Mercury(II)-Catalyzed Cleavage, Isomerization and Depurination of RNA and DNA Model Compounds and Desulfurization of Their Phosphoromonothioate Analogs

: The potential of Hg(II), a metal ion so-far overlooked in the development of artificial nucleases, to cleave RNA and DNA has been assessed. Accordingly, Hg(II)-promoted cleavage and isomerization of the RNA model compound adenylyl-3´,5´-(2´,3´- O -methyleneadenosine) and depurination of 2´-deoxyadenosine were followed by HPLC as a function of pH (5.0–6.0) and the desulfurization of both diastereomers of the phosphoromonothioate analog of adenylyl-3´,5´-(2´,3´-O -methyleneadenosine) at a single pH (6.9). At 5 mM [Hg(II)], cleavage of the RNA model compound was accelerated by two orders of magnitude at the low and by one order of magnitude at the high end of the pH range. Between 0 and 5 mM [Hg(II)], the cleavage rate showed a sigmoidal dependence on [Hg(II)], suggesting the participation of more than one Hg(II) in the reaction. Isomerization and depurination were also facilitated by Hg(II), but much more modestly than cleavage, less than 2-fold over the entire pH range studied. Phosphoromonothioate desulfurization was by far the most susceptible reaction to Hg(II) catalysis, being accelerated by more than four orders of magnitude.


Introduction
Metal ion-catalyzed cleavage of both RNA and DNA phosphodiester linkages has been investigated extensively to understand the mechanism of action of natural nucleases (including ribozymes) [1][2][3][4][5], as well as to develop artificial ones [6][7][8][9][10]. Comparative studies with phosphodiester and phosphorothioate model compounds have been instrumental in revealing coordination of catalytically important metal ions to the scissile linkage [11][12][13][14][15][16]. The catalytic activities of various metal ions reflect their binding affinities to oxygen and sulfur, with hard metal ions being more efficient in the cleavage of phosphates and soft ones in the cleavage of phosphorothioates. Hg(II) lies at the thiophilic end of the spectrum and has indeed been reported to affect very rapid cleavage and desulfurization of RNA phosphorothioate linkages while leaving phosphodiester linkages largely intact [17]. On the other hand, the very low pKa of an aqua ligand of Hg(II) [18] would predict high activity also in the cleavage of RNA phosphodiester linkages [3]. Interestingly, Hg(II) is also the only metal ion reported to accelerate depurination [19], potentially offering a novel approach for the cleavage of DNA. However, to the best of our knowledge, detailed kinetic data on the catalytic effect of Hg(II) on the cleavage, isomerization or depurination of nucleosides and dinucleoside phosphodiesters is not available.
We report herein a detailed kinetic study on the cleavage and isomerization of adenylyl-3´,5´-(2´,3´-O-methyleneadenosine) and the desulfurization of the Rp and Sp diastereomers of its phosphoromonothioate analog, as well as the depurination of 2´-deoxyadenosine in the presence and absence of Hg(II). Adenine was chosen as the base moiety for its relatively rapid depurination. The permanent methylene protection on the 5´-linked nucleoside, in turn, facilitates analysis of the cleavage reaction by ensuring that distinct sets of products would be obtained from the 3´-and 5´linked nucleosides even after possible dephosphorylation. The greatest accelerations by Hg(II) were observed with phosphoromonothioate desulfurization and phosphodiester cleavage, the reactions being four and two orders of magnitude faster in the presence of Hg(II) than in the absence thereof. Much more modest (less than two-fold) accelerations were observed with depurination and isomerization reactions.

Synthesis
Phosphodiester 1a and its phosphoromonothioate analog 2a (as RP and SP diastereomers) were prepared as described in Scheme 1 by displacing the diisopropylamino ligand of an appropriately protected adenosine 3´-(2-cyanoethyl-N,N-diisopropylphosphoramidite) by N 6 ,N 6 -dibenzoyl-2´,3´-Omethyleneadenosine (3), using 5-(benzylthio)-1H-tetrazole as an activator. The phosphite triester formed was oxidized to either phosphate or thiophosphate ester by treatment with either elemental iodine in a mixture of tetrahydrofuran, water and 2,6-lutidine or elemental sulfur in dicholoromethane. The cyanoethyl group was removed in a mixture of pyridine and Et3N and the benzoyl groups in a mixture of aqueous ammonia and methylamine to afford intermediates 4 and 5. Finally, removal of tert-butyldimethylsilyl and dimethoxytrityl groups by treatment with triethylamine trihydrofluoride, followed by RP-HPLC purification, gave the desired products 1a and 2a. The two diastereomers of the phosphorothioate product 2a were separable by RP-HPLC and were assigned absolute configurations based on their 31 P NMR chemical shifts, as described in the literature [20].  Isomerization of Adenylyl-3´,5´-(2´,3´

Cleavage and
Hydrolytic reactions of the phosphodiester model compound 1a were followed by RP-HPLC at 90 °C over a pH range of 5.0-6.0 (30 mM MES buffer) and at a constant ionic strength of 0.10 M (adjusted with sodium nitrate). At pH 6.0, the reactions were also followed at various Hg(II) concentrations ranging from 0 to 5 mM. The narrow pH range was dictated by the low solubility of Hg(II) under more alkaline conditions. Prior to analysis, sodium chloride was added to the samples to precipitate Hg(II) out as the tetrachlorido complex.
k 0 and k Hg are the first-and second-order rate constants for the Hg(II)-independent and Hg(II) catalyzed reactions, respectively, Kd is the dissociation constant of the reactive complex and n is the reaction order with respect to Hg(II) ion. The rate constants obtained by non-linear least-squares fitting of the experimental data to Equation (1) are summarized in Table 1. The rate constants for the Hg(II)-independent reactions were consistent with those reported previously for similar model compounds under the same conditions [21]. The observation that cleavage is catalyzed more efficiently than isomerization (k Hg = 7.0 and 1.3 × 10 −6 M −n s −1 , respectively) is also in line with previous reports on catalysis by other metal ions [11], and suggests that Hg(II) exerts its catalytic effect mainly by assisting in the departure of the 5´-linked nucleoside. This assistance could take the form of either direct coordination of Hg(II) to the departing oxygen or proton transfer from an aqua ligand. The dissociation constants for the reactive complex were very similar for both cleavage and isomerization (Kd = 0.8 and 0.63 mM, respectively) and comparable to the respective value determined for the Hg(II)-adenosine complex [22]. Adenosine 5´monophosphate, on the other hand, has been reported to form indirect chelates with divalent transition metal ions, with an aqua ligand of an N7-coordinated metal ion hydrogen-bonded to the phosphate [23][24][25]. Such a binding mode seems likely also in the present case and would be consistent with the observed catalysis. Finally, fitting of the experimental data suggested a third-order dependence of the rate of cleavage and isomerization on Hg(II). This value is highly uncertain, but the sigmoidal plots obtained for both reactions would seem to indicate the participation of more than one Hg(II) ions. Participation of two metal ions has been demonstrated with several other metals, and the most efficient metal catalysts for the cleavage of RNA phosphodiester linkages are, indeed, dinuclear complexes [26][27][28][29][30][31][32][33].
In the absence of Hg(II), mutual isomerization of 1a and 1b was pH-independent, whereas, cleavage to monomeric products was gradually turning first-order in hydroxide ion on increasing pH ( Figure 2). Isomerization was an order of magnitude faster than cleavage, allowing k2 and k−2 to be determined based on the relative concentrations of 1a and 1b at equilibrium. The ratio of [1a] to [1b] approached unity, corresponding to essentially equal values for k2 and k−2. With cleavage, the contributions of pH-independent and hydroxide ion catalyzed reactions to the observed pseudo firstorder rate constant may be expressed by Equation (2).
KW is the ion product of water under the experimental conditions (6.2 × 10 −13 M 2 ) and k1 W and k1 OH the first-and second-order rate constants for the pH-independent and hydroxide ion catalyzed reactions, respectively. The rate constants obtained (Table 2) were in good agreement with those reported previously for similar model compounds [21].  At a saturating (5.0 mM) concentration of Hg(II), mutual isomerization of 1a and 1b was pHindependent and only marginally faster than in the absence of Hg(II) ( Figure 2). Cleavage, in turn, appeared to be first-order in hydronium ion at low pH and first-order in hydroxide ion at high pH, the rate acceleration by Hg(II) ranging from 140-fold at pH 5.0 to 20-fold at pH 6.0. Comparable acceleration has been reported previously for other divalent transition metal ions, such as Zn(II) [34]. Over the entire pH range studied, cleavage was also significantly faster than isomerization and the 2´-isomer 1b did not accumulate sufficiently for reliable determination of k2 and k−2. The observed pseudo first-order rate constant for the cleavage reaction was broken down to contributions of hydronium and hydroxide ion catalyzed reactions by Equation (3).
KW is the ion product of water under the experimental conditions (6.2 × 10 −13 M 2 ) and k1 H and k1 OH the second-order rate constants for the hydronium and hydroxide ion catalyzed reactions, respectively. Rate constants obtained for all partial reactions are summarized in Table 2.

Depurination of 2´-Deoxyadenosine
Depurination of 2´-deoxyadenosine was studied under the same conditions as the hydrolytic reactions of 1a and 1b. Conversion of 2´-deoxyadenosine to adenine and nonchromophoric products (Scheme 3, k3) was the only reaction over the entire pH range studied and regardless of the presence of Hg(II). The reaction was first-order in hydronium ion both in the absence and presence of Hg(II) (Figure 3), the second-order rate constants being k3 H = 1.16 ± 0.06 and 2.2 ± 0.2 M −1 s −1 , respectively. Similar modest acceleration by Hg(II) has been reportedly previously for 9-(1-ethoxyethyl)purine and attributed to additive electron-withdrawing effects of simultaneous protonation at N1 and Hg(II) coordination at N7 [19].  The rate of desulfurization was determined separately for both diastereomers of adenylyl-3´,5´-(2´,3´-O-methyleneadenosine) phosphoromonothioate (2a) at 0 °C and pH 6.9 and either in the absence or at a saturating concentration of Hg(II). The ionic strength was adjusted to 0.10 M with sodium nitrate. Desulfurization of 2a (Scheme 4, k4) was accompanied with apparent isomerization and cleavage of the immediate product 1a, the observed products being 1a, 1b, cAMP and 6 in a 5:4:1:1 ratio. As discussed in the literature [35], desulfurization of ribonucleoside phosphoromonothioates is initiated by nucleophilic attack of the neighboring OH group to give a pentacoordinate intermediate. Loss of hydrogen (or, in the presence of Hg(II), mercuric) sulfide from this intermediate leads to a cyclic phosphotriester that rapidly decomposes by rupture of either one of the endocyclic P-O bonds, affording 1a or 1b, or the exocyclic P-O bond, affording cAMP and 6. This mechanism also explains the apparent competition between cleavage and desulfurization reported previously [17]-desulfurization always occurs first, and the competition is actually between endo-and exocyclic P-O bond fission. In the absence of Hg(II), desulfurization of both diastereomers of 2a proceeded at nearly equal rates (k4 = 7 ± 2 and 6 ± 2 × 10 −8 s −1 for Sp and Rp diastereomers). The product distribution was unaffected by the presence of Hg(II), but the rate of desulfurization was increased by more than four orders of magnitude (k4 = 4 ± 1 and 1.4 ± 0.1 × 10 −3 s −1 for Sp and Rp diastereomers).

Scheme 4.
Desulfurization of 2a yields both cleavage and isomerization products.

General
Pyridine, CH2Cl2 and MeCN were dried over 4 Å molecular sieves. Et3N and MeNH2 were distilled before use. NMR spectra were recorded on a Bruker Biospin 500 MHz NMR spectrometer (Bruker, Billerica, MA, USA), and the chemical shifts (δ, ppm) are quoted relative to the residual solvent peak as an internal standard. High resolution mass spectra were recorded on a Bruker Daltonics micrOTOF-Q mass spectrometer (Bruker, Billerica, MA, USA) using electrospray ionization.   (Figures S18-S21). For the synthesis of compound 4, a solution of I2 (0.24 mmol) in a mixture of THF (6.9 mL), H2O (3.4 mL) and pyridine (1.7 mL) was added. After stirring at 25 °C for 5 min, the reaction was quenched by addition of 5% aqueous NaHSO3 (100 mL). Phases were separated, the aqueous phase was extracted with CH2Cl2 (2 × 100 mL), the combined organic phases were dried on Na2SO4 and evaporated to dryness. The residue was dissolved in a mixture of pyridine (15 mL) and Et3N (5 mL) and the resulting solution was stirred 25 °C for 16 h, after which it was evaporated to dryness. The crude phosphodiester intermediate thus obtained was dissolved in a mixture of 33% aqueous NH3 (3 mL) and 40% aqueous MeNH2 (3 mL). After being stirred at 25 °C for 1 h, the solution was evaporated to dryness and the residue purified on a silica gel column eluting with a mixture of CH2Cl2, MeOH and Et3N (94:5:1, v/v). Compound 4 was obtained as a solid triethylammonium salt in 25% yield (58.0 mg). 1

Conclusions
Hg(II) accelerates the cleavage of RNA phosphodiester linkages by as much as two orders of magnitude, comparable to other divalent transition metal ions. At least two Hg(II) ions seem to participate in the catalysis, again in line with results obtained with other metal ions. While the observed modest acceleration of isomerization can be explained in terms of electrophilic catalysis by the Hg(II) ions coordinated to the scissile phosphate and/or general base catalysis by a Hg(II) hydroxo ligand, the much more pronounced acceleration of cleavage suggests additional general acid catalysis by a Hg(II) aqua ligand. Together with the unique ability of Hg(II) to accelerate depurination, these findings make Hg(II) an interesting new addition to the repertoire of metal ions usable in the development of artificial nucleases. It is also worth pointing out that while the state-of-the-art artificial metallonucleases are coordination complexes, the high stability of Hg(II)-carbon bonds would allow the design of a novel class of essentially covalent metallanucleases.