Langmuir 1997, 13, 3529-3541
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Electrochemistry of Covalently Mercurated Uridine Nucleotides. Diffusion and Surface-Confined Pathways for the Two-Electron Reduction of the Carbon-Mercury Bond Gregory J. Wagner and James Q. Chambers* Department of Chemistry, The University of Tennessee, Knoxville, Tennessee 37996 Received January 30, 1997. In Final Form: April 14, 1997X The electrochemical reduction of 5-mercuriuridine monophosphate, diphosphate, and triphosphate nucleotides at mercury electrodes in aqueous solution is reported. Both diffusion-controlled and surfaceconfined electrode reactions were observed. In aqueous KCl electrolytes, the diffusion-controlled pathway involved the two-electron reduction of the carbon-mercury bond to give the parent nucleotide. In D2O solution, the corresponding 5-deutero nucleotide was formed. All three mercurated nucleotides formed compact monolayers on the mercury electrode surface in accord with the Langmuir isotherm. Anomalies in the voltammetric behavior and the adsorption isotherm of the 5-mercuriuridine monophosphate in KCl solution are ascribed to the formation of polymeric anions by coordination of the mercury substituent with the nitrogen of the pyrimidine ring. The reduction mechanism of the surface-confined nucleotides is more complex than the diffusion-controlled reaction. For the surface reaction, two one-electron steps (an EE reaction) were observed. The organomercury radical (RHg•) one-electron intermediate formed a compact surface layer that was stable on the voltammetric time frame. This species participates in a surface square scheme that is operative at mercury electrodes over the pH ) 2-12 range. Finally, evidence is presented that implicates kinetically controlled nucleation phenomena in the surface reactions of the adsorbed intermediates in the electrode processes.
A study of the electrochemical reduction in aqueous solutions of the mono-, di-, and triphosphate uridines that are mercurated in the 5-position of the pyrimidine ring is reported in this paper.1 From a mechanistic viewpoint, these molecules are of interest because they possess several different functionalities that determine the course of the electrode reactions. First and foremost is the C-Hg bond which is the locus of the reduction process. Previous studies of simpler organomercury compounds of the type R-HgX have shown that electroreduction proceeds in a two-step process via a radical intermediate
R-HgX + e- f R-Hg• + X-
(1)
R-Hg• + HA + e- f R-H + Hg + A-
(2)
where HA is a proton donor, usually water.2 Second, since the X- moiety can represent both the phosphate group and ring-based nitrogen ligands of the nucleotide, both intra- and intermolecular complexation of the Hg(II) atom are possible. Also it is well-known from double layer capacity measurements and related studies that there are strong adsorption forces between the ring systems of simple pyrimidines and the electrode surface.3 Finally, these molecules are polyprotic acids, a property that impacts all three of the previous factors. Another motivation for this study is that the introduction of the mercury substituent represents a method to tag a complex biomolecule with an electroactive group. If the -HgX substituent does not alter the properties of the molecule to a significant degree, its voltammetric response X
Abstract published in Advance ACS Abstracts, June 1, 1997.
(1) This paper is taken from the Ph.D. thesis of Gregory J. Wagner, Ph.D Thesis, University of Tennessee (Knoxville), 1996. (2) (a) Morris, M. D.; Kok, G. L. In Encyclopedia of the Electrochemistry of the Elements (Organic Sect.); Bard, A. J., Lund, H., Eds.; Marcel Dekker: New York, 1979; Vol. XIII, (b) Benesch, R.; Benesch, R. E. J. Am. Chem. Soc. 1951, 73, 3391. (c) Hush, N. S.; Oldham, K. B. J. Electroanal. Chem. 1963, 6, 35. (d) DeGrand, C.; Laviron, E. Bull. Soc. Chim. Fr. 1968, 2228. (e) Laviron, E.; Roullier, L. Electrochim. Acta 1973, 18, 237. (3) de Levie, R. Chem. Rev. 1988, 88, 599.
S0743-7463(97)00096-6 CCC: $14.00
can then be used as the basis for an analytical procedure in complex systems. An example of this strategy employing an organomercurial tag is the estriol hormone differential pulse polarographic procedure of Heineman et al.4 Thus the present study was undertaken to characterize fully the voltammetric response of the three modified nucleotides; extension to oligo- and polynucleotides is envisioned. Several years ago, Dale and co-workers showed that the pyrimidine base of nucleotides and polynucleotides reacted with Hg(II) acetate to give the 5-mercuripyrimidine product.5-7 These researchers found further that the covalently bound mercury atoms did not alter the polynucleotide structure or the interactions of the polynucleotides with proteins and enzymes. Following Dale and co-workers, we have studied the electrochemical behavior of the 5-mercuriuridine nucleotides prepared from uridine monophosphate (UMP), uridine diphosphate (UDP), and uridine triphosphate (UTP) in aqueous solution over a wide pH range. In this paper, the 5-mercurated derivatives will be designated as UZP-HgX when referred to as a set of nucleotides. However, it will become evident that the monophosphate compound (Z ) M) has some distinctive electrochemical properties not shared by the other two members of the group. The two-electron reduction of the pyrimidine carbonmercury bond takes place at relatively positive potentials at both glassy carbon and mercury electrodes. However, as indicated above, the electrochemical behavior is dependent on the interplay of several chemical factors that relate to the complexity of the molecular structure of the modified nucleotides. The mainly cyclic voltammetric study reported below provides new insight into the mechanistic details of the electrochemical reduction of organomercury compounds. (4) Heineman, W. R.; Anderson, C. W.; Halsall, H. B. Science 1979, 204, 866. (5) Dale, R. M. K.; Livingston, D. C.; Ward, D. C. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 2238. (6) Dale, R. M. K.; Martin, E.; Livingston, D. C.; Ward, D. C. Biochemistry 1975, 14, 2447. (7) Dale, R. M. K.; Ward, D. C. Biochemistry 1975, 14, 2458.
© 1997 American Chemical Society
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Experimental Section Chemicals. The electrolyte and buffer solutions were prepared with reagent grade materials purchased from Sigma or Aldrich Chemical Co. All water was distilled and passed through a Millipore purification filter system. The uridine mono-, di-, and triphosphates were obtained from Sigma. The compressed nitrogen gas (Natl Welders Supply, Co.) used to deaerate the solutions was passed through a column containing MnO dispersed on a vermiculite support.8 The 5-mercurated derivatives of UMP, UDP, and UTP were prepared by the method of Langer et al.9 This method consisted of the addition of 0.368 g of UMP, 0.404 g of UDP, or 0.484 g of UTP (1.00 mmol) to 100 mL of 0.1 M acetate buffer, pH ) 6.0. Then 1.59 g (5.00 mmol) of Hg(II) acetate was added, and the solution was heated for 4 h. After the mixture cooled to room temperature, 0.392 g (9.25 mmol) of LiCl was added and the excess HgCl2 removed by extraction with equal volumes of ethyl acetate. The progress of the extraction was monitored by evaporating each ethyl acetate extractant solution, dissolving the residue in 0.1 M KCl, and determining the amount of Hg(II) present by anodic stripping voltammetry at a glassy carbon electrode. By the fifth extraction, no voltammetric wave assignable to the Hg(0) oxidation was evident. The mercurated nucleotide in the aqueous layer was then precipitated by the addition of three volumes of cold ethanol and collected by centrifugation. In some cases the product was redissolved in LiCl solution and reprecipitated with ethanol. The product was washed with cold ethanol, followed by acetone, and dried under vacuum. For the 5-mercuriuridine monophosphate, elemental analysis (Galbraith) gave: C, 16.94; H, 3.06; N, 4.35; Hg, 32.57. Expected for C9H10N2O9PLi2HgCl‚3H2O: C, 17.32; H, 2.60; N, 4.49; Hg, 32.14. Several experiments were also performed with a small quantity (5 mg) of the 5-mercuriuridine triphosphate obtained from Sigma Chemical Co. The voltammetric results with this material were identical to those obtained with compound prepared by the method of Langer et al.9 The downfield region of the NMR spectra of the mercurated uridines demonstrated that the extent of substitution at the 5-position was complete; see Table 1. The mercurated nucleotides were also analyzed by potentiometric (pH) titration with standard HCl. The titration curve for UMP-HgX, which pertains to the unique behavior of this compound, is shown below (Figure 17). Electrodes. Glassy carbon, dropping mercury, and static mercury drop electrodes were employed as working electrodes. The capillary of the static mercury drop electrode (HDE) was periodically silanized with a 5% solution of (CH3)2SiCl2 in CCl4. The electrode area of an individual HDE was given by a calibration curve that was determined by previously weighing mercury drops formed by reproducible turns of the capillary screw-piston. The relative standard deviation of the electrode area measurement was (2%. All potentials were measured vs a commercial SCE reference electrode (Corning). A coil of Pt wire served as the counter electrode. For the coulometric measurements, the counter electrode was isolated by a glass frit. Most of the experiments were conducted at ambient laboratory temperature, 21 ( 3 °C. Instrumentation. Most of the voltammetric work was carried out using a Model CS-2RA Cypress Systems potentiostat. This instrument applies a staircase waveform in the “CV” mode, so (8) Brown, T. L.; Dickerhoot, C. W.; Bafus, D. A.; Morgan, G. L. Rev. Sci. Instrum. 1962, 22, 491. (9) Langer, R. P.; Waldrop, A. A.; Ward, D. C. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 6633.
Wagner and Chambers the voltammograms in the figures below are staircase voltammograms. A potential step of 1 mV was used throughout these studies. Proton NMR spectra were obtained with a Bruker 250 MHz NMR spectrometer and 31P NMR spectra with a Bruker 400 MHz spectrometer. The solvent for the NMR studies was D2O. For the 31P NMR spectra an external standard of 0.1 M phosphoric acid was assigned a chemical shift of 0.0 ppm. For characterization of the UZP-HgX compounds by proton NMR (see Table 1), equimolar amounts of mercaptoethanol were added to the solutions, which sharpened the down field resonance lines considerably. However, no mercaptoethanol was added in the studies aimed at establishing the complexation state of the mercury atom substituent. A HP Model 8452A diode array spectrophotometer was employed for the UV-vis measurements. Procedures. The concentration of the UZP-HgX solutions was usually determined by measuring the absorbance of the solution. Except for a small shift in the absorption maxima (ca. 5 nm), the spectra of the mercurated and the parent nucleotides were identical: UTP ) 8890, UDP ) 8620, and UMP ) 8560 L mol-1 cm-1. This avoided problems with the absorption of water by the mercurated nucleotide samples. However, solutions prepared from samples, which had been weighed on an analytical balance after being rinsed with acetone and air-dried, typically gave less than a 5% deviation in the concentration from the spectroscopic value. The buffer solutions were prepared from mixtures of the appropriate mono-, di-, and tribasic potassium phosphate compounds. The pH of the solutions measured with a laboratory pH meter was within (0.05 pH units of the values calculated from the Henderson-Hasselbalch equation. Coulometry was perform at both a 10 cm2 Hg pool and a large area glassy carbon electrode. The progress of the reduction was followed by classical polarography; n-values were determined from the plot of the limiting current vs coulombs passed. Calculation of the Voltammograms. The staircase voltammograms were calculated by a program that called Nernst and kinetic subroutines after increments of the electrode potential.10 After each potential step, ∆E, the Nernst condition was satisfied, and then the surface concentrations were adjusted for first-order decay of the intermediate species; i.e., Γi ) Γi° exp(-k ∆t), where Γi is the surface coverage in mol/cm2 of the ith species, k is a first-order rate constant, ∆t is the time increment equal to ∆E/v, and v is the sweep rate. The total surface concentration, i.e., ∑Γi, was assumed to be constant and contributions from diffusing species negligible. The current for each potential step was given by -F(area)∑j∆Γj/∆t, where the summation was taken over the species in the oxidized form. The calculated voltammograms were in agreement with the literature analytical solution of the problem for the simple case of a surface EC reaction.11 For the calculations that assumed a preceding chemical equilibrium step that was sluggish on the voltammetric time scale (see eq 8 below), a perturbation from the equilibrium state was calculated from the initial conditions for the kinetic subroutine, which then relaxed toward the equilibrium condition with a rate constant (kf + kb), where kf and kb are the (pseudo)first-order rate constants for the chemical step. For all of the calculations presented in this paper, the followup chemical reactions were fast on the time scale of the experiment such that the cyclic voltammograms did not exhibit a reverse peak directly coupled to the forward current. In this situation, for the simple EC mechanism, the theoretical peak width is 66 mV for a one-electron process.11 (This is referred to as a ECirr mechanism in the text below.) In all cases, the rate constants used in the calculations for the more complex kinetic schemes were chosen such that agreement with theory was obtained for the EC mechanism at the sweep rate in question. It is of course realized that agreement between an experimental and a calculated cyclic voltammogram cannot prove a mechanism. On the other hand, a lack of agreement indicates that understanding of the process is incomplete.
Results and Discussion Cyclic Voltammetry in 0.1 M KCl. Cyclic voltammograms of 1 mM solutions of UMP-HgX, UDP-HgX, (10) This calculation has been briefly described elsewhere: Chambers, J. Q.; Scaboo, K.; Evans, C. D. J. Electrochem. Soc. 1996, 143, 3039. (11) Laviron, E. J. Electroanal. Chem. 1972, 35, 333.
Electrochemistry of Uridine Nucleotides
Figure 1. Cyclic voltammogram of 1 mM UMP-HgX in 0.1 M KCl: 0.0225 cm2 HDE; sweep rate, 1 V/s.
Figure 2. Cyclic voltammogram of 1 mM UDP-HgX in 0.1 M KCl: 0.0225 cm2 HDE; sweep rate, 1 V/s.
and UTP-HgX in 0.1 M KCl at a static HDE are shown in Figures 1-3. All three CVs exhibit two principal reduction waves, R1 and R2, at ca. -0.5 to -0.6 V and -0.8 to -0.9 V, respectively. Under these conditions, however, the voltammetry of the UMP derivative is different from that of the UDP and UTP derivatives. The latter two compounds produced nearly identical voltammetric behavior under all solution conditions of this study. The voltammetric peaks for the UDP and UTP derivatives are sharper than those of UMP-HgX and the currents return almost directly to the baseline, indications that adsorption phenomena are involved in the redox processes. This observation is reinforced by the semi-integrals of the R2 waves, which exhibit distinct peaks in the region of the peak potentials.12 The UDP and UTP voltammograms exhibited a single sharp oxidation wave (Ox1) at ca. -0.1 V, while the principal anodic current in the CV of UMP-HgX species (12) (a) Bowling, R.; McCreery, R. L. Anal. Chem. 1988, 60, 605. (b) Freund, M. S.; Brajter-Toth, A. J. J. Phys. Chem. 1992, 96, 9400.
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Figure 3. Cyclic voltammogram of 1 mM UTP-HgX in 0.1 M KCl: 0.0225 cm2 HDE; sweep rate, 1 V/s.
was a broad wave at ca. -0.5 V (Ox1′). The CVs of the UDP and UTP species also featured a cathodic peak current (R3) that appeared on the positive-going reverse sweep in the potential region at the foot of wave R2. Coulometry and Product Analysis. Coulometry (at mercury pool and at glassy carbon electrodes) and product analysis by 1H NMR indicated that reduction of all three compounds at -1.0 V proceeded by a two-electron reductive cleavage of the carbon-mercury bond. Thus coulometric n-values for the three nucleotides in 0.1 M KCl were 1.9, 2.1, and 2.3 for UMP-HgX, UDP-HgX, and UTP-HgX, respectively. Electrolyses at potentials between R1 and R2 waves were not successful for reasons that will become evident below. The NMR spectra of the electrolysis products, which were isolated by precipitation with cold ethanol, were especially diagnostic for complete electrolysis. In the presence of mercaptoethanol, the downfield regions of the NMR spectra of the 5-mercuriuridines contained sharp singlets around 8.0 ppm for H6 and a doublet at 6.0 ppm for the H1′ proton in agreement with the literature.6 NMR spectra of the isolated products of exhaustive electrolyses were identical to the spectra of the parent nucleotides, which exhibited doublets for the H5 and H6 protons in addition to the doublet for the enantiomeric H1′ proton; see Table 1. The spectra of products isolated at the midpoint of the electrolyses contained resonance lines for both the mercuriuridines and the parent compounds. Interestingly, in the absence of the mercaptoethanol, the downfield doublets collapsed into broad singlets suggestive of ligand exchange at the mercury heavy metal center. Electrolysis of the UMP-HgX species in D2O further confirmed the two-electron reduction of the uridinemercury bond. The NMR spectrum of the isolated product exhibited a singlet at 8.08 ppm and a doublet at 5.95 ppm, assigned to the H6 and H1′ protons in 5-deuterouridine monophosphate. In view of these observations, the electrode process that takes place in the region of -1.0 V can be written as follows
UZP-HgX + H2O(D2O) + 2e f UZP(D) + Hg + OH-(OD-) + X- (3) where Z ) M, D, or T signifying the mono-, di-, or triphosphate nucleotide. Support for the formation of
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Table 1. Chemical Shifts in Downfield Region of 1H NMR Spectra (D2O) of UZP-HgX and Isolated Electrolysis Products (0.1 M KCl, Hg Pool Electrode, -1.0 V) chemical shifts (δ) UMP UMP-HgX UMP-HgX, 50% electrolysis UMP-HgX, 100% electrolysis UMP-HgX, 100% electrol in D2O UDP UDP-HgX UDP-HgX, 50% electrolysis UDP-HgX, 100% electrolysis UTP UTP-HgX UTP-HgX, 50% electrolysis UTP-HgX, 100% electrolysis
8.10 (d, H6), 5.98 (d, H5), 5.94 (d, H1′) 7.98 (s, H6), 6.05 (d, H1′) 8.10 (d), 7.99 (s), 6.05 (d), 5.96 (d), 5.92 (d) 8.12 (d), 5.97 (d), 5.92 (d) 8.08 (s), 5.95 (d) 8.00 (d, H6), 5.98 (d, H5), 5.95 (d, H1′) 7.89 (s, H6), 6.00 (d, H1′) 8.00 (d), 7.89 (s), 6.02 (d), 5.98 (d), 5.96 (d) 8.00 (d), 5.97 (d), 5.94 (d) 7.95 (d, H6), 5.98 (d, H5), 5.94 (d, C1′) 7.91 (s, H6), 6.00 (d, 6.00) 7.94 (d), 7.90 (s), 6.00 (d), 5.97 (d), 5.95 (d) 7.94 (d), 5.98 (d), 5.94 (d)
Figure 4. Linear sweep voltammograms of 7.0 × 10-5 M UDP-HgX (left) and UTP-HgX (right) in 0.1 M KCl: 0.0225 cm2 HDE; sweep rates, 2, 3, 4, 5 V/s.
metallic mercury in this two-electron process is given by cyclic voltammograms obtained at glassy carbon electrodes which exhibited a large mercury “stripping” wave at +0.15 V. The nature of the ligand species, X-, which plays an important role in the detailed electrode mechanism, is discussed below. Analysis of the Diffusion Wave. Evaluation of the diffusion coefficients for the mercuriuridine species from the peak currents of the R2 wave is difficult due to the contribution of currents due to adsorbed species. However use of a modified Randles-Sevcik equation, Ipk/ACxv ) 298nxD, for an ECirrE reaction,13 indicated a two-electron n-value consistent with the above electrode reaction. Diffusion coefficients at 22 ( 3 °C in 0.1 M KCl were more accurately evaluated from both chronocoulometric transients (Anson plots) and from the chronoamperometric transients on the trailing edges of the R2 waves using the method of Whitson et al.14 These results were also consistent with a two-electron diffusion-controlled reduction and gave the following values for the diffusion coefficients: (2.6 ( 0.3) × 10-6 cm2/s for UMP-HgX, (3.2 ( 0.3) × 10-6 cm2/s for UDP-HgX, and (4.1 ( 0.3) × 10-6 cm2/s for UTP-HgX. (13) (a) Mastragostino, M.; Nadjo, L.; Saveant, J.-M. Electrochim. Acta 1968, 13, 721. (b) Meier, E. P.; Chambers, J. Q. J. Electroanal. Chem. 1970, 25, 435. (14) Whitson, P. E.; Vandenborn, H. N.; Evans, D. H. Anal. Chem. 1973, 45, 1298.
Analysis of Adsorption Currents in 0.1 M KCl. Since the diffusive flux is proportional to the bulk concentration, it is possible to minimize the voltammetric diffusion current by the simple expedient of decreasing the concentration of the mercuriuridine species. Then if the adsorption forces are strong enough to maintain a significant surface coverage (Γ, mol/cm2) of electroactive material, voltammograms can become dominated by the waves due to nondiffusing, surface-confined species. This is the situation for the three mercuriuridine species examined in this study. Figure 4 shows linear sweep voltammograms obtained at moderate sweep rates for the UTP-HgX and UDPHgX species in 0.1 M KCl. (Increasing the sweep rate will also favor adsorption currents at the expense of diffusion currents, which scale with v and v0.5, respectively.) A two-step process with a stable adsorbed intermediate is indicated by these results. Under these conditions (low bulk concentration and moderately fast sweep rates) the Ox1 wave is not present in the CVs if the potential is swept through the R2 wave. However, if the potential is reversed between R1 and R2, wave Ox1 is present as an asymmetric surface wave in the region of -0.1 V. This indicates that an adsorbed intermediate produced in the R1 process is oxidized in the Ox1 wave. The R1/Ox1 waves persist without diminution
Electrochemistry of Uridine Nucleotides
Langmuir, Vol. 13, No. 13, 1997 3533
Table 2. Peak Potential (Epk), Coulombic Charge (Q), and Peak Width at Half-Height (∆E) Data from Cyclic Voltammograms of 7.0 × 10-5 M Mercuriuridines in 0.1 M KCl at a 0.0225 cm2 Static Hanging Mercury Drop Electrode v EpkR1 QR1 ∆ER1 EpkR2 QR2 ∆ER2 EpkOx1 QOx1 ∆EOx1 (V/s) (mV) (µC) (mV) (mV) (µC) (mV) (mV) (µC) (mV) 1 5 10 20 30 40 50
-565 -600 -630 -660 -685 -700 -720
0.42 0.40 0.45 0.40 0.45 0.43 0.44
84 90 85 90 92 95 115
UMP-HgX -740 0.27 -800 0.29 -854 0.32 -880 0.32 -915 0.35 -944 0.37 -962 0.39
110 119 122 123 138
-320 -300 -280 -260 -240 -220
0.08 0.11 0.19 0.19 0.19
1 5 10 20 30 40 50
-610 -670 -690 -720 -740 -750 -760
0.39 0.40 0.39 0.40 0.39 0.40 0.40
62 63 64 64 66 69 76
UDP-HgX -860 0.40 -910 0.40 -930 0.40 -960 0.40 -970 0.39 -990 0.41 -1000 0.40
32 31 40 66 79 80 84
-180 -160 -130 -110 -90 -80 -60
0.35 0.39 0.39 0.40 0.38 0.40 0.40
54 61 63 64 67 67 67
1 5 10 20 30 40 50
-630 -680 -700 -730 -750 -770 -780
0.34 0.34 0.32 0.33 0.34 0.34 0.33
62 62 63 70 68 70 78
UTP-HgX -870 0.35 -920 0.34 -930 0.34 -970 0.34 -980 0.34 -1000 0.36 -1010 0.35
30 34 45 65 79 83 86
-210 -170 -140 -120 -110 100 -80
0.34 0.34 0.31 0.33 0.34 0.35 0.33
27 32 39 46 49 54 56
of peak height or coulombic charge upon repeated potential cycling between ca. -0.05 and -0.70 V. The coulombic charge under the R1, R2, and Ox1 waves was measured as a function of concentration and sweep rate for all three nucleotides. Sweep rate data for the charge values at a concentration of 7 × 10-5 M are collected in Table 2. The Q-values in Table 2 were obtained from two voltammograms at each sweep rate: a linear sweep voltammogram to measure Q(R2) and a cyclic voltammogram with potential reversal between R1 and R2 to measure Q(Ox1). For UDP-HgX and UTP-HgX, the Q-values for waves R1, R2, and Ox1 were independent of sweep rate and equal to each other within experimental error. For UMP-HgX, the ratios of the charge under the R2 and Ox1 waves to the charge under R1 were sweep rate dependent. The situation for the monophosphate species is discussed below. The concentration dependence of the Q-values for the R1 wave indicated that all three species formed a welldefined compact layer of electroactive centers on the mercury surface. Surface coverages calculated from Faraday’s law, Γ ) Q/nFA assuming n ) 1, became independent of concentration above ca. 4 × 10-5 M as shown in Figures 5 and 6 for the three nucleotides. The footprints of the three nucleotides calculated from the limiting values of the saturation surface coverages (Γs) were 0.89, 0.94, and 1.10 nm2/molecule, for UMP-HgX, UDP-HgX and UTP-HgX, respectively. Waiting or accumulation times of up to 10 min were required to obtain the data for the adsorption isotherms at the lower concentrations. Longer accumulation times did not result in significantly increased surface coverages. At the higher concentrations, the Γi values were reached for accumulation times on the order of ca. 1 min. These observations suggest that the adsorption kinetics were diffusion controlled. The molecular footprints of the UZP-HgX species are consistent with the molecule surface areas of simple purines and pyrimidines which are known to form condensed monolayers on mercury electrodes.3 The mo-
Figure 5. Experimental and calculated adsorption isotherms for UDP-HgX and UTP-HgX in 0.1 M KCl at a HDE: (b) circles, experimental data for UTP-HgX; (9) squares, experimental data for UDP-HgX; (2) calculated Langmuir isotherm for UTP-HgX; (1) calculated Langmuir isotherm for UDPHgX; ([) double layer capacity for UTP-HgX.
Figure 6. Experimental (9) and calculated (2) isotherms for UMP-HgX in 0.1 M KCl at a HDE.
lecular footprint area of uracil, which corresponds to a molecular orientation in which the plane of the ring is parallel to the electrode surface, has been reported in the 0.4-0.5 nm2 range.15 The area occupied by a uridine nucleotide with a substituted phosphosugar residue is expected to be significantly larger than this value. Crude calculations using a molecular modeling program16 gave surface areas between 0.76 and 0.84 nm2/molecule for UTP and UDP in a variety of flat orientations. Since hydration was not taken into account in these estimations, the above surface coverages of the mercurated uridines are fully consistent with the one-electron n-values for the R1, R2, and Ox1 waves assumed in the calculation of the molecular areas. Further evidence for the formation of condensed monolayers is the observation that the double layer capacities track the adsorption isotherm data. The capacitance data (15) Brabec, V.; Hirst, G. D.; Hirst, C. J. Electroanal. Chem. 1977, 85, 389; Biophys. 1978, 7, 313. (16) PCModel, Ver. 3.0: Serena Software, Bloomington, IN. Gajewski, J. J.; Gilbert, K. E.; McKelvey, J. MMX An Enhanced Version of MM2. In Advances in Molecular Modeling; Liotta, D., Ed.; JAI Press: Greenwich, CT, 1990; Vol. 2, pp 65-92.
3534 Langmuir, Vol. 13, No. 13, 1997
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Figure 7. Experimental and calculated Cyclic voltammograms of 7 × 10-5 M UTP-HgX in 0.1 M KCl at a 0.0225 cm2 HDE: (A) sweep rate ) 10 V/s, Q ) 0.32 µC; (B) sweep rate ) 30 V/s, Q ) 0.37 µC; (C) sweep rate ) 50 V/s, Q ) 0.34 µC. For the calculations, E1° ) -0.785 V, E2° ) -0.050 V, k1 ) 0.8 × 104 s-1, k2 ) 1 × 104 s-1, CDL ) 13.6 µC/cm2, T ) 21 °C.
for UTP-HgX shown in Figure 5 indicate that the double layer capacity in the potential region just positive of the R1 wave decreased from 29 to 12 µF/cm2 upon formation of the saturated surface layer. (The double layer capacity was evaluated from the voltammetric charging current using the relationship Ichg ) CdlAv, where v is the sweep rate and A is the electrode area.) The adsorption isotherms for the three mercuriuridines were in excellent agreement with a simple Langmuir model, eq 4
Γi/(Γs - Γi) ) βiCbulk
(4)
especially for UTP-HgX and UDP-HgX. The agreement with this equation for UDP-HgX and UTP-HgX is seen in Figure 5 where both the experimental and calculated isotherms are given. For UMP-HgX, there is a positive deviation from Langmuir behavior indicative of some attractive interaction between the adsorbed molecules. The values of β, which were evaluated from the slopes of the plots of Cbulk/Γ vs Cbulk, were 1.03 × 106, 1.10 × 106, and 0.41 × 106 L/mol for UMP-, UDP-, and UTP-HgX, respectively. Square Scheme Behavior for the R1/Ox1 System. The behavior described above for the R1/Ox1 waves is consistent with a mechanistic scheme in which reversible electron transfer steps are coupled to very fast, irreversible follow-up reactions; i.e., a surface square scheme mechanism of the following type. E°1
UZP-HgXads + e \ { } [UZP-HgX•]adsX- vk2
-X- Vk1 E°2
[UZP-Hg+]ads + e \ { } UZP-Hg•ads This observation was noted in a preliminary communication describing the surface voltammetry of these mercuriuridine species.17 Using the limiting saturation values of Γs and constant values of E°1 and E°2, k1, and k2, and the double layer capacity, excellent agreement between experimental and calculated voltammograms was obtained; see Figure 7. At sweep rates above 10 V/s, the peak widths of both wave R1 and Ox1 for the UDP-HgX and UTP-HgX species were 66 ( 1 mV in perfect agreement with the theoretical value for a surface EC reaction. This agreement is strong further support for (17) Wagner, G. J.; Chambers, J. Q. J. Electroanal. Chem. 1996, 408, 243.
the assignment of one-electron n-values for the surface waves in the above voltammograms. In the calculated voltammograms the rate constants k1 and k2 were set equal to ca. 104 s-1. This was a minimum value chosen in order to produce calculated cyclic voltammograms that did not exhibit reverse peak currents at sweep rates up to 50 V/s. Considerable effort was spent, without success, looking for evidence of reverse currents indicative of short-lived species that could be identified with the intermediates in the above square scheme. These experiments included the use of sweep rates up to 50 000 V/s and switching potentials where the forward current was greater than zero. On the basis of these experiments, the lifetimes of the intermediate species were estimated to be less than 10-6 s. With regard to the square scheme behavior in KCl electrolyte, the monophosphate derivative again exhibited behavior different from that of the di- and triphosphate nucleotides. While the CV wave pattern of the R1/Ox1 waves for the latter two compounds was invariant with sweep rate with equal charge under each wave, the UMPHgX species only produced a square scheme pattern at sweep rates of 50 V/s and above. At these sweep rates, the charge under the Ox1 wave was approximately 50% of that under the R1 wave at room temperature. At the temperature of an ice bath (0 °C), the relative charge under the Ox1 increased. This indicates that, in an activation controlled process, the UMP-Hg• species either desorbs from the electrode surface or reacts by a different pathway to produce a product that is electroinactive in this potential range. The behavior of UMP-HgX is discussed further below. Analysis of the R2 Wave. Careful inspection of the CVs in Figure 4 revealed that, in contrast to the R1 wave, the shape of the R2 wave did not conform to the Laviron theory for a simple EC reaction. The most striking deviation from the expected behavior is the variation of the peak width of wave R2 for UDP-HgX and UTP-HgX with sweep rate; see Table 2. Over the sweep rate range from 1 to 50 V/s, the peak width increases from ca. 30 to 86 mV. The Laviron theory for a simple ECirr reaction predicts a constant peak width of 66 mV at 25 °C.11 Furthermore, the plots of Ipk vs sweep rate displayed distinct nonlinearity indicative of a complex surface process. A strong sweep rate, or time, dependence of the voltammetric parameters of this type is an indication that a phase transition/nucleation phenomenon accompanies the electron transfer step. Simple two-dimensional nucleation following a nucleation-growth-collision (NG-C) model predicts that the peak width should scale with
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Figure 8. Experimental current-time transients for 7.0 × 10-5 M UTP-HgX in 0.1 M KCl at a 0.0225 cm2 HDE. The E-step was from -0.60 V to -0.89, -0.88, -0.87, -0.86, and -0.85 V (curves 1-5, respectively).
v0.4 for linear sweep voltammetry.18 Analysis of the data in Table 2 reveals that the peak widths for the R2 wave for UDP- and UTP-HgX scale with v0.29(0.06 and v0.30 ( 0.6 for 0.04, respectively. Likewise, Ipk should scale with v 0.80 ( 0.02 0.78 ( 0.01 and v for the N-G-C model; the result is v UDP- and UTP-HgX, respectively. These sweep rate dependencies are intermediate between theoretical values for an uncomplicated surface wave and a 2-D N-G-C phase transition. Chronoamperometric current transients can be especially diagnostic for the occurrence of nucleation-controlled electrode reactions. Accordingly, current-time transients were obtained for all three mercurated nucleotides for potential steps from a region between waves R1 and R2 to a potential more negative than wave R2. For this potential step, the electrode reaction for UDP- and UTPHgX corresponds to the one-electron reduction of a stable adsorbed monolayer of R-Hg• species.
R-Hg•ads + H2O + e- f R-H + Hg + OH- (5) The results for UDP- and UTP-HgX were nearly identical; both gave current transients that exhibited maxima indicative of nucleation-controlled reactions. A typical result is shown in Figure 8 for UTP-HgX. For successively larger overpotentials, the time and current coordinates of the current maxima varied in accord with expectations for an activation controlled process. After subtraction of the initial charging current, the currents were found to be in good agreement with theory for progressive two-dimensional nucleation theory.19 A typical result for UTP-HgX is shown in Figure 9. The theoretical currents in Figure 9 were calculated from the following equation for progressive 2D nucleation
In ) tn2 exp[(-2/3)(tn3 - 1)]
(6)
(18) Sanchez-Maestre, M.; Rodriguez-Amaro, R.; Mun˜oz, E.; Ruiz, J. J.; Camacho, L. J. Electroanal. Chem. 1994, 373, 31. (19) Harrison, J. A.; Thirsk, H. R. in Electroanalytical Chemistry: A Series of Advances; Bard, A. J., Ed.; Marcel Dekker: New York, 1967; Vol. 5.
Figure 9. Comparison of experimental current-time transient (circles) from Figure 8, E ) -0.60 V f -0.89 V, and calculated currents for both progressive (squares, eq 6) and instantaneous nucleation.
where In and tn are the current and time normalized to the maximum of the transient. The excellent agreement with this theory gives strong support for a two-dimensional progressive nucleation mechanism for the R2 wave. The lack of agreement with the corresponding theory for linear sweep voltammetry noted above is suspected to be a consequence of the staircase waveform employed. At the sweep rates employed (up to 50 V/s) it is possible that a saturation condition of nucleation sites, as assumed by the theory of ref 18, was not reached for the linear sweep voltammograms of Figure 4. It is also suspected that a related nucleation phenomenon is involved in the process responsible for the cathodic wave R3 that appears on the return segments of the CVs of UDP- and UTP-HgX at high concentration. At potentials negative of diffusion wave R2, the x ) 0 concentration of the electroactive species is driven to zero since the two-electron wave is under diffusion control. Thus it is reasonable to assume that the electrode surface is devoid of an adsorbed film of electroactive material. As the potential sweeps positive in this region, two factors come into play for re-establishment of an adsorbed film of UTP-Hg•: (i)Cx)0 increases since the two-electron wave is no longer on the diffusion plateau, and (ii) it is possible that the potential is on the negative edge of a “capacitance pit” of the type that is common for pyrimidines at mercury electrodes.3 Since the potential in the vicinity of wave R3 is negative of R1, the expected reaction is e-
UTP-HgX]x)0 a UTP-HgX]ads 98 UTP-Hg•ads + X- (7) Integration of the charge under wave R3 gave Q-values that were approximately 1.5× the charge under waves R1 and R2 observed at lower bulk concentrations. This suggests that some further reduction to UTP also occurred in the wave R3 process. For UMP-HgX, no maxima were seen in the chronoamperometric transients obtained under conditions equivalent to those of Figures 8 and 9. Furthermore, the shape and the sweep rate dependence of the R2 wave for the UMP-HgX species differed from that seen in Figure 4; see Figure 10. In the voltammograms of Figure 10, the
3536 Langmuir, Vol. 13, No. 13, 1997
Wagner and Chambers Table 4. UTP UTP-HgX UDP UDP-HgX UMP UMP-HgX
Figure 10. Linear sweep voltammograms of 7.0 × 10-5 M UMP-HgX in 0.1 M KCl; 0.0225 cm2 HDE; sweep rates were 2, 3, 4, and 5 V/s. Table 3. Voltammetric Peak Potentials in Different Supporting Electrolytes (0.1 M) for UTP-HgX: Sweep Rate, 5 V/s; concn, 7 × 10-5 M; 0.0225 cm2 HDE electrolyte
EpkR1 (mV)
EpkOx1 (mV)
∆Epk (mV)
NaCl NaBr NaF LiCl KCl KBr KClO4 KNO3
-720 -710 -720 -670 -680 -640 -660 -660
-90 -120 -100 -80 -170 -160 -80 -100
630 590 620 590 510 480 580 560
current does not drop to the baseline between waves R1 and R2. This is further indication that the UMP-Hg• species is either unstable or desorbs from the electrode surface and reacts further. Thus the initial conditions for that chronoamperometric experiment in the case of UMP-HgX are probably quite different from the conditions of Figures 8 and 9. Identity of X in UZP-HgX. Although the above experiments were performed in KCl supporting electrolyte solution, two pieces of evidence suggest that the nucleotide phosphate arm is involved in complexation of the mercury substituent. First, the voltammetric behavior was insensitive to the concentration of the KCl electrolyte. More revealing was the behavior in different supporting electrolytes presented in Table 3, which shows that the voltammetric R1/Ox1 pattern was remarkably insensitive to the nature of the anion of the supporting electrolyte. In noncomplexing media such as KClO4 or NaF, the peak potentials and the peak widths were only slightly different than the values in KCl or NaCl electrolyte solutions, respectively. This is difficult to rationalize with an assignment of X- ) Cl- in the above square scheme. For example, in classic work Hush and Oldham2c many years ago pointed out how the polarography of simple RHgX compounds was related to the dissociation constants for the RHgX a RHg+ + X- process. This observation leads to the hypothesis that the phosphate groups of the UZP-HgX species play a role in the complexation and decomplexation steps in the above square scheme. While both inter- and intramolecular complexation can be envisioned, the insensitivity of the
31P
NMR Chemical Shifts in D2O
δ (R-P) (ppm)
δ (β-P) (ppm)
δ (γ-P) (ppm)
-11.0 -10.5 -10.6 -9.6 +5.0 +5.2
-22.3 -20.5 -7.0 -5.2
-9.1 -5.0
voltammetric parameters to concentration changes (over a range from 10-3 M) suggests that Ox1/R1 square scheme involves complexation of the mercury(II) substituent by an intramolecular phosphate ligand. Below it will be shown that this hypothesis is fully consistent with the pH dependence of the voltammetry over a wide pH range. It is also the basis of our explanation for the difference of the UMP-HgX behavior from that of the UDP-HgX and UTP-HgX species. Further inspection of the data in Table 3 reveals that there is a greater dependence on the cation of the supporting electrolyte than on the anion. This could be related to competing complexation of the negatively charged triphosphate arm of UTP by alkali metal cations, which would lessen the strength of the intramolecular complexation of the mercury substituent. This effect was not investigated further. The second piece of evidence indicating that the di- and triphosphate groups are involved in the complexation of the mercury substituent comes from the 31P and 1H NMR spectra. The chemical shifts of the β and γ phosphorus atoms are sensitive to the presence of a C-5 HgX substituent; see Table 4. For UTP-HgX, the γ-P and the β-P resonances have moved upfield by 4.1 and 1.8 ppm, respectively, relative to the unsubstituted nucleotide. For UDP-HgX, the β-P resonance moves upfield by 1.8 ppm. Although these shifts are expected to be pH-dependent,20 these data are indicative of deshielding of the terminal phosphorus atoms upon phosphate complexation of the mercury substituent group.
Thus these data support the idea that in aqueous electrolyte solutions, and as adsorbed monolayers, the UTP-HgX and UDP-HgX species exist as intramolecular complexes. One possible structure for UTP-HgX is shown above; charges are omitted in this drawing. pH Dependence of the Cyclic Voltammetry. Profound changes in the CV behavior were noted when the pH was varied by addition of buffer components to the electrolyte solutions. The results are best summarized by “kinetic” Pourbaix diagrams of the type shown in Figure 11 for UTP-HgX. In these diagrams, the peak potentials of the R1 and R2 surface waves are plotted vs pH. Since the peak potentials contain kinetic terms for the chemical reactions that are coupled to the electron transfer steps, exact thermodynamic information cannot be gleaned from (20) Robitaille, P.-M. L.; Robitaille, P. A.; Brown, G. G., Jr.; Brown, G. G. J. Magn. Reson. 1991, 92, 73.
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The dependence of the new R1′ wave on pH and chloride concentration suggests that Cl- can compete for coordination sites on the mercury substituent as the coordinating strength of the phosphate arm is weakened upon protonation. k3
UTP-Hg(triphos) + Cl- y\ z UTP-HgCl k
(8)
-3
Figure 11. Kinetic peak potential diagram for 7.0 × 10-5 M UTP-HgX in 0.1 M potassium phosphate buffers containing 0.2 M KCl: 0.0225 cm2 HDE; sweep rate, 10 V/s.
the diagram. However, if the rate constants of the coupled reactions remain approximately constant with pH, the inflection points in the plots can give approximate pKa values if the electron transfer steps are reversible. At any rate, the diagrams provide a basis for a qualitative understanding of the UTP-HgX redox system. At pH values greater than ca. 9, the peak potential of the R1 wave became independent of pH for UDP- and UTP-HgX. The pKa value of 8.9 estimated from the intersection of the two straight line segments in the diagram is intermediate between pKa values for the triphosphate proton and for the ionizable ring proton of UTP.21 On the basis of the properties of UMP-HgX, the mercuro substituent is expected to increase these values slightly. The 60 mV/pH slope in the pH region between 5 and 9 indicates that the first step in the square scheme above involves the reversible transfer of one proton and one electron. In order to understand the pH dependence vis-a-vis the above results in KCl electrolyte solution, it is important to realize that the UZP-HgX compounds were isolated as the alkali metal ion salts, i.e., in their stable basic form. The pH of the solutions of these compounds, in the absence of added buffer components, was in the region of pH ) 8.5 to 9.0. Thus the data for UZP-HgX in KCl solutions reported above pertain to the pH region around 9 in these diagrams. There are two significant observations concerning the Pourbaix diagram of Figure 11 that relate to the components of the buffer/electrolyte solution. A phosphate buffer medium was used to vary the pH and the solution also contained 0.2 M KCl. In the presence of KCl, as the pH was lowered the R1 wave split into two surface waves, R1′ and R1, where R1′ appeared at the more positive potential. In the absence of chloride ion, wave R1′ was not present. Furthermore, the general behavior of the Figure 11 Pourbaix diagram was also obtained in more cursory studies in both acetate and HEPES buffer solutions. Very similar kinetic Pourbaix diagrams were obtained for both the UMP-HgX and UDP-HgX nucleotides. (21) Saenger, W. Principles of Nucleic Acid Structures; SpringerVerlag: Berlin, 1984; pp 107-110.
In this case, voltammetry in the shaded region of the Pourbaix diagram corresponds to a six-component ladder scheme. Calculated and experimental voltammograms assuming this mechanism and reversible electron transfers are shown in Figures 12 and 13 as a function of chloride concentration and sweep rate. Generally good agreement is seen. Especially interesting from a diagnostic viewpoint is the potential region between the waves that are assigned to the reduction of the intramolecular complex and the chloromercury complex. The “leakage” current in this region can be simulated in the calculation by adjustment of the rate constants for displacement of the phosphate arm by Cl-. The rate constants used in the calculations of the voltammograms in Figure 12 give an equilibrium constant equal to 1.2 M-1 for the reaction of eq 8 at pH ) 5.5. The pH dependence of the voltammetric behavior also could be correlated with the pH and electrolyte dependence of the chemical shift of the C6 proton on the pyrimidine ring in the NMR spectra of UTP-HgX. At pH 11 in phosphate buffer, the value (δ) was 7.82 ppm in the presence of either 0.2 M KCl or 0.2 M KClO4. At pH 3 in KCl solution, this resonance shifted downfield to 8.25 ppm, while at pH 3 in the absence of chloride the value was 8.39 ppm. At intermediate pH values two resonance lines assignable to the C6 proton were present in the NMR spectra. This is further evidence for the existence of different complexation states of the 5-mercuriuridines that are connected by a chemical equilibrium which is frozen on the NMR time scale. While the agreement between experimental and calculated CVs in Figures 12 and 13 is good, it is not perfect and brief comment on several features is appropriate. (i) As the Cl- concentration was increased, the “background” current in the region of 0.0 V increased. This is probably related to calomel formation that is known to occur at mercury in KCl electrolyte solution;22 mercurous phosphate formation is also possible.23 (ii) Regardless of how the thermodynamic and kinetic parameters were manipulated in the calculations, seldom was a good fit obtained for the Ox1 wave except at the faster sweep rates. This was also the case for the CVs in KCl solution shown in Figure 7. It is suspected that a kinetically controlled nucleation phenomenon, similar to the one described above for the R2 wave, is at play for the Ox1 wave. Due to the proximity of Ox1 to the background oxidation of mercury, this was not investigated further. (iii) Finally in order to match the non-Faradaic background, different double layer capacities were assumed for the oxidized and for the reduced states of the surface layer. In addition a very small amount of Faradaic background current was included (as an additive current with a linear dependence on potential). A major disagreement between the calculated and the experimental CVs was seen at the lowest sweep rate of Figure 13. In this case the calculated CV, using the (22) Wrona, P. K.; Galus, Z. In Encyclopedia of the Electrochemistry of the Elements; Bard, A. J., Lund, H., Eds.; Marcel Dekker: New York, 1982; Vol. IX. (23) Armstrong, R. D.; Fleischmann, M.; Oldfield, J. W. J. Electroanal. Chem. 1967, 14, 235.
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Wagner and Chambers
Figure 12. Variation of 7 × 10-5 M UTP-HgX cyclic voltammetry with [Cl-], 0.1 M pH 5.5 phosphate buffer, 0.0199 cm2 HDE; sweep rate, 5 V/s. For the calculations, E1° ) -0.515 ( 0.024 V, E2° ) -0.078 ( 0.020 V, E3° ) -0.280 ( 0.003 V (R1′ wave), k1 ) 8000 s-1, k2 ) 10 000 s-1, k3 ) 49.5 L mol-1 s-1, k-3 ) 42.5 s-1, CDLOx ) 13 ( 2 µF/cm2, CDLRed ) 6 ( 2 µF/cm2, and T ) 21 °C. The total charge decreased as [Cl-] increased, QTot ) 0.22, 0.19, 0.16, and 0.10 µC.
parameters that gave a good fit at 5 and 10 V/s, indicated that the forward reaction of eq 8 was fast on the CV time scale. However, the experimental result clearly indicated that wave R1, and presumably the adsorbed phosphate complex, was still present. The reason for this discrepancy is not understood. Perhaps, the surface layer is resupplied by diffusion of bulk component species under these conditions, a complexity not taken into account in the calculations. At low pH (pH ) 2-4), two significant changes in the surface voltammetry were noted for all three UZP-HgX species. At these pH values the R1 wave was no longer present and, in the presence of Cl-, the R1′ wave appeared to be reversible. Typical behavior is shown in Figure 14. Second, the charge under the R1′ wave decreased as the pH was lowered. This latter behavior is shown in Figure 15, which shows the adsorption isotherms calculated from the charge under the R1′ + R1 waves again assuming a one-electron n-value. At pH ) 3, the saturation value of the surface coverage was approximately one-third the
value at pH 9. Thus the apparent molecular footprint of UTP-HgX on the mercury surface has increased 3-fold upon protonation of the phosphate arm. In addition, the data at intermediate pH in Figure 15 do not correspond to simple Langmuirian behavior, which may be related to kinetically slow establishment of the surface layers under these conditions. The CV behavior in Figure 14 suggests that the first electron transfer step was reversible at low pH. However, this interpretation is difficult to reconcile with the chemistry of the square scheme put forth above to explain the behavior at higher pH. In addition at pH ) 3, the peak width of the R1′ wave for all three UZP-HgX species was considerably less than the theoretical value of 90 mV for a reversible one-electron wave. A conceptually more satisfying explanation is that at low pH the square scheme is still operative with X- ) Cl-. The calculated voltammograms in Figure 14, which assumed this mechanism, are in good agreement with experiment. Although the ∆E° values are on the order of 100 mV in these calculations,
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Langmuir, Vol. 13, No. 13, 1997 3539
Figure 13. Variation of 7 × 10-5 M UTP-HgX cyclic voltammetry with sweep rate, 0.2 M KCl, 0.1 M pH 5.0 potassium phosphate buffer, 0.0199 cm2 HDE. For the calculations, E1° ) -0.485 V, E2° ) -0.050 V, E3° ) -0.274 V (R1′ wave), k1 ) 8000 s-1, k2 ) 10 000 s-1, k3 ) 77.5 L mol-1 s-1, k-3 ) 14.5 s-1, CDLOx ) 22 ( 7 µF/cm2, CDLRed ) 9 ( 6 µF/cm2, QTot ) 0.17 µC, and T ) 21 °C.
Figure 14. Experimental (circles) and calculated (squares) cyclic voltammograms for UMP-HgX (left), UDP-HgX (middle), and UTP-HgX (right) in 0.2 M KCl, 0.1 M pH 3.0 potassium phosphate buffer, 0.0199 cm2 HDE: sweep rate, 5 V/s; concn, 7 × 10-5 M. For the calculations: E1° ) -0.250, -0.230, -0.235 V; E2° ) -0.060, -0.130, -0.138 V; k1 ) 8000 s-1; k2 ) 10 000 s-1; CDLOx ) 17, 17, 14 µF/cm2; CDLRed ) 7, 7, 5 µF/cm2; QTot ) 0.11, 0.12, 0.07 µC; T ) 21 °C. (Values are given for UMP-HgX, UDP-HgX, and UTP-HgX, respectively, where appropriate.)
Figure 15. Adsorption isotherms for UTP-HgX in 0.1 M phosphate buffer at pH ) 3 (O), 5 (b), 7 (0), 9 (9).
the kinetic terms in the peak potential expressions shift both cathodic and anodic waves to sub-Nernstian potentials such that the waves appear to be reversible images. Significantly, the peak widths approach the theoretical 66 mV value for an ECirr surface wave.
pH Dependence of the UMP-HgX Voltammetry. When the pH was lowered by addition of buffer components to KCl solutions of UMP-HgX, the voltammetric behavior of the monophosphate was more in accord with that of the di- and triphosphate species. This is shown, for example, by the CV of UMP-HgX in Figure 14 at pH 3 that is in agreement with a square scheme mechanism. At pH 7 in phosphate buffer, the square scheme mechanism also appears to be operative as indicated by the CV of Figure 16. Significantly, in contrast to the behavior in KCl solution, the adsorption isotherm of UMP-HgX at pH 7 was in excellent agreement with the simple Langmuir model, eq 4. The “kinetic” Pourbaix diagram for UMPHgX was also in agreement with the previously discussed behavior of the di- and triphosphate species. Thus, it is at higher pH values, the pH of the UMPHgX solutions in the absence of buffer components, that there is a significant difference between the voltammetry of the three species. An explanation of this behavior can again be found in the acid-base chemistry of these multifunctioned compounds. For the monophosphate nucleotide, at pH ca. 9, dissociation of the proton on the ring nitrogen is expected. Complexation of the soft mercury atom by the ring nitrogen atom now becomes a distinct possibility.24 As previously noted by Dale et al.,6 this leads to formation of polymeric organomercury (24) (a) Mansy, S.; Tobias, R. S. Inorg. Chem. 1975, 14, 287. (b) Marzelli, L. G.; de Castro, B.; Solorzano, C. J. Am. Chem. Soc. 1982, 104, 461. (c) Guay, F.; Beauchamp, A. Inorg. Chim. Acta 1982, 66, 57.
3540 Langmuir, Vol. 13, No. 13, 1997
Figure 16. Experimental (squares) and calculated (circles) cyclic voltammograms of 7 × 10-5 M UMP-HgX in pH 7.00, 0.1 M potassium phosphate buffer; 0.0225 cm2 HDE; sweep rate, 5 V/s. For the Calculation: E1° ) -0.535 V, E2° ) -0.078 V; k1 ) k2 ) 10 000 s-1; CDL ) 5 µF/cm2; T ) 21°C.
Wagner and Chambers
Figure 18. Variation of the R1 wave for UMP-HgX with pH: concentration, 7 × 10-5 M UMP-HgX; 0.0225 cm2 HDE; sweep rate, 5 V/s; (1) pH 7.0, (2) pH 8.5, (3) pH 10.0.
calculated from the titration curve. These titration data indicate that as HCl is added to the solution the reaction that takes place is X-
(1/n)(-UMP-Hg-)n + H+ y\z UMP-HgX
Figure 17. Titration of 0.0223 g of UMP-HgX plus NaOH in ca. 10 mL of H2O with 0.00961 M HCl.
nucleotide anions in the bulk of the solution. In this view the HgX bond in UMP-HgX is actually an intermolecular Hg-N bond and (-UMP-Hg-)n is a better formulation of the compound at high pH. It is tempting to ascribe the positive deviation of UMP-HgX from Langmuir behavior in Figure 6 to the formation of a polyanionic surface layer of this nature. This possibility is supported by the pH titration curve of “UMP-HgX” shown in Figure 17. The initial pH of the solution of the lithium salt of “UMP-HgX” was approximately 8.5; additional NaOH was added to the solution before titration with HCl in the experiment of Figure 17. The shape of the titration curve between the breaks at pH ca. 9 and 4 indicates that two protons are required, in accord with the stoichiometric ratio of 7.21 × 10-5 mol of HCl/3.57 × 10-5 mol of “UMP-HgX”
(9)
where X is either a buffer component or the monophosphate group of UMP-HgX intramolecularly complexed to the mercury atom. Then the second step would involve protonation of the monophosphate group. Subtle changes in the shape of the R1 surface wave for UMP-HgX took place when the pH was varied around the initial pH ) 8.5 value; see Figure 18. At pH ) 8.5, where in the above view the principal species is a polymeric (-UMP-Hg)n- anion, the peak width was 91 mV, which is significantly greater than the expected value of 66 mV. However, at pH ) 7.0 the peak width was 60 mV and at pH ) 10 it was 65 mV. These data suggest that at pH ) 8.5 the R1 wave for “UMP-HgX” is due to a ladder scheme where the coupled components of the above equilibrium, eq 9, are the electroactive species being reduced. In this view, at pH ) 7, the current is due to reduction of adsorbed UMP-HgX which is in rapid equilibrium with (-UMPHg)n- on the time scale of the voltammograms of Figure 18. Not all features of the UMP-HgX voltammetry can be rationalized in terms of the above mechanistic arguments however. As noted above, even at the fastest sweep rates of this study, the charge under the Ox1 wave in KCl electrolyte solution did not match the charge under the R1 waves. At slower sweep rates, anodic currents are evident in the voltammograms in the region of -500 mV that cannot be explained in the context of the square scheme behavior postulated above. Inspection of Figure 1 reveals the presence of these anodic currents which are not present in the voltammograms of UDP-HgX or UTPHgX. One possibility is that one-electron reduction of (-UMP-Hg)n does not follow the square scheme behavior but that the (-UMP-Hg•-)n species remains intact, at least in part, and is oxidized in the region of -500 mV. Conclusions The mechanistic arguments advanced above tie together the voltammetric behavior of the three mercurated
Electrochemistry of Uridine Nucleotides
uridines over a wide range of concentrations and solution pH values. The key component of the electrode mechanisms for all three species is the surface square scheme given above. Reversible electron transfer steps are assumed in all cases considered in this paper. For these UZP-HgX nucleotides, and probably for other RHgX organomercury compounds as well, the role of coupled chemical reactions that involve reaction with the mercury center is critical to an understanding of the electrode process. Only for the monophosphate nucleotide at high pH, where formation of polymeric (-UMP-Hg-) anions is possible, was the square scheme insufficient to rationalize the voltammetric behavior. In light of this, the reason that different voltammetric behavior is seen for the diand triphosphate nucleotides under these conditions is that polyphosphate arms have prevented the formation of the polymeric species. In a sense, the polyphosphate moieties are internal intramolecular buffer components that determine the course of the reduction. As the pH is lowered, the phosphate groups are protonated and the reduction mechanism proceeds via a new square scheme giving rise to the R1′ wave. Another novel feature of this study is the suspected role of nucleation phenomena coupled to the electron
Langmuir, Vol. 13, No. 13, 1997 3541
transfer steps. The microscopic nature of the nucleation process of course cannot be determined from these studies. It can be speculated, however, that the R2 wave involves replacement of the adsorbed UZP-Hg• film with a solvent dipole structure. In this mechanistic view, initial electron transfer to an adsorbed UTP-Hg• radical in the surface film is an activation-controlled slow step. However, subsequent electron transfer is fast to UTP-Hg•ads centers located on the periphery of the growing solvent dipole layer. The persistent voltammetry of the R1/Ox1 couple, which is fundamentally based on the above surface square scheme, provides a pattern that can be used to identify the pyrimidine nucleus tagged with a mercury substituent. Finally, it can be noted that reductive electrolysis at more negative potential provides a convenient synthetic method both to remove the tag and to label the parent compound with deuterium. Acknowledgment. This research was supported by a grant from the NSF (CHE-9220930) and by The University of Tennessee, Knoxville. LA970096J
