466 Volume 57, Number 4, 2003 APPLIED SPECTROSCOPY0003-7028 / 03 / 5704-0466$2.00 / 0q 2003 Society for Applied Spectroscopy

Sugar Interaction with Metals in Aqueous Solution: IndirectDetermination from Infrared and Direct Determinationfrom Nuclear Magnetic Resonance Spectroscopy

PHILIPPE RONDEAU, SANDRINE SERS, DHANJAY JHURRY, andFREDERIC CADET*Laboratoire de Biochimie et Genetique Moleculaire, Faculte des Sciences et Technologies, Universite de la Reunion, BP 715197715 Saint-Denis, France-DOM (P.R., S.S., F.C.); and Department of Chemistry, Faculty of Science, University of Mauritius,Reduit, Mauritius (D.J.)

In this article, mid-infrared Fourier transform (Mid-FT-IR) andcarbon thirteen nuclear magnetic resonance (13C NMR) spectros-copy have been used to determine possible interactions between su-crose and various alkali or alkaline earth metals in aqueous solu-tion. In the presence of these metals, signi� cant shifts in the ab-sorption bands of sucrose were noted by mid-FT-IR coupled withprincipal component analysis (PCA). These shifts were explained onthe basis of weakening of the H-bond network between sucrose andwater and possible interactions between sucrose and the metal ion.Factorial maps were established and the spectral patterns obtainedshow that these interactions vary according to the nature of themetal ion. 13C NMR analysis showed that the carbon atoms of su-crose undergo shielding or deshielding in the presence of metal ionsin aqueous solutions. Two factors were invoked to account for thevariation of chemical shifts: the rupture of hydrogen bonds due tohydration of the metal ion and the possible coordination of the met-al ion to the oxygen atoms of sucrose.

Index Headings: Mid-infrared Fourier transform; Mid-FT-IR; Ion–sucrose interactions; Principal component analysis; PCA; 13C NMR.

INTRODUCTION

Sugar is a component part of nucleic acids and as such,sugar–metal ion interactions bear signi� cant biochemicalimportance.1 The magnesium–sugar interaction has a sta-bilizing effect on the DNA double-helix structure.2

Therefore, complexation between the ATP molecule andmagnesium ion is a necessary co-factor for phosphoryland nucleotidyl transfer enzymes, as well as a major en-ergy storage method for cells.3 Calcium ions and sugarare involved in biological adhesion and agglutination pro-cesses.4 Several studies about sugar–ion interactions, par-ticularly with alkali and alkaline earth metals, have beenreported.5,6

Structural studies in the solid state have suggested thatthe metal ion interaction with sugar can affect the con-formations of the latter.7 Moreover, in the solid state, in-frared absorption bands of sugar are more detailed andimportant spectral shifts induced by the metal interactionscan be more easily observed.8 However, from the biolog-ical point of view, it is important to understand the typeof interactions between sugar and metal ions in aqueoussolution. According to Tajmir-Riahi,9 such interactionsare not obvious to detect in aqueous solution, since thereis no signi� cant difference between the infrared spectrum

Received 11 September 2002; accepted 2 December 2002.* Author to whom correspondence should be sent. E-mail: cadet@

univ-reunion.fr.

of pure sucrose solution and the spectrum of solutioncontaining an alkali metal ion. The Fourier transform in-frared (FT-IR) analysis of sucrose in solution by the at-tenuated total re� ectance (ATR) technique has been car-ried out by Kodad et al.10 The authors have made a thor-ough assignment of the sucrose bands. More recently, theprincipal component analysis (PCA) technique applied tomid-infrared spectra has proved to be ef� cient for quan-tifying shifts induced by sugar–metal interactions in so-lution.1,11 Our group has also reported previously thequantitative mid-infrared spectroscopic analysis of aque-ous sucrose in limited concentration ranges.12 Carper andco-workers13,14 have used both the FT-IR and nuclearmagnetic resonance (NMR) techniques to investigate var-ious divalent metal ion interactions with gluconic acid orsodium gluconate or gluconolactone. They have shownfor example that the sodium ion interacts with all thegluconate OH groups except for the one in the 3 positionwhile the magnesium ion binds only at OH 1 and 6 toform a 1:1 complex. In the present article, we study in a� rst instance the nature of interactions between sucroseand various alkali and alkaline earth metals in solutionby mid-infrared (MIR) spectroscopy using the principalcomponent analysis (PCA) technique. In a second part,we attempt to identify such interactions by 13C NMRspectroscopy. The results of these two techniques arecompared and discussed in the present article. In previouswork, Bock and Lemieux15 have shown that the confor-mation of sucrose is approximately the same in dimeth-ylsulfoxide and water solutions and that this conforma-tion is similar to that observed in the solid state. In ad-dition, SIMPLE 1H-NMR isotope-shift measurements16,17

of aqueous sucrose solutions have shown the existenceof an equilibrium between two intramolecular hydrogen-bonded conformations, in which the 2-oxygen of the glu-cose moiety acts as the acceptor for either the 1 9-O or3 9-OH of the fructose moiety, as illustrated in Fig. 1.

EXPERIMENTAL

Material. Sucrose and anhydrous metal halide com-pounds were all purchased from Sigma (St. Louis, MO).They were used without further puri� cation.

Mid-Fourier Transform Infrared Attenuated TotalRe� ectance Spectra. Mid-infrared analyses were run onaqueous solution of pure sucrose (0.6 M) and on aqueoussucrose solu tion in which were dissolved variab leamounts of solid metal salts: NaCl (0 –3.4 M), KCl (0 –

APPLIED SPECTROSCOPY 467

FIG. 1. Conformations of sucrose in solution showing two intramolecular hydrogen-bonded forms.

FIG. 2. Mid-FT-IR spectrum (900–1250 cm21) of (a) pure MgCl2 (2M), (b) pure sucrose (0.6 M), and (c) sucrose (0.6 M) with MgCl2 (2M).

2.6 M), CaCl2 (0 –1.8 M), and MgCl2 (0 –2 M). The con-centration of sucrose is maintained at 0.6 M. The spectrahave been recorded on a Nicolet 460 Fourier-TransformMichelson I.R . spectrophotometer, equipped with aDTGS–KBr detector and a KBr beam-splitter, having aspectral resolution of 2 cm21. Attenuated total re� ectancespectra were obtained with an ARK ATR system. Thecrystal of re� ectance, which is made of germanium, hasa high refractive index (n 5 4 at 1000 cm21) and is quiteinert to water. With 408 bevels, the IR beam re� ects 14times along the crystal. The combination of eight scansresulted in an average spectrum.

Carbon Thirteen Nuclear Magnetic ResonanceAnalysis. Solutions of sucrose (0.3 M) with or withoutmetal salts were prepared in distilled/deionized water andplaced in 5 mm tubes. A few drops of D2O were addedprior to running the NMR experiment for lock purposes.The solution containing a metal salt was prepared by add-ing the solid salt to a 0.3 M solution of sucrose in dis-tilled/deionized water. Spectra were recorded on a BrukerFT 250 MHz spectrometer (acquisition time 5 2.37 s;recycle delay 5 2 s). It is important to note that the spec-trum of pure sucrose was recorded several times and novariation was observed in the chemical shifts. Moreover,since no internal reference was added to the solutions,the spectrum of pure sucrose was recorded systematicallyprior to that of an aqueous solution containing a metalion.

Mathematical Treatment. All statistical calculationswere performed on a Compaq personal computer. The

software, written in C language and developed in ourlaboratory, was used for principal component analysis(PCA) and extraction of spectral patterns. The Unscrum-bler, a more powerful analytical software developed andpublished by Camo ASA, was also used for describingvariation in multidimensional data.

A multidimensional statistical analysis such as PCA isa method that optimizes data description with minimalloss of information.18 Each spectrum can be seen as apoint in n-dimensional space, where each dimension cor-responds to an axis or wavenumber at which the sampleis measured. However, this space, which uses the originaldata, is not appropriate to characterize a collection ofspectra.19 Indeed, in the mid-infrared range, data may behighly correlated. Thus, the information has a large re-dundancy, which can be eliminated by creating more suit-able variables.20 The new variables obtained by PCA area linear combination of all the original variables and haveno correlation with each other. The new axes correspond-ing to these variables are called ‘‘principal components’’and have the advantage of being orthogonal.

The PCA technique was applied to the MIR spectra ofaqueous sucrose salt solution in the region from 900 to1250 cm21 (with 362 data points used as principal vari-ables). It is good to point out that no subtraction of thesalt solution spectra has been carried out because of theabsence of a spectral � ngerprint of the salt in that region.Spectra were centered prior to PCA according to:

X ij 5 A ij 2 A j 2 A i 1 A

where X ij 5 centered data; A ij 5 spectral data (log R0 /R)of spectrum i and wavenumber j; R0 corresponds to thebackground obtained without sample; R is the re� ectedintensity from the sample; A j 5 the mean value of spec-tral data at wavenumber j for every spectrum; A i 5 themean value of spectral data of spectrum i for every wave-number; and A 5 the average mean of all spectral datain the collection. PCA treatment was used for morpho-logical analysis of spectra21 and for graphical represen-tation of spectra similarity.22

RESULTS AND DISCUSSION

Fourier Transform Infrared Study. The MIR spectraof aqueous solutions of sucrose in the presence and ab-sence of metal salts under our conditions have been re-corded in the region 900–1250 cm21, as shown in Fig. 2.This region is characteristic of C–O, C–O–H, and C–Cvibrations due to the sucrose molecule.23,24 Both spectraare quite similar and no important change or displace-

468 Volume 57, Number 4, 2003

FIG. 3. (a) First principal component (PC 1) pattern and second prin-cipal component (PC 2) patterns for sucrose (0.6 M) mixed with: (b)Na1 (0.3–3.4 M), (c) K1 (0–2.6 M), (d ) Mg 21 (0–2 M), and (e) Ca 21

(0–1.8 M).

TABLE I. PC 1 and PC 2 spectral patterns with absorption bands (s) and amplitude of shfts (Ds) for Na1, K1, Mg21, and Ca21.

Sucroses

PC 1 (cm21)s

PC 2/Na1 (cm21)

s Ds

PC 2/K1 (cm21)

s Ds

PC 2/Mg21 (cm 21)

s Ds

PC 2/Ca21 (cm21)

s Ds Assignments10

1137 1138 1134 24 1134 24 1130 28 1132 26 dC–O endo fru1110 1109 1102

107725 1102

107825 1097

1076212 1100

107729 dC–O endo glu

1054 1054 1050 22 1051 23 10481031

26 10501032

24 dC–O exo

1017 1017 1014 23 1013 24 1012 25 1013 24998928

998928

992 26 992 26 984922

214 986 212 dC–O–CnC–C fru

ment of bands can be noted in the presence of metal saltsunder our conditions.

Morphological Analysis of Spectral Patterns. The IRspectral data of sucrose solutions, in the presence ofgroups I and II metal halides, were subjected indepen-dently to PCA. The major advantage of PCA is that itprovides principal spectra and hence, extracts the majorcharacteristic wavelengths.22,25 The spectral pattern (Fig.3a) illustrated in Fig. 3 corresponds to the � rst principalcomponent (PC 1) and it is the same for all ions (Na1,K1, Mg 21, Ca 21). It features absorption bands due to puresucrose and does not reveal any difference between themetal ions. On the other hand, the spectral patterns forthe second principal component (PC 2) indicate shifts inthe absorption bands of sucrose towards smaller wave-numbers and is characteristic of each ion, as depicted bypro� les (Figs. 3b–3e). Moreover, the bands at 1031 and1076 cm21 are not distinct in the pure sucrose solutionspectrum, while they appear as clear and distinct bandson the PC 2 plot. At this stage, these bands cannot beassigned.

Similar band shifts were previously reported in the sol-id state for Na1 and Mg 21,9 which was accounted for bya weakening of the OH bond in crystalline sucrose dueto interaction between the metal ions and the oxygen at-oms of sucrose. The same type of interaction may alsooperate in solution. However, there are differences be-

tween alkali and alkaline earth metal ions regarding theamplitude of shifts. As summarized in Table I, all stretch-ing (n) and bending (d) vibrations of sucrose experiencevery small shifts (between 2 and 6 cm21) in the case ofalkali ions whereas, a more signi� cant displacement ofbands is noted with the alkaline earth ions (between 5and 14 cm21). It is worth observing that the absorptionband at 998 cm21 is the most shifted for all the ions. Ina previous paper, this band was assigned to the glycosidelink (C–O–C).26,27 Table I also presents a possible assign-ment of the various bands based on previous data re-ported by Kodad.10

Factorial Map. All the MIR spectra obtained in thepresence of the different metal ions at various concentra-tions were gathered and subjected to PCA. A factorialmap is then set up from the PCA using PC 1 (78% oftotal variance) and PC 2 (21% of total variance) as co-ordinate axes. These two PCs, which describe almost99% of the variation in the spectral data, are the mostin� uential PCs. The factorial map in Fig. 4a shows thatthe spectra are distributed along a concentration gradient.A correlation can be established between both PC 1 andPC 2 and the metal ion concentrations.28 The same fac-torial map is presented differently in Fig. 4b, where it ispossible to de� ne two distinct classes, I and II, corre-sponding respectively to monovalent and divalent cat-ions. The factorial maps con� rm the difference in bandshifts detailed in the previous paragraph between the al-kali and alkaline earth metal ions.

A more in-depth analysis of the factorial map relatedto class II also enables the differentiation between Mg21

and Ca 21 (Fig. 4c), while such a distinction is not possiblefor the alkali cations. This suggests that the nature of theinteractions between the ion and sucrose may differ fromone class to another.

The PCA technique was also applied to the IR spectraof aqueous sucrose solutions containing metal salts in theOH-stretching region (3600–2700 cm21). As depicted inthe PC 1 plot in Fig. 5, a displacement of the OH bandtowards higher wavenumber is observed in the presenceof NaCl, KCl, and CaCl2, thus indicating a weakening ofthe H-bond network and a strengthening of the originalO–H bond. In a previous article,29 the only presence ofsalts in water was shown to blue-shift the OH-stretchingband. However, with MgCl2, the OH band is pushed tolower wavenumber compared to that of an aqueous so-lution of sucrose, indicative this time of a weakening ofthe O–H bond. This characteristic behavior of Mg21 maypossibly be explained on the basis of preferential inter-actions between sucrose and the metal cation, although

APPLIED SPECTROSCOPY 469

FIG. 4. (a, b, and c) Factorial maps de� ned by PC 1 (78% of total variance) and PC 2 (21% of total variance). ( v ) Na1, ( V ) K1, ( n ) Ca 21, and( m ) Mg 21.

FIG. 5. First principal component (PC 1) patterns for (a) pure sucrose(0.6 M) and sucrose mixed with: (b) Na1 (0–3.4 M), (c) K1 (0–2.6 M),(d ) Mg21 (0–2 M), and (e) Ca21 (0–1.8 M) in the 3600–2700 cm21

region.

this is dif� cult to con� rm by the IR technique. In an FT-IR study of aqueous sucrose solutions containing metalions, Kodad et al.30 have proposed the existence of spe-ci� c metal–cation interactions. They observed signi� cantchanges in the (COH) deformation and (C–O) stretchingbands with Ca 21 and Mg 21 but only minor changes withNa1 and K1. According to these authors, this is due topreferential interactions between sucrose and the divalentcations.

Carbon Thirteen Nuclear Magnetic ResonanceStudy. In order to further the results obtained with theFT-IR technique, it was of interest to investigate whetherthe presence of metal ions would induce any shift in theNMR signals due to the different carbon atoms of su-crose.

In a preliminary study, we have compared the 13CNMR spectrum of an aqueous solution of sucrose, de-picted in Fig. 6, with the spectra recorded in the presenceof an alkali or alkaline earth metal salt. Table II presentsthe chemical shifts for a 0.3 M aqueous solution of su-crose and the corresponding assignment reported byLemieux,15 as well as the variation in chemical shifts ob-served for all the signals of sucrose (0.3 M solution) inthe presence of NaCl (5.81 M), KCl (4.56 M), MgCl2

(3.68 M), and CaCl2 (2.70 M).

470 Volume 57, Number 4, 2003

FIG. 6. 13C NMR (D 2O) spectrum of 0.3 M aqueous sucrose solution.

TABLE II. 13C NMR (D2O) chemical shifts and assignments of sucrose carbon atoms and variation in chemical shifts for the carbon atomsof sucrose in the presence of metal ions.

d (ppm)C-atom

60.2C6

61.4C 19

62.4C 69

69.3C4

71.1C2

72.4C5

72.7C3

74.1C49

76.5C 39

81.4C59

92.2C1

103.7C29

Variation in chemical shifts (ppm)[NaCl]/[Sucrose] 5 16[KCl]/[Sucrose] 5 16[MgCl2]/[Sucrose] 5 16[CaCl2]/[Sucrose] 5 16

0.60.50.20.4

0.60.600.5

0.80.70.20.5

0.50.5

20.10.2

0.40.4

20.40

0.40.4

20.40.1

0.30.3

20.40

0.60.6

20.10.2

10.700.6

0.20.3

20.720.1

0.50.4

20.40.1

0.20.3

20.50

Several comments can be made from Table II:

(1) In the case of the alkali metals Na1 and K1, thechemical shifts for all the atoms are displaced to-wards a lower � eld, which corresponds to a de-shielding of the carbon atoms. The signal corre-sponding to C 3 9 undergoes the most pronouncedvariation.

(2) In the presence of Ca 21, the trend is also towardspositive variation in chemical shifts but to a lesserextent compared to the alkali metals.

(3) The variation depicted with Mg 21 differs markedlyfrom the other ions, as a quite distinct displace-ment towards a higher � eld is observed for thesecondary carbon atoms.

(4) In a general manner, it is noted that the variationin chemical shifts of almost all carbon atoms de-creases according to the following order: NaCl [KCl . CaCl2 . MgCl2. This tendency seems to bein agreement with results obtained by the FT-IRtechnique coupled with PCA as illustrated in Fig.4c.

These trends were further con� rmed by analyzing su-crose solutions containing various concentrations of met-al salts. The variations in chemical shifts for some carbonatoms were then plotted as a function of the concentrationof metal salt, as shown in Figs. 7a–7d. As can be seen,the shielding or deshielding of the carbon atoms varieslinearly with concentration. Figure 7a con� rms the pos-itive variation of chemical shifts noted previously withNa1, K1, and Ca 21. The only negative displacement ob-served in the presence of Ca 21 ions corresponds to C 5 9.In the case of Mg21 (Fig. 7c), a slightly higher deshield-ing is observed at the two primary carbon atoms (6 and6 9) upon increasing the salt concentration. On the otherhand, the shielding effect experienced by the secondarycarbon atoms and, in particular by C5 9, appears to be morepronounced.

A Possible Explanation for the Shielding or De-shielding of Carbon Atoms of Sucrose. Before attempt-ing to provide an explanation of the shielding/deshieldingof the carbon atoms in the presence of metal ions, let ushave a closer look at the dissolution of sucrose in water.

APPLIED SPECTROSCOPY 471

FIG. 7. Variation in chemical shifts as a function of metal salt concentration and sucrose (0.3 M) mixed with: (a) Na1 (0–5.8 M), (b) K1 (0–4.6M), (c) Mg 21 (0–3.7 M), and (d ) Ca 21 (0–2.7 M).

This occurs via the formation of an extensive network ofhydrogen bonds between sucrose hydroxyl groups andwater molecules and accounts for the very high solubilityof sucrose in water. When a metal salt is added to anaqueous solution of sucrose, hydration occurs wherebythe separated ions are surrounded by water molecules.The hydrate numbers for Na1, K1, and Mg 21 were pre-viously reported 29 to be 5, 5, and 4, respectively. Thus,the H-bond network between sucrose and water mole-cules is expected to be reduced in the presence of metalsalts, more so with Na1 and K1.

It is well established that hydrogen bonding leads todeshielding and to an increase in the frequency of theNMR signal of the hydrogen-bonded proton.31 The effecton the adjacent carbon atom appears to be less clear-cutand is more dif� cult to predict. In the presence of Na1,

K1, and Ca 21, almost all the carbon atoms experience adeshielding effect, that is, the electron density aroundthese carbons is decreased. On the basis of our IR study,this deshielding is related to a weakening of hydrogenbonds and is the opposite of what is expected in 1H NMR.

With Mg21, we � nd that the primary carbons of su-crose are slightly deshielded, implying a lesser extent ofdisorganization of H-bonds around these sites as com-pared to the other ions. On the other hand, all the sec-ondary carbons are shielded as a result probably of agreater polarization and subsequent weakening of the O–H bonds of sucrose. As we proposed in the section onIR analysis, the interaction or possible coordination ofMg 21 directly to the oxygen atoms of sucrose seems tobe a plausible explanation supporting the displacement toa higher � eld in 13C NMR. This may occur preferentially

472 Volume 57, Number 4, 2003

with Mg 21 due to its higher charge density (charge-to-size ratio) compared to Na1, K1, and Ca 21 and probablyoutweighs the shielding effect as a net shift to lower vi-bration frequency is noted by IR.

CONCLUSION

We showed in this present study that it is possible bythe FT-IR technique in solution coupled with principalcomponent analysis to put into evidence the interactionbetween a metal ion and sucrose. From the setting up offactorial maps, it was possible to establish three types ofpatterns corresponding to the alkali metal ions Ca 21 andMg 21. These distinct trends probably suggest a speci� ctype of interaction for each metal ion.

In addition, we demonstrated by 13C NMR spectros-copy that the presence of metal ions in aqueous sucrosesolutions leads to variation of chemical shifts of carbonatoms. The displacements towards higher frequencies ordeshielding effect were quite pronounced for the alkalimetals. The presence of Mg 21 ions gave rise to quite animportant shielding effect mainly at the secondary carbonatoms while the primary carbon atoms underwent a slightpositive variation. The behavior of Ca 21 lies betweenthese two tendencies. An attempt was made to explainthese two effects: hydration of the metal ions causesweakening of the H-bond network and leads to deshield-ing of the carbons of sucrose, whereas the possible in-teraction between Mg 21 and the oxygen atoms adjacentto the secondary carbons of sucrose brings about a shield-ing effect.

Finally, it is noteworthy that the results derived by FT-IR coupled with the PCA technique give quite a largeamount of information on the nature of interactions be-tween sucrose and metal ions in aqueous solution. The13C NMR study complements the IR study and providesa better insight into the sites of interaction.

ACKNOWLEDGMENTS

We thank Prof. Bernard Vidal (Universite de La Reunion) for hiscomments and suggestions on this article. This work was supported inpart by grants from Le Conseil General de La Reunion.

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