of selectivity. Crescenzi et al. E, have already shown that SCLOX is able to give a conformational transition in the presence of Ca(I1) ions. In this paper we showed that, among the three cations investigated, lead ions are the most efficiently bound by the carboxylate polysaccharide. In addition to that, a c parameter much lower than


2 100

g 80 W u

0, II

0

- 60 r

LO

2 20

0 0

C W

Q

c

:

100

80 s W II

0

- 60 r

2 LO C W

Q

c 2 20

0 0 E

0.4 0.8 3.6 L.0 R=lCul/Ipoll

g 100 W 0

0. 2 80 W

f 60 c 0

2 LO

z - 20 E

W C W

0.

0 0 0.4 0.8 3.2 3.1

R=IZnl/lpoll

Fig. 6. Percent of the completeness of the process (see text) as detected by means of polarography (0 ) and circular dichroism ( 0 ) . Solid lines represent the result of the best fitting procedure. R represents the ratio of [M”] added (moles) to the polymer concentration (equivalents)

unity indicates that the binding process is cooperative, due to the onset of a simultaneous conformational transition. On the contrary, the binding of either Cu(I1) or Zn(I1) does not show any co-operative behaviour since the (T parameter is very near to unity.


’kb. 1. Binding constants (K*) , cooperative parameters (a), and stoichiometry coeffi- cients (n), for Pb(II), Cu(I1) and Zn(I1) complexation, as obtained from polarographic (pol) experimental data

~ o - ~ . K * / ( L . ~ o ~ - ~ ) 7.0 k 0.2 1.6 f 0.1 0.46 f 0.02 a 0.07 k 0.01 0.6 0.1 0.81 k 0.07 n a) 0.51 f 0.02 0.33 f 0.01 0.49 k 0.01

a) n = moles of binding sites per moles of carboxylic groups.

The Italian Ministry for University and Scientific and Technological Research is kindly acknowledged for financial support. Dr. R Zunetti and Dr. R. Toffanin from POLY-bibs Research Center are acknowledged for molecular weight measurements and for NMR spectra, respectively. One of the authors (M. B.) is grateful to the Italian National Research Council for a fellowship.

G. 0. Aspinal, Ed., “ThePolysaccharides’: vol. 1-111, Academic Press, New York 1983 ’) D. J. Candy, “Biological Function of Carbohydrates’: Wiley, New York 1980 3, M. Yalpani, Tetrahedron 41, 2957 (1985) 4, M. Bosco, E. Reisenhofer, R. Rizzo, Bioelectrochem. Bioenerg. 32, 181 (1993)

6, J. Johnson Jr., S. Kirkwood, A. Misaki, T. E. Nelson, J. V. Scaletti, F. Smith, Chem.

’) T. L. Bluhm, Y. Deslandes, R. H. Marchessault, S. Perez, M. Rinaudo, Carbohydc Res.

8, V. Crescenzi, A. Gamini, G. Paradossi, G. Torri, Carbohydr. Polym. 3, 273 (1983) ’) B. T. Hofreiter, I. A. Wolff, C. L. Mehltretter, J Am. Chem. SOC. 79, 6454 (1957)

’O) T. Norisuye, T. Yanaki, H. Fujita, J Polym. Sci., Part B: Polym. Phys. 18, 547 (1980) ‘ l ) D. Lecacheux, Y. Mustiere, R. Panaras, G. Brigand, Carbohydr. Polym. 6 , 477 (1986) 12) V. Crescenzi, A. Gamini, R. Rizzo, V. S. Meille, CarbohydE Polym. 9, 169 (1988) 13) A. Cesaro, F. Delben, A. Flaibani, S. Paoletti, Carbohydr. Res. 181, 13 (1988) 14) G. Schwarz, Eur. J Biochem. 12,442 (1970)

M. Bosco, A. Gamini, R. Rizzo, Carbohydr. Polym. 25, 71 (1994)

Ind. (London) 820 (1963)

100, 117 (1982)

Documents

Divalent cation interactions with a carboxylated derivative of scleroglucan