This article was downloaded by: [Rutgers University] On: 05 April 2015, At: 12:32 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20

Proton and Multinuclear N.M.R. Studies of Cyclogentiotetraose Peracetate-Alkali Cation Complexation a

G. Bonas & M. R. Vignon

a

a

Centre de Recherches sur les Macromolécules Végétales , CNRS , B.P. 53 X, 38041 , Grenoble cedex , France Published online: 21 May 2012.

To cite this article: G. Bonas & M. R. Vignon (1991) Proton and Multinuclear N.M.R. Studies of Cyclogentiotetraose Peracetate-Alkali Cation Complexation, Journal of Biomolecular Structure and Dynamics, 8:4, 781-791, DOI: 10.1080/07391102.1991.10507844 To link to this article: http://dx.doi.org/10.1080/07391102.1991.10507844

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.

Downloaded by [Rutgers University] at 12:32 05 April 2015

This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Journal of Biomolecular Structure & Dynamics, /SSN 0739-1102 Volume 8, Issue Number 4 (1991), "'Adenine Press (1991).

Proton and Multinuclear N.M.R. Studies of Cyclogentiotetraose Peracetate-Alkali Cation Complexation

Downloaded by [Rutgers University] at 12:32 05 April 2015

G. Bonas and M.R. Vignon * Centre de Recherches sur les Macromolecules Vegetales CNRS, B.P. 53 X 38041 Grenoble cedex, France Abstract Complexation of alkali cation picrates with cyclogentiotetraose peracetate (CGD4Ac) have been studied by 1H-N .M.R. spectroscopy in acetone d 6 and nitromethane d 3. We determined the stability constants directly from the observed change of the chemical shifts ofH-4 and H-6 proS protons of CGD4Ac at constant ligand concentration with increasing amounts of alkali salt. The stability constants have also been determined by multinuclear n.m.r. spectroscopies, from the observed change of the chemical shifts ofLithium-7, Sodium-23, Potassium39, Rubidium-87 and Cesium-133 at constant alkali salt concentration with increasing amountofCGD4Ac. The stabilities of the complexes varied in theorderCs+ > Rb+ > K+ > Na + > Li+. The complexation of CG D4Ac with Cs + induced conformational change, the gg conformer being predominent at the complexed state. In most cases the cationic exchanges between the free and complexed sites were rapid. However in the CsPic-CGD4Ac-Acetone system the exchange was slow enough to observe below 288 K two 133Cs + resonances.

Introduction During the last two decades the studies of alkali metal ions complexes with macrocyclic ligands have been investigated by numerous authors (1) using several methods. However n.m.r. spectroscopy is one of the most powerful technique, in particular multinuclear n.m.r. in non aqueous solvents. In this paper we described the complexation of alkali cations with a new ligand, cyclogentiotetraose peracetate (2) (CGD4Ac) either by proton or multinuclear n.m.r. spectroscopies, in deuterated acetone and nitromethane solvents, and we measured the stability constants. These results showed that the ligand affinity decreased in both solvents from Cesium to Lithium. In the case of Cesium complexation in acetone we observed by proton n.m.r. a conformational change of the ligand. We also studied this equilibrium at different temperatures by 133 Cs n.m.r. and the coalescence temperature was reached at 288 K; below288 K, two absorptions corresponding to the free and complexed 133Cs species were observed. *Author to whom correspondence should be addressed.

781

782

Banas & Vignon

Materials and Methods Alkali picrate salts were a generous gift of Dr J.P. Dutasta. The ligand and the alkali salts were dried at 50oC under vacuum (0.1 mm) during 24 h. The solutions were prepared with fully deuterated nitromethane d 3 or acetone d 6, from "Commissariat a l'Energie Atomique, Centre d'Etudes Nucleaires de Saclay, France". The solvents were directly used from the manufacturer's sealed vials. All n.m.r. spectra were taken on a Bruker AM 300 spectrometer in the pulsed Fourier Transform mode. 1

Downloaded by [Rutgers University] at 12:32 05 April 2015

H NMR. Experiments

1

H n.m.r. experiments were performed at 300,13 MHz. The sample solutions were placed in 5 mm o.d n.m.r. tubes. Assignments ofthe 1H spectra (in acetone d 6 or nitromethane d 3) were made previously by 2D n.m.r. experiments (3). The chemical shifts (8, p.p.m.) were measured relative to TMS as internal standard. All theM+ I CG D4Ac proton n.m.r. binding curves were determined at constant ligand concentration by varying the concentration of the corresponding salt. Thus, a serie of 11 samples were prepared as described below in order to obtain the complexation curves of the corresponding alkali cation. In each different n.m.r. tube, to 0.1 ml of standard ligand solution [CGD4Ac] 0, were added 0 to 0.4 ml of an alkali salt solution: [M+] 0 = A orB = N4; the solution was completed up to 0.5 ml by addition of pure solvent. a - Preparation of n.m.r. tubes in acetone d 6. Cesium: [CGD4Ac] 0 = 5 X 10- 3 M; [CsPic] 0 = A= 7 X 10- 3 M; dilution B = N4. Samples (tube number; salt solution in ml; solution: A or B): (0; 0; B); (l;O.ll;B); (2;0.20;B); (3;0.29;B); (4;0.37;B); (5;0.ll;A) (6;0.14;A); (7;0.19;A); (8;0.24;A); (9;0.31;A); ( 10;0.40;A). Rubidium: [CGD4Ac] 0 = 6X 10- 3 M; [RbPic] 0 =A= 5 X 10- 3 M;dilution:B = N4. Samples: (O;O;B); (l;O.lO;B); (2;0.24;B); (3;0.34;B); (4;0.ll;A); (5;0.13;A); 6;0.15;A); (7;0.18;A); (8;0.23;A); (9;0.30;A); (10;0.40;A). Potassium: [CGD4Ac] 0 = 6 X 10- 3 M; [KPic] 0 =A= 8 X 10- 3 M; dilution B = N4. Samples: (O;O;B); (1;0.12;B); (2;0.24;B); (3;0.33;B); (4;0.11 ;A); (5;0.13;A); (6;0.15;A); (7;0.19;A); (8;0.25;A); (9;0.32;A); (l0;0.40;A).

b - Preparation of n.m.r. tubes in nitromethane d 3. Cesium: [CGD4Ac] 0 = 5 X 10- 3 M; [CsPic] 0 = A= 4 X 10- 3 M; dilution: B = N4. Samples: (O;O;B); (l;O.lO;B); (2;0.25;B); (3;0.32;B); (3;0.40;B); (4;0.13;A); (5;0.15;A); (6;0.17;A); (7;0.20;A); (8;0.25;A); (9;0.40;A).

Complexation of Sugars with Cations

783

Rubidium: [CGD4Ac] 0 = 2 X.5.10- 3 M; [RbPic] 0 =A= 4.5 X 10- 3 M; dilution: B = N4. Samples: (O;O;B); (l;O.ll;B); (2;0.20;B); (3;0.27;B); (4;0.08;A); (5;0.10;A); (6;0.12;A); (7;0.16;A); (8;0.2l;A); (9;0.29;A); (10;0.40;A). Sodium: [CGD4Ac] 0 = 5 X 10- 3 M; [NaPic] 0 = A= 15 X 10- 3 M. Samples: (0;0); (1;0.03); (2;0.07); (3;1.10); (4;0.13); (5;0.17); (6;0.20); (7;0.23); (8;0.27); (9;0.33); (10;0.40). Lithium: [CGD4Ac] 0 = 15 X 10- 3 M; [LiPic] 0 =A= 75 X 10- 3 M; dilution: B = N4. Samples: (O;O;B); (1;0.08;B); (2;0.16;B); (3;0.24;B); (4;0.32;B);(5;0.40;B); (6;0.12;A); (7;0.16;A); (8;0.22;A); (9;0.30;A); (10;0.40; A).

Downloaded by [Rutgers University] at 12:32 05 April 2015

Alkali Cation NMR. Experiments

The multinuclear measurements were performed in a 10 mm o.d. n.m.r. tube, at 116.64 MHz for 7 Li, 79.39 MHz for 23 Na, 14.0 MHz for 39 K, 98.20 MHz for 87 Rb and 39.36 MHz for 133Cs. The chemical shifts were expressed in p.p.m. relative to the signal of the free alkali cation as standard, measured before any addition of ligand. All the CGD4Ac/M+ alkali n.m.r. binding curves were determined at constant concentration of the salt ([M Pic] 0 ; 2 ml of deuterated solvent, acetone d 6 or nitromethane d 3 by varying the concentration of the ligand CGD4Ac (x = total amount in mg ofligand added) as indicated below. a - Preparation of n.m.r. tubes in acetone d 6. Cesium 133: [CsPic] 0 =4.4 X 10- 3 M;x = 0; 1.2; 3.9;6.2; 8.4; 10.4; 12.7; 17.3; 22.1; 28.6; 38.7. Rubidium 87: [RbPic] 0 = 10 X 10- 3 M; x = 0; 8.4; 20.2; 28.8; 38.1; 47.7; 57.1; 74.8; 114.8. Lithium 7: [LiPic] 0 = 5.1 X 10- 3 M; x = 0; 0.7; 2.1; 2.6; 5.3; 7.2; 9.8; 11.4; 13.1; 18.1; 26.1; 40.9; 60.6; 87.0; 135.2.

b - Preparation of n.m.r. tubes in nitromethane d 3. Cesium 133: [CsPic] 0 = 5 X 10- 33 M; x = 0; 1.4; 2.4; 4.5; 6.9; 9.2; 11.5; 23.0; 34.5. Rubidium 87: [RbPic] 0 = 10.1 X 10- 3 M; x = 0; 1.9; 5.1; 8.5; 13.0; 16.1; 23.8; 52.2. Sodium 23: [NaPic] 0 = 5 X 10- 3 M; x = 0; 9.7; 22.3; 48.8; 79.9; 101.9; 126.6; 182.0; 243.6. Lithium 7: [LiPic] 0 = 5 X 10- 3 M; x = 0; 1.3; 3.2; 5.9; 10.1; 20.9; 35.5; 77.8; 122.6.

c - Variable temperature studies. For these experiments, 13.9 X 10- 3 mmole ofCsPic (5.02 mg) and 8.4 X 10- 3 mmole

784

Banas & Vignon

of CG D4Ac (9.68 mg) were dissolved in 2 ml of acetone d 6, and the recorded at 323, 303, 278, 263,244, 233,223,213 and 203 K

133

Cs spectra

The n.m.r. spin lattice relaxation times (T 1) of 133Cs were performed on the same tube by using the inversion recovery method, at 323 K before the addition of the ligand and at 203 K after the addition of the ligand.

Results and Discussion Proton NMR. Study Downloaded by [Rutgers University] at 12:32 05 April 2015

1- Uncomplexed cyclogentiotetraose peracetate (CGD4Ac). This cyclic oligosaccharide has been prepared by internal cyclisation ofbifonctional linear gentiotetraose (2,4) as shown below.

oJ-o'--x Ac;xcr OAc

(X=13CI

or

or.Br)

The proton spectra ofCGD4Ac in different deuterated solvents (CDC1 3, CD 3COCD 3, CD3N02) showed seven signals in agreement with a C4 symetry axis of this molecule. The assignments have been done by selective decoupling and 2D cosy n.m.r. experiments and the corresponding chemical shifts are reported in Table I. 2- Complexation of CGD4Ac with alkali cations.

785

Complexation of Sugars with Cations Table I Proton Chemical Shifts ofUncomplexed CGD4Ac in Various Solvents" H-6proSh

H-6proRh

Solvent

HI

H2

H3

H4

CDC13

4.73

5.02

5.24

5.02

CD 3N02

4.74

4.95

5.17

5.09



CD 3COCD 3

4.89

4.97

5.20

4.96

< --------------3.85-4.02------------- >

H5

< ---------------3.5-4.0-------------- > 3.88

Downloaded by [Rutgers University] at 12:32 05 April 2015

"All chemical shifts are given in parts per million and are referred to internal TMS. hH-6proR and H-6proS as defined by K.R. Hanson (5).

CsPic I CGDIJAc ( 111 J

3

1

2 S , 6proR

I

I 1 1

I I

I

CsPic I CGD"Ac 10.511 I I I I

I

I

CGDIIAc

I

I

I I

I

1 I

I

I

eproS S..&proR I

5.0

4.5

I

4.0

"" Figure 1: 1H n.m.r. spectra of CGD4Ac in nitromethane-d 3 at 323 K for different Cesium Picrate/ CGD4Ac molar ratio.

The addition of alkali salt to CGD4Ac in solution induces significant modifications in the proton spectra, as shown on Figure 1. Two protons are strongly affected, H-4 and H-6proS which are downfield shifted when the alkali cation-ligand molar ratio increases. We used the 1H chemical shift variations of these two protons as a function of the [M+]I[CG D4Ac) molar ratio to calculate the formation constants of the complex.

Banas & Vignon

786 Considering that we have a 1:1 complex formation CGD4Ac + M+

CGD4Ac - M+

¢

[1]

due to a fast exchange, the weight average chemical shift can be expressed according to AI. Popov and coworkers (6) as shown below (7).

Downloaded by [Rutgers University] at 12:32 05 April 2015

In this procedure we input the chemical shift (Or) and the experimental ligand and cation concentrations (L,M). A least square curvefitting program calculates the complexation constant by successive iterations ofK and oc, in order to obtain the best fit between experimental and calculated chemical shifts as shown on Figure 2.

1

i•

.=•

5

0

[ Cs-t] ll CG04Ac]

Figure 2: Normalized cesium induced shifts of CGD4Ac protons at various relative concentrations (Cs +]/[CGD4Ac) in acetone-d 6. The average of the normalized shifts for H4 and H6 proS protons is plotted. Solid line is computer fitted curve and dots are experimental points.

All the calculated formation constants K in L mole -I of CGD4Ac-alkali cation complexes are reported in Table II and clearly indicate that the log K values increase continuously from Lithium to Cesium in both solvents. These results show that CGD4Ac complexes preferentially with Cesium, in good agreement with the ionic diameter of Cesium (3.40 A) (8) and the cavity size of the ligand (3.30 A), calculated by molecular modelling) (9). Furthermore, the addition of cesium picrate to CGD4Ac in acetone solution induces a variation of the 3J-H5,H-6proR coupling constant of CGD4Ac, which continuously decreases from 4.6 to 2.0 Hz, as the [Cs +]I[CGD4Ac] molar ratio increases. From these data the gg and gt rotamer populations along C5-C6 bond, as defined by M. Sundaralingam (10) and shown in Figure 3 were calculated with the following equations: 1.3 gg

+ 11.5 gt =

gg+gt=l

3 JH-5,H-6proR

787

Complexation of Sugars with Cations Table II Log K" Values of CG D4Ac-Alkali Cation Complexes Calculated From Proton N.M.R. Experiment at 323 K

Ionic Radius (in A)

0.74

1.02

1.70

2.04

1.38

1.49

1.70

2.48

3.19 3.93

3.43 3.96

Downloaded by [Rutgers University] at 12:32 05 April 2015

Solvent

"Average values in L mol- 1 calculated from H-4 and H-6 proS proton chemical shifts variations; the estimated errors are 0.10 on the log K values, even though the computer fit indicates smaller errors. hLog K could not be determined accurately, due to weak complex formation. 'Log K could not be determined, due to the low solubility of the potassium picrate in CD 3N02•

Q*"'

pi'DS05

'praR

01

HI

HI

115

Figure 3: gg, gt and tg conformations along C5-C6 bond. Table III Rotamer Populations About C5-C6 Bound of CGD4Ac During Cesium Complexation in Acetone d 6 at 323 K [Cs+]![CGD4Ac]

%complex"

JJ H·l.H·6proR

J JH·l.H·6proS

%gg

% gt

0 0.3 0.53 0.8 1.2 1.8 4.15

0 15 35 50 62 75 90

4.6 4.4 4.0 2.8 2.5 2.2 2.0

1.7 1.7 1.7 1.7 1.7 1.7 1.7

67.5 69.5 73.5 85 88 91 93

32.5 30.5 26.5 15 12 9 7

"Deduced from Figure 2, where the lioh/li, ratio indicates directly the percentage of the complex.

derived from W. Saenger and coworkers ( 11) equations in which the tg conformer contribution has been neglected, according to D. Gagnaire and coworkers (12) who found that the tg conformation cannot yield the cyclic tetrasaccharide. All these results are reported in Table III, and indicate clearly a conformational change of the ligand due to the complexation. The conformation along the C5-C6 bond in the uncomplexed CG D4Ac in acetone solution appears as a mixture of 67.5% of gg and 32.5% of gt. The Cesium complexation induces a significant modification of the conformer

Downloaded by [Rutgers University] at 12:32 05 April 2015

788

Banas & Vignon

Figure 4: A schematic representation of cyclogentiotetraose CGD4Ac (acetate groups being not visualized).

population, the gg conformer becoming largely predominant (when 90% of CGD4Ac is complexed, we have only 7% of the gt conformer). According to preliminary molecular modelling calculation (9), in the gg conformer the four intra-residue oxygen atoms are coplanar (Figure 4) and this could explain the better affinity of this conformer for Cesium. Multinuclear NMR. Studies

The complexation reaction between alkali picrates (MPic) and CGD4Ac has been studied by 7Li, 23 Na, 39 K, 87 Rb and 133 Cs n.m.r. in nitromethane d 3 and acetone d 6. During the addition of CGD4Ac in the picrate salt solution we observed a downfield shift of the n.m.r. resonance. In the case of 7Li and 23 Na we measured a variation of less than I p.p.m., whereas we found a downfield shift of approximatively 100 p.p.m. for 87 Rb and 133 Cs. We used the variations of the alkali cation chemical shifts with the [CGD4Ac]IM+ molar ratio to calculate the stability constant K, according to the procedure previously described in the proton study. All the data are reported in Table IV and are in good agreement with the results obtained by 1H n.m.r., showing an increasing affinity of the li~and from Lithium to Cesium in both solvents, with the exception of 87 Rb and 1 Cs in nitromethane which presented the same log K value towards CGD4Ac, within the experimental error.

789

Complexation of Sugars with Cations Table IV Log K• Values ofCGD4Ac-Alkali Cation Complexes Calculated From Multinuclear N.M.R. Experiments at 323 K cation solvent 1.54 1.70

2.63 3.38

2.40

3.44 3.32

Downloaded by [Rutgers University] at 12:32 05 April 2015

•Estimated errors ± 0.10 on the log K values. hLog K could not be determined accurately due to the low magnitude of the chemical shifts variations. cLog K could not be measured due to the line broadening and the low variation of the 39 K n.m.r. signal during the addition of the ligand, the low sensitivity of 39K nucleus; furthermore its picrate salt has a low solubility, particularly in CD 3N0 2•

In the case of Cesium we have also studied the complexation reaction as a function of the temperature. Thus the 133Cs spectra were recorded on a mixture ofCGD4Ac (9.68 mg) and CsPic (5.02 mg) (0.6/1 molar ratio) in acetone d 6 solution. At 323 K we observed only one population-averaged signal (half-height line width: ~v 112 = 127 Hz), indicating a fast exchange of the metal ion between the two cationic sites. Lowering of the temperature induced a line-broadening (~v 112 = 473Hz at 303 K; ~v 112 = 860Hz at 293 K), until the coalescence temperature was reached at 288 K. When the temperature was further lowered, two absorptions corresponding to the free and comp1exed 133Cs species were observed. These two signals became narrow when the temperature further decreased (~v 112 Cs +free = 175Hz, ~v 112 Cs +complexect = 150Hz at 263 K; ~v 112 Cs +free ~ ~v 112 Cs +complexed = 10Hz at 203 K), as shown on Figure 5. This behaviour indicated a slow exchange, between the free and complexed cesium cation, on the 133 Cs n.m.r. time scale, below 230 K. From fully relaxed spectra (13) recorded below the coalescence temperature, it is possible to calculate the complexation constant of equilibrium [1], at a given temperature, by measuring directly the peak areas of the two signals (14) as shown in Table V. From the log K values obtained at different temperatures, and according to the equation [2]: log K

= - ~

it is possible by plotting log K constants:

=

Ho /(2.3 RT)

+~so /(2.3

R)

[2]

f(l/T) to deduce the following thermodynamic

~ Ho = 1.2 ± 0.3 Kcal mol-l ~so = 21 ± 2 cal.K- 1 mol- 1

From the~ Ho and~ So contribution to the~ Go, it can be concluded that the complex is entropy stabilized in acetone.

790

Banas & Vignon

Downloaded by [Rutgers University] at 12:32 05 April 2015

CCD.. Ac +

Ce .. lc

I0 • • /1 I

ACETONE

c.·.r.. ..,..

d

20S K

... Figure 5: Cesium-133 n.m.r. spectra ofCG D4Ac/CsPic (0.6/ 1 molar ratio) in acetone-d 6 at various temperatures (the scale is graduated every p.p.m.); a: rapid exchange; b,c: intermediate exchange; d: slow exchange. Table V Log K Values of CGD4Ac-Cs+ Complex at Different Temperatures T(K)

263

253

243

233

223

213

203

logK

3.80

3.74

3.67

3.62

3.56

3.54

3.47

Conclusion We have examined the complexation of cyclogentiotetraose peracetate CGD4Ac, with alkali cations by proton or multinuclear n.m.r. in deuterated acetone and nitromethane solvents. The stability constants were calculated and the values reported in Tables II and IV showed that there is a good agreement between the K values determined by proton and multinuclei n.m.r. spectroscopies. The log K values increase continuously from Lithium to Cesium in both solvents. These results show that CGD4Ac complexes preferentially with Cesium, in good accord with the relative

Complexation of Sugars with Cations ionic diameter of cations and the cavity size of CGD4Ac (3.3 molecular modelling.

791

A) obtained by

The conformation along the C5-C6 bond in the complexed CGD4Ac in acetone appears as a mixture of 67.5% of gg and 32.5% of gt. The Cesium complexation induces a significant modification of the conformer population, the gg conformer becoming largely predominant.

Downloaded by [Rutgers University] at 12:32 05 April 2015

We also studied the equilibrium at different temperatures in acetone-d 6 solution by 133 Cs n.m.r. and the coalescence temperature was reached at 288 K. A slow exchange on the 133 Cs n.m.r. time-scale between the free and complexed Cesium cation is observed below 230 K. References and Footnotes

I. Izatt, R.M .. Bradshaw, J.S .• Nielsen, SA, Lamb, J.D .. Christensen, J.J. and Sen, D., Chern. Rev. 85, 271-239 ( 1985) and references therein. 2. Excoffier, G., Paillet, M. and Vignon, M.R., Carbohydr. Res. 135, ClO-Cll (1985). 3. Gey, C. and Vignon, M.R.. Compt. Rendus Acad. Sci. 302, 1051-1056 (1986). 4. Bonas, G., Excoffier, G., Paillet. M. and Vignon, M.R., Recueil des Travaux Chimiques des Pays Bas 108.259-261 (1989). 5. Hanson, K.R.,J Am. Chern. Soc. 88,2731-2742 (1966). 6. Bodner, L., Greenberg, M.S. and Popov, AI .. Spectroscopy Letters 5, 489-495 (1972). 7. The following equation correlates the chemical shift variations (oohs) to the formation constant of 112 thecomplexK: oohs = ((KL-KM-1) + (K2M 2+K2L 2-2K2ML+2KM+2KL+ 1) ) X ((or-oc)/2KL) + oc where, for a given proton of the ligand CG D4Ac, oohs is the observed chemical shift. oc is the limiting chemical shift for the complexed CGD4Ac; Land M being the total ligand and cation concentrations, respectively; Kin L mol- 1• 8. Shannon, R.D. and Prewitt, C.T .. Acta Cryst. B25, 925-946 (1969). 9. Vergelatti, C .. Personal communication. 10. Sundaralingam, M., Biopolymers 6, 189-213 (1968). 11. Manor, P.C., Saenger, W., Davies, D. B.. Jankowski, K. and Rabczenko, A, Biochim. Biophys. Acta 340,472-483 (1974). 12. Gagnaire, D., Perez, S. and Tran, V., Carbohydr. Res. 78,89-109 (1980). 13. T 1 values of0,4 and 0,07 s at203 K for free and complexed mcs respectively, and T 1of3.1 s for a sample of pure CsPic at 323 K were obtained. In order to estimate quantitatively the amount of the two mcs species, the n.m.r. spectra were recorded by using relaxation delay of24 s (8 X T 1 of the free mcs at 323 K). 14. SF and Sc being the free andcomplexed mcs peak areas, MandL being the total cation and ligand concentrations, the complex concentration C can be deduced from the following equation: C = M[Sc/(SF+Sc)]. From the mass law it follows: K = C/((L-C)(M-C)). Date Received: September 27, 1990

Communicated by the Editor Dino Moras