Phosphodiester cleavage by yttrium(III) peroxide complexes

www.elsevier.com/locate/ica Inorganica Chimica Acta 328 (2002) 241– 246

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Phosphodiester cleavage by yttrium(III) peroxide complexes Yamilet Mejia-Radillo, Anatoly K. Yatsimirsky * Facultad de Quı´mica, Uni6ersidad Nacional Auto´noma de Me´xico (UNAM), Mexico DF 04510, Mexico Received 17 May 2001; accepted 5 September 2001

Abstract Potentiometric titrations of hydrogen peroxide in the presence of Y(III) revealed formation of dinuclear Y2(O2)22 + and Y2(O2)2(OH)2 complexes. Kinetics of the cleavage of bis(4-nitrophenyl) phosphate (BNPP) in the presence of Y(III) and H2O2 was studied at 25 °C in pH range 6–8 and at variable metal and H2O2 concentrations. Comparison of the pH-dependence of the reaction rate with the species distribution diagram shows that Y2(O2)2(OH)2 is the reactive species. The reaction kinetics is second-order in Y(III) at low metal concentration, but is of a ‘saturation’ type at high metal concentrations. A reaction mechanism, which agrees with such kinetics, involves intermediate reversible dimerization of Y2(O2)2(OH)2 to a tetranuclear complex capable to bind BNPP anion and to cleave it intramolecularly. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Yttrium(III) complexes; Peroxide complexes; Bis(4-nitrophenyl) phosphate; Phosphodiester cleavage; BNPP anion

1. Introduction Metal complex catalysis in the cleavage of phosphoric acid esters is an area of active research [1]. The hydrolysis of phosphodiesters, which are extremely resistant to the nucleophilic attack even with activated 4-nitrophenolate leaving groups [2], is especially important because phosphodiester bonds form the backbone of DNA and RNA macromolecules and sufficiently active catalysts of their hydrolysis can find important applications as artificial nucleases in biochemical and medicinal researches. Combinations of lanthanides and hydrogen peroxide are among the most active systems for the cleavage of activated phosphodiesters [3 – 5] and RNA [6]. Takasaki and Chin demonstrated by using 18O labeling studies [4] that the peroxide acts as a nucleophile in the cleavage of diesters and proposed as the active species a dinuclear complex La(O2)2La (1) on basis of potentiometric titration and kinetic experiments [3,4]. However, more recent potentiometric titrations of H2O2 – La(III) mix* Corresponding author. Fax: + 52-5-616 2010. E-mail address: [email protected] (A.K. Yatsimirsky).

tures showed formation of a different neutral dinuclear complex with three instead of two peroxide anions: La(O2)3La [6]. In addition, the kinetic results with RNA as a substrate were interpreted in terms of a mechanism involving equilibrium formation of a hexanuclear complex (La(O2)3La)3 as an active species [6]. The reason(s) of this discrepancy is not clear, but known low stability of the H2O2 –La(III) mixture [3,4] may be a serious complicating factor. In addition, well-known tendency of lanthanides(III) to form polymeric species in solution may contribute to low reproducibility of both kinetic and equilibrium results. Testing different lanthanides showed similar phosphodiesterase activities for La(III), Pr(III), Nd(III) and Eu(III) in combination with hydrogen peroxide [4]. In this paper we report the use of Y(III) in a similar combination. An advantage of Y(III) is generally higher stability of its solutions, which are not undergo ‘aging’ in contrast to lanthanides [7]. Cation Y3 + by its size (ionic radius ri = 104 pm in 6-coordination) is similar to Ho3 + (ri = 104.1 pm in 6-coordination) and by its acidity (pKa = 8.61 at ionic strength I=0.3 M) [8] is similar to Gd3 + (pKa = 8.62 at I= 0.3 M) [8], but the solubility product for Y(OH)3 6.3×10 − 24 M4 is con-

0020-1693/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 0 - 1 6 9 3 ( 0 1 ) 0 0 6 7 3 - 9

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siderably larger than for both lanthanides (approximately 1.5× 10 − 26 M4) and therefore it is less susceptible to precipitation in basic solutions. Polymeric Y(III) hydroxide complexes of general composition [Y(OH)2]nn + , stabilized by 1.25 equivalents of anthranilic acid, were found to catalyze the hydrolysis of 4-nitrophenyl methylphosphonate [7]. Comparison of catalytic activities of different metal cations in the hydrolysis of this substrate shows that Y(III) is 100 times more active than La(III) and only 10 times less active than highly electrophilic cations like Th4 + , Ce4 + and Zr4 + [9]. Therefore, we expected Y(III) to be at least as active as lanthanides in a combination with hydrogen peroxide, but more stable and therefore more suitable for equilibrium and kinetic studies. We employed as the substrate for this study bis(4-nitrophenyl) phosphate (BNPP), which is often used for studies of the phosphodiesterase activity of metal complexes.

2. Experimental

2.1. Materials Bis(4-nitrophenyl) phosphate (Aldrich) was recrystallized from ethanol– water. Imidazole from Sigma, NaNO3 and reagent grade yttrium(III) nitrate from Aldrich were used as supplied. The concentration of metal ions in stock solutions was determined by adding excess of ethylenediaminetetraacetic acid and then back titrating with ZnCl2 with Eriochrome Black T as indicator [10]. Distilled and deionized water (Barnstead nanopure system) was used.

mM of Y(III). In order to avoid complications due to formation of Y(III) hydroxide complexes the titrations were terminated at pH about 7.5, well below pKa of the aquo-ion. Species distribution diagrams were calculated by using SPECIES Ver. 0.8 Academic Software 1999.

2.3. Kinetics Kinetic measurements were performed by using a Hewlett– Packard 8453 diode array spectrophotometer equipped with a Peltier thermostatted cell compartment. Reaction solutions were prepared by combining appropriate amounts of Y(III) and H2O2 stock solutions to the desired volume and pH was adjusted by adding small volumes of strong acid or base. Reactions were initiated by adding an aliquot of the substrate solution. Solution pH was measured after each run and all kinetic runs in which pH variation was larger than 0.1 were excluded. In order to have sufficiently high buffer capacity of the solution, the reaction kinetics was studied in the presence of 50 mM imidazole. The course of all hydrolysis reactions was monitored spectrophotometrically by appearance of 4-nitrophenolate anion at 400 nm. Kinetic measurements used 50 mM substrate and varied concentrations of metal and hydrogen peroxide at 25 °C. The observed first-order rate constants (kobs) were calculated by the integral method typically over 5 half-lives or, for slow reactions, from initial rates. 3. Results and discussion

3.1. Complexation equilibria in the Yttrium(III) – H2O2 system

2.2. Potentiometry Potentiometric titrations were performed following general recommendations given in Ref. [11]. All titrations were performed in a 75-ml thermostated cell kept under nitrogen at 25 °C. The initial volume of titrating solution was 50 ml. The ionic strength was kept constant with 0.1 M NaNO3. Measurements of pH were taken on an Orion Model 710-A research digital pH meter as NaOH solution was added to the system in small increments. The electrode was calibrated with standard buffer solutions. A titration of dilute (approximately 0.01 M) standardized strong acid solution with NaOH with the same background electrolyte was performed to check the electrode calibration and the presence of carbonates in base solution. The pKw =13.799 0.04 was found under our conditions from the strong acid titration experiment. The program HYPERQUAD 2000 Version 2.1 NT was used to calculate all equilibrium constants [12]. Titrations of hydrogen peroxide mixtures with yttrium nitrate were performed in the concentration range 6– 20 mM of H2O2 and 2–6

Attempts to isolate complexes of Y(III) with H2O2 were unsuccessful and the information regarding the stoichiometry and stability of these complexes in solution was obtained by potentiometric titrations. Fig. 1(a) shows the pH titration curve for the mixture of 20 mM hydrogen peroxide and 5.7 mM yttrium(III) nitrate (see Supporting information). The titration curve shows the consumption of 3 equivalents of NaOH per 1 equivalent of metal in accordance with results of Komiyama and coworkers [6] for the La(III)–H2O2 mixture. Also the fitting to the model involving formation of Y(O2)3Y species, shown by the dashed line in Fig. 1(a), is quite satisfactory and gives the equilibrium constant [Y2(O2)3][H]6/[Y]2[H2O2]3 = 9.0× 10 − 33 M2 close to the value of 3.3× 10 − 32 M2 reported for the respective complex of La(III) [6]. However, in the titration of the 1:1 H2O2 –Y(III) mixture, shown in Fig. 1(b), also 3 equiv. of NaOH per 1 equiv. of metal are consumed, which means that at least 1 equiv. of ionizable hydrogens proceeds from coordinated water rather than from hydrogen peroxide.

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Fig. 1. Titration curves of: (a) 20.0 mM H2O2 and 5.7 mM Y(NO3)3; (b) 6.0 mM H2O2 and 6.1 mM Y(NO3)3; (c) 6.0 mM H2O2 and 1.8 mM Y(NO3)3 in 0.1 M NaNO3. a is the number of equivalents of NaOH per 1 equiv. of Y(III). Solid lines are the fitting curves generated by HYPERQUAD 2000 to the Scheme 1 with the equilibrium constants given in Table 1. Dashed lines are the respective fitting curves to a scheme involving formation of Y2(O2)3, (a) and (c) or Y(O2)(OH), (b).

A simplest model, which agrees with the titration stoichiometry involves the formation of a mononuclear complex Y(O2)(OH). The fitting curve for the formation of such a complex is shown in Fig. 1(b) by the dashed line. It obviously is not satisfactory and the shape of the titration curve indicates that more suitable would be a model with a respective dinuclear complex, Y2(O2)2(OH)2. Fitting to such a model (see Supporting information) was indeed better, but the best fit was obtained for a more complex model shown in Scheme 1, which involved also the formation of Y(O2)2Y, similar to 1 (solid line in Fig. 1(b)). The solid line in Fig. 1(a) illustrates the fitting of the results obtained with excess of H2O2 to this model.

Scheme 1.

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Table 1 Stability constants for the formation of Yttrium(3+) complexes with H2O2(25 °C, 0.1 M NaNO3) Equilibrium

[Y2(O2)2(OH)2][H]6/[Y]2[H2O2]2 [Y2(O2)2][H]4/[Y]2[H2O2]2

log K

a

Mean b

[Y(III)]T = 5.7 mM [H2O2]T = 20.0 mM

6.1 mM 6.0 mM

1.8 mM 6.0 mM

−32.200 90.002 −20.0990.02

−32.143 90.008 −20.199 0.12

−31.792 90.008 −18.7190.02

−32.049 0.13 −19.669 0.48

a

Errors are the standard fitting errors calculated by the HYPERQUAD 2000 after the refinement procedure. Errors are the standard errors of the mean values of the equilibrium constants calculated by averaging the values determined in all titrations of Y(III)–H2O2 mixtures. b

The third titration was performed with more diluted metal and hydrogen peroxide solutions in order to assure that the stability constants for the model given in Scheme 1 are really independent of the total reactant concentrations. Fig. 1(c) shows the titration curve for the mixture of 1.8 mM yttrium nitrate and 6 mM hydrogen peroxide. Fitting to the model involving the Y(O2)3Y complex is shown by a dashed line and fitting to the model in Scheme 1 by the solid line. Evidently, in this case the models can be clearly discriminated. Stability constants obtained from all three titrations in accordance with the model in Scheme 1 are collected in Table 1. By analogy with the structure 1 we suppose that Y(O2)2Y2 + also have two bridging peroxide anions. The stability constant for the formation of Y(O2)2Y2 + 2.2× 10 − 20 M is reasonably close to the reported stability constant for the formation of 1 1.4× 10 − 23 M [3]. Binding of two additional hydroxides in Y2(O2)2(OH)2 probably leaves intact bridging peroxide ligands.

3.2. Kinetics of BNPP hydrolysis The absorbance A vs. time t profiles monitored at the absorption maximum of 4-nitrophenolate anions followed simple first-order kinetics with simultaneous liberation of both nitrophenol groups of the substrate. The simultaneous liberation of two nitrophenol groups occurs also in the reaction of BNPP with hydrogen peroxide in the absence of metal ions [13]. Similarly to the situation in the absence of metal ions, no cleavage of mono 4-nitrophenyl phosphate was observed in the presence of Y(III) and hydrogen peroxide. Therefore, in both cases the reaction does not involve formation of a kinetically significant half-cleaved intermediate. The pH-dependence of the observed first-order rate constant (kobs) is shown in Fig. 2 together with the species distribution diagram. Evidently the increase in kobs parallels the increase in fractions of peroxide complexes and the major contribution belongs to Y2(O2)2(OH)2. The concentration dependences of kobs were studied at pH 7 when the dominant species is Y2(O2)2(OH)2.

The reaction rate was essentially independent of H2O2 concentration in the range 5–50 mM in the presence of 1–5 mM Y(III). Indeed, in accordance with stability constants given in Table 1, under these conditions the degree of formation of Y2(O2)2(OH)2 is close to 90% already with 5 mM H2O2. Dependence of kobs on total Y(III) concentration ([Y(III)]T) is shown in Fig. 3. Under these conditions Y2(O2)2 constitutes less than 10% of total Y(III) and the concentration of dominating Y2(O2)2(OH)2 complex, shown by the dashed line, linearly depends on the total Y(III) concentration. However, the dependence of kobs on [Y(III)]T is not linear: it is quadratic when [Y(III)]T is below 2 mM, but tends to ‘saturate’ at higher metal concentrations. The ‘saturation’ kinetics was observed also for the cleavage of BNPP in the La(III)–H2O2 system [3,4]. It was interpreted as being due to the complexation of BNPP by 1 with unusually high for such a poor ligand stability constant 1.3× 103 M − 1 [4]. Interestingly, ‘kinetic’ stability constants of BNPP complexes with lanthanide aquo-ions [14] as well as complexes of the 4-nitrophenyl phosphate ester of propylene glycol with some lan-

Fig. 2. Observed first-order rate constants for the BNPP hydrolysis at 25 °C (solid squares) and species distribution diagrams for hydroxocomplexes (Scheme 1, dashed lines) for 1.7 mM yttrium(III) and 30 mM H2O2 as a function of pH.

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{(Y2(O2)2(OH)2)2(BNPP)} “products

245

(4)

one obtains the following expression for kobs (under conditions of high excess of Y(III) over BNPP): kobs = k4K2K3[Y(III)]2T/(1+ K2K3[Y(III)]2T)

Fig. 3. Observed first-order rate constants for the BNPP hydrolysis vs. Y(III) concentration in the presence of 5 mM (solid squares) and 50 mM (open squares) H2O2 at pH 7.0 and 25 °C. Solid line is the fitting curve in accordance with the Eq. (1) with parameters given in the text.

thanide macrocyclic complexes [15] estimated from the ‘saturation’ kinetic data are of the same order of magnitude, although the equilibrium stability constants of phosphodiesters with such metals as Co(III) [16], Cu(II) [17], and Zn(II) [1a] are below 10 M − 1. These cations are generally better coordinating species than lanthanides(III) and strongly enhanced stability constants of BNPP with lanthanides found from kinetic data most probably reflect more complex mechanism of hydrolysis than the usually assumed Michaelis– Menten type kinetics. An empirical rate equation, which takes into account both the second-order kinetics at low concentrations of Y(III) and a ‘saturation’ at higher concentrations and satisfactorily fits the results in Fig. 3 has the form: kobs =k[Y(III)]2T/(1+ K[Y(III)]2T)

(1)

s and K =(2.2 9 0.3) × 10 where k = 4309 50 M M − 2. Note, that the second-order kinetics at low [Y(III)]T does not result from the formation of the Y2(O2)2(OH)2 dinuclear complex because the degree of its formation is approximately 85% even at lowest total Y(III) concentration. There are several kinetic schemes, which lead to the Eq. (1). Assuming, as in Ref. [6], that the actual reactive form is an aggregate produced by dimeric complexes, in our case a tetranuclear complex −2

−1

2Y2(O2)2(OH)2 = (Y2(O2)2(OH)2)2

5

(5)

where k4 is the rate constant for the reaction Eq. (4), K2 and K3 are the equilibrium constants of the reactions Eqs. (2) and (3), respectively. The Eq. (5) coincides with the empirical Eq. (1) provided k=k4K2K3 and K= K2K3. The dimerization constant K2 cannot be large because the potentiometric titration data obtained with [Y(III)]T up to 6 mM do not indicate a presence of any aggregates. Therefore, K2 should be lower than approximately 20 M − 1. This means that the binding constant of BNPP to the tetranuclear complex K3 Eq. (3) should be of the order of 104 M − 1. We again see, therefore, that the interpretation of the ‘saturation’ kinetics in terms of intermediate complexation of BNPP leads to unreasonably large value of the binding constant. No simple explanation of this phenomenon can be proposed at the moment, however. In conclusion, our results for the Y(III)–H2O2 system confirm the presence of M2(O2)22 + species previously reported for La(III) and show the formation of new M2(O2)2(OH)2 complexes. Formation of M2(O2)3 complexes is disproved by performing titrations in a wide range of reactant concentrations. Kinetic results indicate participation of two Y2(O2)2(OH)2 complexes in the cleavage of BNPP and a ‘saturation’ behavior at higher metal concentrations. The latter probably reflects a complex reaction mechanism rather than real complexation of BNPP with Y(III) peroxide complexes.

4. Supporting information available Supporting material is available from the authors upon request.

Acknowledgements The work was supported by CONACYT (Project 25183-E) and DGAPA-UNAM (Project IN 214998).

References

(2)

which can bind the substrate in accordance with the Eq. (3) (Y2(O2)2(OH)2)2 + BNPP = {(Y2(O2)2(OH)2)2(BNPP)} (3) with subsequent intramolecular cleavage of the phosphodiester bond,

[1] (a) Recent reviews: (a) E. Kimura, in: K.D. Karlin (Ed.), Progress in Inorganic Chemistry, vol. 41, Wiley, 1994, pp. 443 –491; (b) E. Kimura, T. Koike, in: A.G. Sykes (Ed.), Advances in Inorganic Chemistry, vol. 44, Academic Press, New York, 1997, p. 229; (c) E.L. Hegg, J.N. Burstyn, Coord.Chem. Rev. 173 (1998) 133; (d) M. Komiyama, N. Takeda, H. Shigekawa, Chem. Commun. (1999) 1443; (e) R. Kra¨ mer, Coord. Chem. Rev. 182 (1999) 243;

246

[2] [3] [4] [5] [6] [7] [8]

Y. Mejia-Radillo, A.K. Yatsimirsky / Inorganica Chimica Acta 328 (2002) 241–246 (f) N.H. Williams, B. Takasaki, J. Chin, Acc. Chem. Res. 32 (1999) 485; (g) A. Blasko´ , T.C. Bruice, Acc. Chem. Res. 32 (1999) 475; (h) J.K. Bashkin, Curr. Opin. Chem. Biol. 3 (1999) 752; (i) P. Molenveld, J.F.J. Engbersen, D.N. Reinhoudt, Chem. Soc. Rev. 29 (2000) 75. A.J. Kirby, M. Younas, J. Chem. Soc., Sect. B (1970) 510. B.K. Takasaki, J. Chin, J. Am. Chem. Soc. 115 (1993) 9337. B.K. Takasaki, J. Chin, J. Am. Chem. Soc. 117 (1995) 8582. R. Breslow, B. Zhang, J. Am. Chem. Soc. 116 (1994) 7893. J. Kamitani, J. Sumaoka, H. Asanuma, M. Komiyama, J. Chem. Soc., Perkin Trans. 2 (1998) 523. F.McC. Blewett, P. Watts, J. Chem. Soc., Sect. B (1971) 881. (a) R.M. Smith, A.E. Martell, Critical Stability Constants, vol. 4, Plenum Press, New York, 1976; (b) C.F. Baes Jr., R.E. Mesmer, The Hydrolysis of Cations, Wiley, New York, 1976;

[9] [10] [11]

[12] [13] [14]

[15] [16] [17]

(c) R.M. Smith, A.E. Martell, Critical Stability Constants, vol. 2, Plenum Press, New York, 1975. R.A. Moss, K.G. Ragunathan, Lamgmuir 15 (1999) 107. S.J. Lyle, Md.M. Rahman, Talanta 10 (1963) 1177. A.E. Martell, R.J. Motekaitis, in: P.S. Mahe (Ed.), Determination and Use of Stability Constants, 2nd ed., John Wiley & Sons, New York, 1992. P. Gans, A. Sabatini, A. Vacca, Talanta 43 (1996) 1739. Y. Mejia-Radillo, A.K. Yatsimirsky, H.J. Foroudian, N.D. Gillitt, C.A. Bunton, J. Phys. Org. Chem. 13 (2000) 505. (a) H.-J. Schneider, J. Rammo, R. Hettich, Angew. Chem., Int. Ed. Engl. 32 (1993) 1716; (b) A. Roigk, R. Hettich, H.-J. Schneider, Inorg. Chem. 37 (1998) 751. K.O.A. Chin, J.R. Morrow, Inorg. Chem. 33 (1994) 5036. J.H. Kim, J. Chin, J. Am. Chem. Soc. 114 (1992) 9792. P.E. Jurek, A.E. Martell, Inorg. Chem. 38 (1999) 6003.