Vibrational spectroscopic study of the interaction of metal ions with diethyl phosphate, a model for biological systems

Transcription

1 Vibrational spectroscopic study of the interaction of metal ions with diethyl phosphate, a model for biological systems JANUSZ STANGRET' AND RODRIGUE SAVOIE' Department of Chetnistty. Lavnl University, Qutbec (Q~lt.), Canada GlK 7P4 Received June 3, 1991 JANUSZ STANGRET and RODRIGUE SAVOIE. Can. J. Chem. 70, 2875 (1992). We have studied, using Raman and infrared spectroscopy, the interaction of various monovalent, divalent, and trivalent metal cations with the PO, group of diethyl phosphate in aqueous solution, at meta1:phosphate ratios ranging from 1 :40 to 6: 1. The results show that the metal cations interact with the phosphate groups in various ways and to different extents. Spectral characteristics for some specific types of interactions are presented, namely, for direct metal binding by one of the oxygen atoms in the PO; group, the formation of water-separated ion pairs, and non-specific electrostatic interactions. JANUSZ STANGRET et RODRIGUE SAVOIE. Can. J. Chem. 70, 2875 (1992). Nous avons etudii par spectroscopie Raman et infrarouge l'interaction de divers cations mttalliques monovalents, divalents et trivalents avec le groupe charge PO, du phosphate de diethyle en solution aqueuse, a des rapports metal : phosphate variant de 1 : : 1. Les resultats demontrent que les ions se distinguent tant par leur affinite pour le groupement phosphate que par leur fa~on d'interagir avec celui-ci. On prtsente diverses caracttristiques spectrales permettant d'identifier certains types d'interaction, notamment la fixation directe par l'un des atomes d'oxygkne du groupement phosphate, la formation de paires d'ions stparees par molccules d'eau et les interactions non specifiques de nature electrostatique. Introduction Phosphate groups are of prime importance in biological systems, as they are present in major classes of compounds, such as nucleic acids, phospholipids, etc. In DNA, they play a structural role in the linking of subunits through a phosphodiester bridge of the type R,O-PO;-OR2. They can also interact with positively charged species, such as basic proteins and metal ions, through the negative charge on the PO; groups. Although many metal cations can interact with the bases as well as with the phosphate groups in nucleic acids, the latter type of interaction is particularly important as it stabilizes the DNA double helix (1, 2). From theoretical considerations (3), phosphate groups also appear to be the most likely association sites for water in nucleic acids. Furthermore, the manner in which water molecules are organized around DNA through PO;-metal-water interactions seems to be an important factor in the stabilization of specific DNA conformations (4). Vibrational spectroscopy, particularly Raman spectroscopy, has been used extensively for the study of nucleic acids (5-8). The symmetric stretching vibration of the PO; groups gives a moderately strong,and sharp band near 1090 cm-' in the Raman spectrum of DNA. This peak is relatively insensitive to the conformation of the polynucleotide and many investigators have followed the suggestion of Tsuboi et al. (9) that it be used as an internal standard in conformational studies. On the other hand, Raman studies of aqueous DNA solutions have shown that this band can be affected in intensity as well as in frequency when metal ions are present in the solution (10-12). Just how these changes are related to the extent and type of metal binding by the phosphate groups is not completely understood. It is the purpose of the present study to investigate this behavior with 'Permanent address: Department of Physical Chemistry, Institute of Inorganic Chemistry and Technology, Technical University of Gdadsk, Gdansk, Poland. 2~uthor to whom correspondence should be addressed. a very simple model compound, the diethyl phosphate anion ((C2H50)2PO;), in the presence of different types of metal cations. The vibrational spectra of various salts of DEP have been reported previously (13-16) and they have been the subject of force constant calculations (14, 15, 17). This molecule has also been used as a model for the calculation of the vibrational frequencies of the DNA backbone (14, 18) and of the phosphate group in phospholipids (2). Experimental Diethyl phosphate (DEP, acid form) was purchased from Eastman Kodak and used without further purification. Metal salts were reagent grade and used as supplied. Stock solutions were prepared by mixing weighted amounts of diethyl phosphate in demineralized water and neutralizing with aqueous NaOH (or LiOH or KOH). The ph was measured with a micro-electrode and an Orion Research Model 721 ph-meter. These DEP solutions, at 0.1 or 0.3 M concentration, were used to obtain the reference spectra. Weighted amounts of the solid metal chlorides were dissolved in the reference solutions to prepare mixed samples at various metal:dep ratios. The series of NiC1, solutions at appropriate Ni2+ concentrations were prepared by mixing equal volumes of a DEP reference solution and solutions of NiCl, (from the same original stock solution) at the appropriate molar concentration. The ph was not adjusted following the mixing; it varied from 7.5 for the pure DEP solution to 5.5 for a solution at 6: 1 Ni:DEP molal ratio. The Raman spectra were obtained from the aqueous samples contained in sealed capillary cells placed in a temperature-regulated holder (19). These spectra were recorded on a microcomputer-controlled Spex Model 1400 spectrometer, with either a conventional photomultiplier detection system or a multichannel detector (CCD9000 system from Photometrics Ltd., with 1152 x 298 pixels Thompson TH7895 detector). The spectra were excited by the nm line from a Spectra Physics Model 2020 argon ion laser at an average power of 300 mw at the sample. The dispersive spectra were obtained at a spectral resolution of 5 cm-i, the data being collected at 2 cm-' intervals. The total integration time was 25s per point, resulting from the summation of 5 spectra obtained at 5s per point. The spectra obtained with the multichannel detector (1 152 data points) covered a spectral region of ca. 500 cm-i with 1200 grooves/mm gratings or ca cm-' with

2 2876 CAN. J. CHEM. \ FREQUENCY (cm-1) FIG. 1. (A) Raman and (B) infrared spectra of an aqueous solution of diethyl phosphate at 20 C; (-) 0.3M solution at ph 7 and (- - -) 1M solution in 4M HC1 (ph < 0). Insert: variation in intensity of the 1081 cm-i Raman band of the anion as a function of ph. 300 grooves/mm gratings. Intensity corrections were made for the transmission of the spectrometer and of the filter (holographic edge filter from Physical Optics Corporation, Torrance, CA, U.S.A.). The frequency calibration was achieved using emission lines from a neon spectral lamp (20), with an estimated accuracy of 1 cm-' or better. The entrance slit of the spectrometer was adjusted to yield the maximum resolution consistent with channel separation in the detector (ca. 0.5 and 2 cm-i, respectively, with the two types of gratings). The acquisition time was 10 min on the average. The infrared spectra were obtained from samples contained between two BaF2 windows separated by a 10 km-thick Mylar spacer. They were recorded on a commercial BOMEM Model DA3.02 interferometer with MCT detector. These spectra were typically taken at a spectral resolution of 2 cm-i by summation of 1000 interferograms and using a Bartlett apodisation function. Spectral manipulations were performed with the SpectraCalc program (Galactic Industries Corporation, Salem, New Hampshire, U.S.A.). Results The Raman and infrared spectra of neutral (sodium salt) and strongly acidic aqueous solutions of diethyl phosphate are reproduced in Fig. 1. At neutral ph, the phosphate group of this molecule is ionized, giving the (C,H,O),PO; anion. Previous studies (13, 17) have shown that the stretching vibrations of the phosphate group in this species occur in two different regions: the charged PO; group gives a symmetric mode (v,) responsible for the strong Raman band and medium intensity infrared peak near 1080 cm-', whereas the antisymmetric mode (v,) gives a strong infrared band, with little corresponding Raman activity, at ca cm-'. The phosphodiester -0-P-0- group is also characterized by symmetric and antisymmetric components, which give, respectively, a strong maximum near 750 cm-' and a weaker band at 810 cm-' in the Raman spectrum. The two strong bands at 1038 and 1055 cm-' in the infrared, with relatively weak Raman counterparts, are usually assigned to C-0 vibrations, and that at 953 cm-' to a C-C stretching mode (2, 13, 14, 16). The weak band at 1105 cm-', present in both types of spectra, has generally been identified as a compo- nent of the v,(po;) vibration in the solid phase spectra. Its presence in the solution spectra, its absence in the spectra of dimethyl phosphate, and the fact that it is only weakly active in the spectra of methyl ethyl phosphate (15, 16) suggest that the band corresponds to a fundamental vibration of the ethyl group. Most of the weaker bands at frequencies higher than 1100 cm-' arise from vibrations of alkyl groups. Normal coordinate calculations have indicated that vibrational frequencies of the phosphate group in DEP vary with the molecular conformation (14, 15, 17). However, these effects are small and we have assumed that the spectral changes resulting from the addition of metal cations to DEP were not caused by changes in the distribution of these structures in the solutions. Protonation of DEP causes important changes in the vibrations of the PO; group. The infrared and Raman bands at 1080 cm-i, caused by the v, vibration of this group, are practically absent from the spectra of a strongly acidic solution (Fig. I). Similarly, the strong infrared band at 1201 cm-i, assigned to v,, and that at 1055 cm-' show an -pp a reciable loss in intensity, whereas the band at 1105 cm in the Raman spectrum displays the opposite behaviour. It seems that the addition of another single P-0 bond to the system affects the mixing of P-0, C-0, and C-C stretching vibrations, displacing the 1080 and 1055 cm-i bands of the anion to 1030 cm-i, while the C-C vibrational band at 953 cm-i loses most of its intensity. The composite band at 1029 cm-' in the infrared spectrum of the acid solution has appreciable intensity, but the corresponding band in the Rarnan spectrum is quite weak. In a previous study on the interaction of A~' ions with dialkylphosphates (13), it was proposed that the v, vibrational band of the anion at 1200 cm-i in neutral solution is displaced to ca cm-i upon covalent neutralisation of the phosphate group. We rather believe that the 1200 cm-i band is displaced, with reduced intensity, to ca cm-' in the infrared spectrum of the acid solution, the increased intensity in the 1150 cm-' region being due, at least in part, to the v, vibration of H,O+ ions (21). One also notes that the Raman band due to the symmetric stretching mode of the phosphodiester --O--P-0- group is shifted to 746 cm-' at low ph, its intensity being enhanced in the process. The relative height of the 1081 cm-i Raman band of the DEP anion shows that this species remains ionized in the ph range (see insert, Fig. 1). The changes occurring in the infrared and Raman spectra of DEP as a result of interaction with metal ions are usually small, especially at low metal concentration. Minor spectral modifications are more easily seen in the difference spectra, which are obtained by subtracting the spectrum of DEP from that of the corresponding solutions in the presence of metal ions. This can be done in a straightforward manner with infrared spectra if the sample thickness and concentration are very accurately known, which is seldom the case. In Rarnan spectroscopy, to determine the proper value of the constant to be used in the subtraction operation ([DEP + metal] - Kx [DEP]), a band present in both spectra and insensitive to the effect of the metal ions is needed as a reference intensity standard. We have used the Raman bands of the alkyl groups in the cm-i range for this purpose, since they should not be affected by perturbations of the PO; group. In what follows, we will use a normalized multiplication constant, K, which is chosen in each case to yield the value

3 STANGRET AND SAVOIE 2877 FREQUENCY (crn-1) FIG. 2. (A) Raman spectrum of an aqueous solution of diethyl phosphate (0. lm, ph 7.5) at 20 C. (B-F) Difference spectra (intensity multiplied by 6) obtained in the presence of NiCll at a Ni : DEP molar ratio of (B) 0.025, (C) 0.25, (D) 1.5, (E) 3.0, and (F) (100%) when the reference bands used as intensity standards are completely annulled in the difference spectrum. As an example of the use of difference spectra, the curves obtained from aqueous solutions of DEP in the presence of Ni(I1) at metal : DEP ratios varying from : 1 to 5 : 1 are given in Fig. 2. These difference spectra show that the spectral changes depend on the relative metal concentration. At very low metal :DEP ratio, one observes a slight shift to lower frequency of the v,(po,) band at 1081 cm-i and a weak perturbation in the phosphodiester vibrational region near 800 cm-i. At higher metal concentration, on the other hand, the v,(po;) band is shifted to higher frequency, with some loss in intensity. Band shifts and intensity changes are also apparent in the cm-' region (C-0 stretching vibrations). Another way in which spectral changes can be more easily visualized is through the "affected spectrum" (22-24), obtained by incompletely subtracting the reference spectrum to yield a positive difference curve in the region of interest. This is illustrated in Fig. 3, which shows the difference spectra obtained by subtracting the spectrum of an aqueous solution of DEP (O.1M) from'that of the same solution in the presence of Ni(I1) at a Ni: DEP molar ratio of 6: 1, for various values of the normalized multiplication constant K. In the present study, the retained affected spectrum for the mixed solution with Ni(I1) was the positive difference spectrum, obtained with the maximum value of K, which could be fitted with calculated band components. The program used for curve fitting was the FIT program from Spectrum Square Associates, Inc. (Ithaca, NY, U.S.A.), run under SpectraCalc (Galactic Industries Corporation, Salem, NH, U.S. A.). The difference between calculated and experimental spectra was taken to be: N 2 - i=l X - [(data (vi) - calc (vi))/~rnsl2 N-F FREQUENCY (cm-1 ) FIG. 3. (A) Raman spectrum of an aqueous solution of (-) diethyl phosphate (DEP) (O.lM, ph 7.5) and (---) the same solution with added NiC1, (DEP + metal) at a Ni:DEP molar ratio of 6: 1. (B) Subtracted spectra (intensity multiplied by 5) ([DEP + metal] - K X [DEP]) for different values of the normalized multiplication constant K (see text). in which N is the number of data points, F is the number of degrees of freedom, v is the frequency, and Rms is a measure of the noise in the spectrum. Taking Fig. 3 as an example, it was observed that negative peaks occurred in the difference curves for values of K approximately higher than To fit the difference spectra for this value of K and lower, five components (of mixed Gaussian and Lorenztian shape) were needed. The fit between calculated and experimental spectra, as determined from the value of x2, decreased by an order of magnitude as K was lowered from 0.88 to At this point the value of x2 as a function of K passed through a minimum, and the difference spectrum obtained with K = 0.85 was therefore taken as the affected spectrum. This procedure worked well in general, although in some cases the function x2 = f (K) gave a very broad minimum or merely levelled off with decreasing K. The calculated spectrum for the example given above is compared with the affected spectrum in Fig It is obvious that the manipulations of the spectral data needed for the complete analysis of the effect of a particular type of metal ion on the spectrum of DEP were very time-consuming, so that the whole procedure was used only in the case of Ni(I1) in the present study. In the other cases, qualitatively affected spectra chosen by visual inspection were retained. The affected spectrum can be very meaningful for a system limited number of discrete components. In this case, the above example is interpreted as follows. Since the final value of K was 0.85, we surmise that 85% of the spectrum of DEP remained unaffected by the metal ions under our experimental conditions. We also conclude that the affected spec-

4 CAN. J. CHEM. VOL. 70. I992 I I I FREQUENCY (cm-1) FREQUENCl (em-') I I I I FREQUENCY (cm-') FREQUENCY (cm-1) FIG. 4. Band fitting of (A) the Raman spectrum of aqueous diethyl phosphate (DEP) (0. lm, ph 7.5) and of (B-D) the affected spectra obtained from solutions of DEP containing NiCl, at a Ni: DEP molar ratio of (B) 0.1 : 1, (C) 2: 1, and (D) 6: 1. (-) Calculated spectra. trum obtained by the subtraction procedure (at K = 0.85) represents 15% of DEP molecules. This interpretation can be questionable, particularly in the case of weak band shifts caused by bulk effects in the solution and affecting all solute molecules. It remains, however, that the affected spectrum in these conditions constitutes a very sensitive indicator of the band shift. The affected Raman spectra in the cm-' of solutions of DEP in the presence of Ni(I1) at various metal : DEP ratios (Fig. 4) are particularly revealing. Theoretical band fitting of the spectrum in aqueous DEP alone necessitates four components, the major one being associated to the v,(po;) vibration at 1081 cm-i (Fig. 4-A). For the affected spectrum from the solution at Ni:DEP ratio of 1 :40 (Fig. 4-B), the same number of components is needed, but the main band is shifted to a lower frequency by 3 cm-', which is consistent with the effect observed in the difference spectrum (Fig. 2-B). At higher metal :DEP ratio (Fig. 4-C, D), an extra component is needed at 1089 cm-' to fit the affected spectra. This extra band becomes progressively more important as the metal concentration is increased. A shift to lower frequency of the 1037 and 1056 cm-' bands of DEP, with enhancement of the lower frequency peak, is also observed in the affected spectra, in accordance with the derivative features present in the different spectra for these solutions (Fig. 2). This behavior, which will be discussed more fully below, indicates that DEP interacts with Ni(I1) in different ways, depending on the concentration of the metal cation in the solution. In an attempt to determine the number of components required to explain the spectral data, we have performed a factor analysis using the cm-' region of the Raman szectra of 16 solutions of DEP (0.1 M) in the presence of Ni-, at metal: DEP ratios ranging from 6: 1 to 0 (pure DEP). (The analysis was done using the PLSPLUS option of SpectraCalc; see ref. 25 for details on factor analysis.) The results of this analysis suggested that four major components are necessary to adequately describe the chosen ensemble. Difference and affected Raman spectra for solutions of DEP (0.3M) in the presence of the cations K+, cap, and CU'+, at a meta1:dep ratio of 1 : 1, are illustrated in Fig. 5. The corresponding curves for a solution at ph = 1 (effect of the H,O+ ion) are also reproduced. (The affected spectra for these mixtures are qualitative only, as they were not subjected to the band fitting procedure used with the Ni(I1) solutions.) The subtracted spectra ~enerally consist of a differential feature in the 1080 cm- region, indicative of a shift to higher frequency of the v,(po;) vibrational mode, and a slight positive peak at 750 cm-', resulting from an increased intensity of the band due to the symmetric phosphodiester stretch. The difference spectra of the divalent metal cations also show a decreased intensity in the 1081 cm-' region and extra components on the low-frequency side of the main peak. The difference spectrum obtained with a solution at ph 1 versus that at ph 7, which shows the effect of protonation, is very similar to that obtained from a CaCl, solution, although the intensity loss at 1080 cm-' is much larger for the acid solution. The affected spectrum, on the other hand, closely resembles the raw spectrum of a DEP solution at very low ph (Fig. l), as expected.

5 STANGRET AND SAVOIE - m - m '2 FREQUENCY (cm-i) FIG. 6. (A) Infrared spectrum of an aqueous solution of diethyl phosphate (DEP) (O.lM, ph 7). Affected spectra obtained from solutions of DEP in the presence of (B) CaCl, and (C) NiCl? at a metal : DEP ratio of 1 : 1. The information provided by the affected spectra in infrared are complementary to those provided by the Raman technique. An example of application is shown in Fig. 6, which shows the PO; stretching vibrational region of the affected spectra obtained from DEP (O.1M) in the presence of ca2+ and Ni2+ at a metal : DEP ratio of 1 : 1. The ~a'+-affected spectrum is typical in that it shows a shift to lower frequency of the bands at 1038 and 1055 cm-', whereas the peaks at 1080 and 1201 cm-', arising from the stretching vibrations of the PO; group, are shifted in the opposite direction. The effect of Ni2+ is clearly different, the v,(po;) mode at 1080 cm-i remaining essentially invariant, while the v,(po,) mode at 1201 cm-' is shifted to a lower frequency. The ph of the solution has some effect on the frequency and intensity of the bands of DEP within the range in which the phosphate group is ionized. The effect of metal ions also FREQUENCY (crn" ) FREQUENCY (crn-1) FIG. 5. (A) Raman spectrum of an aqueous solution of diethyl phosphate (DEP) (0.3M, ph adjusted to 7 with NaOH). Difference (left) and affected (right) spectra (intensity multiplied by 3) obtained from solutions of DEP in the presence of (B) KCl, (C) CaCl?, and (D) CuC1, at a cation: DEP ratio of 1 : 1. (E) Corresponding curves (intensity multiplied by 1) for a solution of DEP at ph 1. The % values indicate the amount of affected spectrum (see text). differ from that at neutral ph. This is illustrated in Fig. 7, where the spectra of solutions brought to ph near 12 by addition of LiOH, NaOH, and KOH are compared with a solution adjusted to ph 7 with the same base. Increasing the ph to 12 results in a shift to lower frequency of the v,(po;) band. Discussion As shown in Fig. 5, the difference and affected spectra obtained from a series of solutions of DEP in the presence of different metal ions indicate that the complexation of this type of cations by ionized phosphate groups depends on the nature of the metals. As our measurements have been particularly extensive on the DEP-N~'+ system, this particular case will be discusseci first, so that the conclusions can be extended to other systems. DEP - ~i'+ system The vibrational spectroscopic results gathered in this study indicate that the interaction of Ni2+ cations with the phosphate group of DEP is a function of metal concentration. This is particularly evident when one compares the difference and affected spectra (Figs. 2 and 4) obtained from solutions at metal : DEP ratios of : 1 and 6: 1. We believe that the four spectral components indicated by the factor analysis for this ensemble reflect the existence of phosphate groups of DEP in four different types of environment, which we tentitavely identify as follows: Species A: These are ionized PO; groups as they are found in neutral aqueous DEP (Naf salt). They are for the most part hydrogen-bonded to water molecules and they interact electrostatically with their neighboring Na+ counterion. These interactions are, on average, symmetrical with respect to the two oxygen atoms of the charged phosphate groups. Such a system is characterized by an intense v,(po;) Raman band at 1081 cm-' (Fig. 4-A). Species B: These species consist of ionized PO, groups interacting with Ni2+ ions through water molecules (outer-sphere inter-

6 CAN. J. CHEM. VOL. 70, 1992 r [NOOH, p ~ 51-[NOOH = ~ neutral] ~ D KOH, ph- I; I FREQUENCY (crn-0 FREQUENCY (crn-0 FIG. 7. (A) Raman spectrum of an aqueous solution of diethyl phosphate (DEP) (0.3M, ph 7). B-D (left) difference and (right) affected spectra (intensity multiplied by 6) obtained from solutions of DEP at high ph (conditions indicated). The % values indicate the amount of affected spectrum (see text). actions or, more generally, formation of water-separated ions pairs). The effect of the metal cation in this ~i'+ -.. O(H)-H -0,P(OEt)2 type of aggregate is to increase the acidity of the water molecule and to strengthen the hydrogen bond affecting the phosphate group, causing a decrease in frequency of the v,(po,) vibration. Typically, the spectrum of such species corresponds to the affected spectrum obtained at very low metal : DEP ratio, such as in Fig. 4-B. Note that this spectrum is very similar to that of aqueous DEP alone, except for the displacement of the v,(po;) vibrational band to 1078 cm-i. Another noteworthy difference is the absolute intensity of this peak, which is increased considerably in the presence of the metal cation. This causes a positive peak to appear in the difference spectrum, as in Fig. 2-B. The effect is also apparent when the intensity of the v,(po;) band of DEP near 1080 cm-' is plotted as a function of the relative metal concentration, which shows a maximum at a Ni : DEP ratio of 0.25 : 1 (Fig. 8-B). The increased intensity of the 1078 cm-' band for the B- type species, compared to that of the corresponding band at 1081 cm-i for the A-type variety, has to be taken into account when attempts are made to relate the intensity of the 1078 cm-i component (see Fig. 8-C) to the actual concentration of the B-type species in solutions of various meta1:dep ratios. Even so, this curve displays a very peculiar shape in lowinefa1 concentrations, an effect which is reflected in the graph giving the total proportion of DEP affected by ~i,' ions (Fig. 8-A), as determined by the bandfitting procedure described in the "results" section above. These findings, which are even more striking when one takes into account the total amount of affected DEP (Fig. 8-D), clearly show that the number of DEP molecules linked to ~ i'+ ions by outer-sphere coordination is proportionally much larger at low metal concentrations. This can be explained by the fact that this type of bonding is favored on an entropy basis: when the metal concentration is small, it is relatively easy for the ~ i'+ ions to find, within a few layers of water molecules, one or several phosphate groups with which it can interact through these solvent molecules. As the metal concentration is increased, more and more metal ions find their way close to the phosphate groups, and more direct inter- actions take place, at the expense of outer-sphere coordination. Species C: These are Ni+-0-P(=O)(O-~t), species, analogous to protonated DEP, with the acid proton replaced by the metal cation. The Rarnan spectrum of these unsymmetrically bound ion pairs are likely similar to that of protonated DEP (see Fig. l), which is characterized by a 90% reduction in intensity at 1081 cm-i and a 40% reduction at 1055 cm-i, a shift to 1031 cm-' of the band at 1038 cm-' in DEP solution at ph 7, and an increase by a factor of 2.3 of the intensity of the peak at 1105 cm-l, which is shifted to 1100 cm-i. The frequency shift of the low-frequency band from 1039 to 1031 cm-i, and the increased intensity of this band and of that near 1100 cm-i when the Ni : DEP ratio is raised from to 1 (Fig. 4-B, C), are likely due to the formation of these metallated species. Even more characteristic is the negative component at ca cm-i in the difference spectra (Fig. 2), which shows the decrease in intensity of the v,(po,) band as the meta1:dep ratio is raised. Also apparent in these spectra are the reduced intensity at 1055 cm-' and the shift to lower frequency of the 1038 cm-' band. Species D: The existence of these species has to be inferred to account for the component growing at 1090 cm-i in the affected spectra at high metal concentrations (Fig. 8-C, D). The intensity of this band grows more slowly than that of the other components of the affected spectra at low metal: DEP ratios, but it does not level off at Ni:DEP ratios >3 as the others do. This suggests that the concentration of the species responsible for the 1090 cm-' band become proportionally more important at high metal concentrations. We believe that species of this type are ionized phosphate groups which interact in a non-specific electrostatic way with metal cations. A partial disruption of the hydrogen bond network with water molecules in the neighborhood of the phosphate groups probably results from these interactions at increased metal concentrations. The effect is then equivalent to a partial dehydration of the PO, groups, which is known to displace the v,(po,) vibrational frequency to higher values (17, 26). It is also possible that the PO, angle of the charged I

7 STANGRET AND SAVOIE - 0,99h Ni / DEP FIG. 8. Variation of spectral parameters obtained from a O.1M aqueous solution of DEP with added NiCl, as a function of the Ni:DEP ratio. (A) Fraction of the spectrum affected, (B) relative peak-height intensity of the 1081 cm-' Raman band, (C) intensity of calculated spectral components, and (D) intensity of calculated spectral components (given in C) divided by the fraction of affected spectrum (given in A). phosphate group in DEP is modified by the presence of the metal cations, causing a modification of the vibrational frequencies of this group (17). Note that type-a species are equivalent to those of type D, except that the statistically symmetrical interactions involved in the latter case are more important or consequential than with the ~ a counterions + used in the reference solution (DEP-Na solution at ph 7). DEP - other metal-ion systems The conclusions from our analysis of the ~i'+-dep spectra can now be extended to the other metal-dep systems. Unless otherwise specified, the spectra discussed below are for a 1 : 1 metal ch1oride:dep ratio; indicated ph values refer to the cation/dep solutions. The simplest metal ions to be considered are those of the alkaline metals: Li+ (ph = 6.4), Na+ (ph = 7.4) and K+ (ph = 7.2). As shown in Fig. 5, the difference and affected Raman spectra for K+ are indicative of a mere shift, with no loss in intensity, of the v,(po,) vibrational band. This is corroborated by the shape of the affected spectrum, which is very similar to that of the original DEP spectrum. The frequency shift is rather small (ca. 2 cm-i) for K+ and Na+ (as determined from a Na+ solution at 2: 1 Na:DEP ratio), and slightly larger (ca. 4 cm-i) for Li+. These results show that the alkaline-metal ions interact electrostastically in a general manner with charged phos- phate groups, these interactions being essentially symmetrical with respect to the two oxygen atoms. It is interesting to note that in a solution at a much larger Na+ : DEP ratio of 18, the frequency of the v,(po;) band (spectrum not shown) is essentially identical to that at a metal: DEP ratio of 2: 1 (ca cm-i). The affected spectrum at high metal ion content, however, shows an increased scattering at cm-', with corresponding intensity decrease at 1055 cm-i. This observation, together with the decrease in intensity (by approximately 5%) at 1081 cm-', suggests that unsymmetrical interactions (analogous to those yielding type-c species) take place to some extent at high salt concentrations. Alkaline-earth metal ions appear to have an intermediate behavior towards DEP, by comparison with alkaline and transition metal ions. As shown in Fig. 5-C for ca2+ (ph = 6.7), the frequency shift of the v,(po;) band is positive, but larger than with K+ (1088 versus 1083 cm-'), indicating generally stronger interactions with the PO; groups. A slight overall decrease in intensity is also revealed in the v,(po,) region of the difference spectrum, suggesting some degree of asymmetrical interaction with the oxygen atoms of the phosphate groups. The decrease intensity at 1055 cm-' in the affected spectrum supports this conclusion. Note that the difference spectrum in this case bears some resemblance to that for a solution of DEP at ph 1 (Fig. 5-E), for which a

8 2882 CAN. J. CHEM. \ good proportion of the phosphate groups are protonated. The affected spectrum (not shown) for a M~"-DEP solution (ph = 6.8) shows an extra component at 1077 cm-', indicating that this metal cation is more involved than ca2+ ions in the formation of water-separated ion pairs with the phosphate groups. Transition metal ions, such as ~ i'+ ( p H = 6.2) (Figs. 3 and 4), CU" (ph + = 3.4) (Fig. 5-D), Zn (ph = 5.l), and ~ d "(ph = 6.3) (spectra not shown) behave in the same manner. Their affected spectrum contains a high-frequency component at ca cm-i, characteristic of symmetric electrostatic interactions with the PO; groups, as well as a low-frequency band resulting from outer-sphere coordination. Their difference spectrum further shows a decrease in intensity in the v,(po;) region, indicating asymmetric interactions with the oxygen atoms of the phosphate groups. This contrasting behavior compared to alkaline-earth metal ions is especially obvious in the v,(por) region of the affected infrared spectrum (Fig. 6), where the band at 1201 crn-i in aqueous DEP is shifted to 1231 cm-' in the presence of Ca2' and 1190 cm-i in a ~ i solution. ~ + The affected spectra (not shown) of DEP solutions in the presence of ~ g (from + AgClO,; ph = 6) and pb2+ (ph = 4.2) unexpectedly display a low-frequency component only, at ca cm-'. This is particularly surprising in the case of Ag+, as the infrared spectrum for the solid silver salt of DEP is suggestive of the formation of a partly covalent (therefore asymmetrical) bond with PO; groups (13). The present results suggest that both Ag+ and pb2+ in solution have a relatively high affinity for water molecules, and that they interact with the phosphate groups through the solvent molecules, forming water-separated ions pairs (type-b species). The trivalent cations ka3+ (ph = 3.5) and A?+ (ph = 6,l) also behave in a peculiar way, their affected spectrum (not shown) containing mainly a high-frequency component at 1090 cm-' with very weak accompanying peak at 1078 cm-i. However, the fraction of affected spectrum with these cations (25-30%) is much higher than with monovalent and divalent metal ions. Furthermore, a considerable intensity loss occurs at 1081 cm-i, suggesting that these ions can also interact directly, in an asymmetric way with the oxygen atoms of the PO,- groups. Otherwise, the interactions of the phosphate groups with these highly charged species, which are no doubt extensively solvated, appear to be non specific. It is also worth mentioning that the mixture with A1C13 at ph = 3.3 yields anaffected spectrum which is quite similar to that obtained with DEP at ph 1 (Fig. 5-E). This probably results from a partial protonation of DEP under these circumstances. Effect of high ph The neutralization of aqueous solutions of DEP with either LiOH, NaOH, or KOH has the same effect on the vibrational spectra of this compound. In particular, the frequency of the v,(po;) in the resulting solutions is the same (within 1 crn-i). However, raising the ph of such solutions to ca. 12 has a marked influence on this frequency, even though the concentration of OH- ions is much lower than that of DEP. As shown in Fig. 7, the effect depends on the nature of the cation. The frequency shift of the v,(po;) vibrational mode in the affected spectrum from the LiOH solution is quite large (14 cm-i), suggesting a strong perturbation of the phosphate groups in the ca. 5% affected DEP molecules. We tentatively explain this behavior by the formation of OH-. Li+--O-P(=O)(O-Et)2 species, in which a LiOH molecule comes in close contact with one of the two oxygen atoms of the charged phosphate group. The effect is different in the KOH solution, which causes only a small frequency shift (3 cm-l) of the v,(po;) mode. This minor effect probably results from small changes in the distribution of charged species in the vicinity of phosphate groups, as the affinity of K+ cations for OH- ions is not expected to be exactly the same as that for the negatively charged phosphate groups. The affected spectrum from the NaOH solution shows peaks at both 1067 and 1078 cm-i, indicating that both types of structures are present in the mixture. Conclusion The present results indicate that metal cations have different affinities for charged phosphate groups and also that they interact in different ways with these groups. The vibrational spectra show that such interactions can have different and even opposite effects on the intensity and frequency of the vibrational bands of the PO; unit. These changes can be interpreted in terms of direct metal binding by one of the oxygen atoms of the PO; groups, non-specific electrostatic interactions, and formation of water-separated ion pairs with the phosphate groups. It can be concluded from these observations that the decrease in intensity of the band due to the symmetrical stretching mode of charged phosphate groups, which is prominent near 1080 cm-' in the spectra of several types of biomolecules (phospholipids, nucleic acids...), is only partially characteristic of the extent of metal binding by these groups, as it arises mainly from interactions of the first kind. Acknowledgements We are indebted to the Natural Sciences and Engineering Research Council of Canada and to the Ministkre de 1'~ducation du Qukbec for financial support. 1. J. K. Barton and S. J. Lippard. In Nucleic acid - metal ion interactions. Edited by T. G. Spiro. Wiley, New York pp L. G. Marzilli, T. J. Kistenmacher, and G. L. Eichhorn. 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