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

Abstract

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.

1. 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 pK_a 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 R_p and S_p 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.

2. Results and Discussion

2.1. Synthesis

Phosphodiester 1a and its phosphoromonothioate analog 2a (as R_P and S_P 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 N⁶,N⁶-dibenzoyl-2′,3′-O-methyleneadenosine (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 ³¹P NMR chemical shifts, as described in the literature [20].

2.2. Reaction Kinetics

2.2.1. Cleavage and Isomerization of Adenylyl-3′,5′-(2′,3′-O-Methyleneadenosine) (1a)

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.

Over the pH and [Hg(II)] range studied, the expected reactions, namely, cleavage to adenosine 2′,3′-cyclic monophosphate (cAMP) and 2′,3′-O-methyleneadenosine (6) and isomerization to adenylyl-2′,5′-(2′,3′-O-methyleneadenosine) (1b), predominated (Scheme 2, k₁, k₂ and k₋₂, respectively). cAMP was further converted to adenosine without accumulation of 2′- and 3′-monophosphate intermediates. Finally, depurination of the various adenosine derivatives was also observed. The rates of these last two reactions were, however, too slow to be quantified reliably.

The dependence of the rate of cleavage and isomerization of 1a on [Hg(II)] was studied over a concentration range of 0–5 mM at pH 6.0 (Figure 1). Both reactions exhibited a similar pattern of [Hg(II)]-dependence of rate at low [Hg(II)] and leveling-off at high [Hg(II)], and in both cases, the dependence of the observed pseudo first-order rate constant on [Hg(II)] may be expressed by Equation (1).

$$k^{obs}=k^0+\frac{k^{Hg}{\left[\mathrm{Hg}\right]}^n}{K_d{}^n+{\left[\mathrm{Hg}\right]}^n},$$

(1)

k⁰ and k^Hg are the first- and second-order rate constants for the Hg(II)-independent and Hg(II) catalyzed reactions, respectively, K_d 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. Kinetic parameters for Hg(II)-catalyzed cleavage (k₁) and isomerization (k₂ + k_-2) of 1a and 1b; T = 90 °C; pH = 6.0; [buffer] = 30 mM; I(NaNO₃) = 100 mM.

	k0/10−7 s−1	kHg/10−6 M−n s−1	n
k1	2.8 ± 0.1	7 ± 2	3 ± 10
k2 + k−2	14 ± 1	1.3 ± 0.2	3 ± 6

.

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 (K_d = 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 first-order rate constant may be expressed by Equation (2).

$$k_1^{obs}=k_1^W+k_1^{OH}\frac{K_W}{\left[{\mathrm{H}}^{+}\right]},$$

(2)

K_W is the ion product of water under the experimental conditions (6.2 × 10⁻¹³ M²) 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. Rate constants for the partial reactions contributing to the cleavage and isomerization of 1a and 1b; T = 90 °C; [Hg(II)] = 0/5 mM; [buffer] = 30 mM; I(NaNO₃) = 100 mM.

Hg(II)	kH/M−1 s−1		kW/10−8 s−1		kOH/M−1 s−1
	−	+	−	+	−	+
k1	n.a.	1.0 ± 0.3	9 ± 2	n.a.	0.30 ± 0.04	6 ± 1
k2 + k−2	n.a.	n.a.	107 ± 8	140 ± 10	n.a.	n.a.
k2	n.a.	n.a.	59 ± 4	n.a.	n.a.	n.a.
k−2	n.a.	n.a.	48 ± 4	n.a.	n.a.	n.a.

) 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 pH-independent 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).

$$k_1^{obs}=k_1^H\left[{\mathrm{H}}^{+}\right]+k_1^{OH}\frac{K_W}{\left[{\mathrm{H}}^{+}\right]},$$

(3)

K_W is the ion product of water under the experimental conditions (6.2 × 10⁻¹³ M²) 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.

2.2.2. 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].

2.2.3. Desulfurization of Adenylyl-3′,5′-(2′,3′-O-Methyleneadenosine) Phosphoromonothioate (2a)

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 S_p and R_p 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 S_p and R_p diastereomers).

3. Materials and Methods

3.1. 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.

3.2. Materials

2′-O-(tert-Butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)-N⁶-benzoyladenosine 3′-(2-cyanoethyl-N,N-diisopropylphosphoramidite) was a product of Innovassynth Technologies (Khopoli, MH, India). 2′-Deoxyadenosine, I₂ oxidizer solution, HEPES (free acid and sodium salt) and Et₃N•3HF were products of Sigma-Aldrich (Saint Louis, MO, USA). 5-(Benzylthio)-1H-tetrazole was a product of Glen Research (Sterling, VA, USA). Et₃N and MES monohydrate were products of Acros Organics (Geel, Belgium) and MeNH₂ and S₈ of Fluka (Seelze, Germany) and Merck (Darmstadt, Germany), respectively.

3.3. Kinetic Measurements

Reactions of 2′-deoxyadenosine and adenylyl-3′,5′-(2′,3′-O-methyleneadenosine) (1a) were carried out in sealed tubes immersed in a thermostated water bath (90 °C ± 0.1 °C), while the desulfurization of adenylyl-3′,5′-(2′,3′-O-methyleneadenosine) phosphoromonothioate (2a) was carried out in an ice/water bath (0 °C). The hydronium ion concentration of the reaction solutions was adjusted with 2-(N-morpholino)ethanesulfonic acid (MES) and N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffers. The buffer concentration used was 0.030 M. The ionic strength of the solutions was adjusted to 0.1 M with NaNO₃. Hg(II) ions were added as mercuric acetate. After addition of mercuric acetate, the pH of the reaction solutions was adjusted back to the value observed in the absence of Hg(II) by titrating with 1.00 M NaOH.

The aliquots (200 µL) withdrawn from the reaction solutions at appropriate time intervals were cooled in an ice/water bath to quench the reactions. Before analysis, Hg(II) was precipitated out as the tetrachloride complex by the addition of 10 μL of 2.0 M NaCl. Composition of the samples was determined by RP-HPLC on a Thermo Scientific Aquasil column (4 × 150 mm, 5 μm) using UV detection at λ = 260 nm and a flow rate of 1.0 mL min⁻¹. For depurination of 2′-deoxyadenosine, isocratic elution with 6% MeCN in 50 mM aqueous NH₄OAc was used, the retention times of adenine and 2′-deoxyadenosine being 8.1 and 12.7 min, respectively. For the reactions of 1a and 2a, the samples were eluted with linear gradients (3–15% over 30 min and 3–19% over 40 min, respectively) of MeCN in 50 mM aqueous NH₄OAc. The observed retention times (min) for the starting materials and products were 9.7 (2′,3′-cAMP), 14.6 (adenosine), 19.9 (1b), 26.1 (6), 26.8 (1a), 29.1 (2a, S_p diastereomer) and 34.5 (2a, R_p diastereomer). The products were identified by spiking with authentic samples and by HPLC/MS analysis. Representative examples of the chromatograms of the product mixtures can be found in the Supporting Information (Figures S18–S21).

3.4. 2′-O-(tert-Butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)-adenylyl-3′,5′-(2′,3′-O-methyleneadenosine) (4)

N⁶,N⁶-Dibenzoyl-2′,3′-O-methyleneadenosine (200 mg, 0.410 mmol) was co-evaporated three times from anhydrous pyridine, after which it was dried in a vacuum desiccator over P₂O₅ for 16 h. The dried N⁶,N⁶-dibenzoyl-2′,3′-O-methyleneadenosine was dissolved in anhydrous MeCN (3.3 mL) and a solution of 2′-O-(tert-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)-N⁶-benzoyladenosine 3′-(2-cyanoethyl-N,N-diisopropylphosphoramidite) (405 mg, 0.410 mmol) in anhydrous MeCN (3.3 mL) was added, followed by a solution of 5-(benzylthio)-1H-tetrazole (0.82 mmol) in anhydrous MeCN (3.3 mL). The reaction mixture was stirred at 25 °C under N₂ for 15 min, after which it was divided into two equal portions for the synthesis of compounds 4 and 5.

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). ¹H NMR (500 MHz, CDCl₃) δ: 8.39 (s, 1H, H8), 8.32 (s, 1H, H2), 8.22 (s, 1H, H2), 8.06 (s, 1H, H8), 7.46 (m, 2H, DMTr), 7.35 (m, 4H, DMTr), 7.27–7.18 (m, 3H, DMTr), 6.80 (m, 4H, DMTr), 6.30 (br, 2H, NH₂), 6.20 (d, J = 3.0 Hz, 1H, H1′), 6.13 (d, J = 5.5 Hz, 1H, H1′), 6.01 (br, 2H, NH₂), 5.23 (s, 1H, OCH₂O), 5.16 (dd, J = 6.0, 3.0 Hz, 1H, H2′), 5.06 (s, 1H, OCH₂O), 5.00 (m, 1H, H2′), 5.04 (dd, J = 6.5, 2.5 Hz, 1H, H3′), 4.78 (m, 1H, H3′), 4.59 (m, 1H, H4′), 4.40 (m, 1H, H4′), 4.02 (m, 1H, H5′), 3.97 (m, 1H, H5′), 3.756 (s, 3H, OCH₃), 3.755 (s, 3H, OCH₃), 3.55 (dd, J = 10.5, 3.0 Hz, 1H, H5′), 3.46 (m, 1H, H5′′), 0.77 (s, 9H, SiC(CH₃)₃), 0.04 (s, 3H, SiCH₃), −0.08 (s, 3H, SiCH₃). ¹³C NMR (126 MHz, CDCl₃) δ: 158.5 (DMTr), 155.6 (C6), 155.4 (C6), 153.2 (C2), 152.9 (C2), 150.1 (C4), 149.7 (C4), 144.6 (DMTr), 139.7 (C8), 139.1 (C8), 135.7 (DMTr), 130.2 (DMTr), 128.3 (DMTr), 127.9 (DMTr), 126.9 (DMTr), 119.9 (C5), 119.4 (C5), 113.2 (DMTr), 96.4 (OCH₂O), 89.3, (C1′), 87.9 (C1′), 86.6 (DMTr), 84.4 (d, J = 7.2 Hz, C4′), 84.2 (C2′), 83.2 (d, J = 7.2 Hz, C4′), 81.6 (C3′), 75.2 (d, J = 5.3 Hz, C2′), 74.7 (d, J = 5.3 Hz, C3′), 65.6 (d, J = 5.0 Hz, C5′), 63.7 (C5′), 55.2 (OCH₃), 25.6 (SiC(CH₃)₃), 18.3 (SiC(CH₃)₃), −4.8 (SiCH₃), −5.2 (SiCH₃). ³¹P NMR (202 MHz, CDCl₃): δ = −1.6. HRMS (ESI) m/z: [M − H]⁻ calcd for C₄₈H₅₆N₁₀O₁₂Psi^- 1023.3592, found 1023.3625.

3.5. 2′-O-(tert-Butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)-adenylyl-3′,5′-(2′,3′-O-methyleneadenosine) phosphoromonothioate (5)

For the synthesis of compound 5, S₈ (20 mg, 0.62 mmol) and CH₂Cl₂ (5.0 mL) were added to the other half of the MeCN solution containing the crude phosphite triester (approximately 0.205 mmol) prepared above. The resulting mixture was stirred at 25 °C for 16 h, after which H₂O (100 mL) and CH₂Cl₂ (100 mL) were added. Phases were separated and the organic phase 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 phosphorothioate diester 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 (97:2:1, v/v). Compound 5 was obtained as a solid triethylammonium salt in 27% yield (65.0 mg, mixture of two diastereomers in approximately 2:3 ratio). ¹H NMR (500 MHz, CDCl₃, major diastereomer) δ: 8.42 (s, 1H, H8), 8.31 (s, 1H, H2), 8.18 (s, 1H, H2), 8.04 (s, 1H, H8), 7.47 (m, 2H, DMTr), 7.36 (m, 4H, DMTr), 7.26 (m, 2H, DMTr), 7.18 (m, 1H, DMTr), 6.80 (d, J = 8.5 Hz, 4H, DMTr), 6.46 (br, 2H, NH₂), 6.28 (br, 2H, NH₂), 6.21 (d, J = 3.0 Hz, 1H, H1′), 6.14 (d, J = 6.5 Hz, 1H, H1′), 5.25 (s, 1H, OCH₂O), 5.22 (dd, J = 6.5, 3.0 Hz, 1H, H2′), 5.12 (s, 1H, OCH₂O), 5.11–5.06 (m, 2H, 2 × H3′), 4.97 (dd, J = 6.0, 2.5 Hz, 1H, H2′), 4.70 (m, 1H, H4′), 4.52 (m, 1H, H4′), 4.20 (m, 2H, H5′ & H5′′), 3.75 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 3.52–3.40 (m, 2H, H5′ & H5′′), 0.68 (s, SiC(CH₃)₃), −0.06 (s, 3H, SiCH₃), −0.20 (s, 3H, SiCH₃). ¹H NMR (500 MHz, CDCl₃, minor diastereomer) δ: 8.38 (s, 1H, H8), 8.28 (s, 1H, H2), 8.23 (s, 1H, H2), 8.05 (s, 1H, H8), 7.47 (m, 2H, DMTr), 7.36 (m, 4H, DMTr), 7.26 (m, 2H, DMTr), 7.18 (m, 1H, DMTr), 6.33 (br, 2H, NH₂), 6.16 (d, J = 2.5 Hz, 1H, H1′), 6.10 (d, J = 6.0 Hz, 1H, H1′), 5.99 (br, 2H, NH₂), 5.17 (s, 1H, OCH₂O), 5.13 (m, 1H, H2′), 5.11–5.06 (m, 3H, H2′ & 2 × H3′), 5.01 (s, 1H, OCH₂O), 4.52 (m, 1H, H4′), 4.36 (m, 1H, H4′), 4.07 (m, 1H, H5′), 3.99 (m, 1H, H5′′), 3.75 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 3.52–3.40 (m, 2H, H5′ & H5′′), 0.71 (s, SiC(CH₃)₃), 0.03 (s, 3H, SiCH₃), −0.11 (s, 3H, SiCH₃). ¹³C NMR (126 MHz, CDCl₃, major diastereomer) δ: 158.5 (DMTr), 155.6 (C6), 155.5 (C6), 153.0 (C2), 152.7 (C2), 150.1 (C4), 149.6 (C4), 144.7 (DMTr), 139.7 (C8), 139.4 (C8), 135.8 (DMTr), 130.2 (DMTr), 128.3 (DMTr), 127.9 (DMTr), 126.5 (DMTr), 119.8 (C5), 119.6 (C5), 113.3 (DMTr), 96.4 (OCH₂O), 89.9 (C1′), 87.5 (C1′), 86.7 (DMTr), 84.5 (d, J = 9.1 Hz, C4′), 84.3 (C2′), 83.7 (C4′), 82.0 (C3′), 75.7 (d, J = 5.2 Hz, C2′), 75.1 (d, J = 8.7 Hz, C3′), 66.2 (d, J = 5.6 Hz, C5′), 64.0 (C5′), 55.2 (OCH₃), 25.5 (SiC(CH₃)₃), 17.9 (SiC(CH₃)₃), −4.8 (SiCH₃), −5.4 (SiCH₃). ¹³C NMR (126 MHz, CDCl₃, minor diastereomer) δ: 158.5 (DMTr), 155.6 (C6), 155.4 (C6), 153.0 (C2), 152.7 (C2), 150.0 (C4), 149.5 (C4), 144.6 (DMTr), 139.6 (C8), 139.0 (C8), 135.7 (DMTr), 130.2 (DMTr), 128.3 (DMTr), 127.9 (DMTr), 126.5 (DMTr), 119.7 (C5), 119.4 (C5), 113.2 (DMTr), 96.3 (OCH₂O), 89.4 (C1′), 87.6 (C1′), 86.7 (DMTr), 84.4 (C4′), 84.1 (C2′), 83.1 (C4′), 81.7 (C3′), 75.2 (d, J = 7.6 Hz, C2′), 75.4 (d, J = 6.9 Hz, C3′), 65.6 (d, J = 5.6 Hz, C5′), 63.8 (C5′), 55.2 (OCH₃), 25.6 (SiC(CH₃)₃), 18.0 (SiC(CH₃)₃), −4.6 (SiCH₃), −5.3 (SiCH₃). ³¹P NMR (202 MHz, CDCl₃, major diastereomer): δ: 57.8. ³¹P NMR (202 MHz, CDCl₃, minor diastereomer): δ: 58.3. HRMS (ESI) m/z: [M − H]⁻ calcd for 1039.3363 C₄₈H₅₆N₁₀O₁₁PSSi^-, found 1039.3397.

3.6. Adenylyl-3′,5′-(2′,3′-O-methyleneadenosine) (1a)

Compound 4 (37.0 mg, 0.0361 mmol) and triethylamine trihydrofluoride (44.0 µL, 0.270 mmol) were dissolved in anhydrous CH₂Cl₂ (2.0 mL). After stirring for 72 h, the precipitation formed was recovered and washed with CH₂Cl₂ (3 × 5 mL). The crude product was purified by reverse phase chromatography on a ThermoHypersil Hyperprep column (10 × 250 mm, 8 µm) eluting with a mixture of MeCN and 50 mM aqueous ammonium formate (11:89, v/v). Compound 1a was obtained as a solid ammonium salt in 31% yield (6.8 mg). ¹H NMR (500 MHz, D₂O) δ: 8.25 (s, 1H, H8), 8.17 (s, 1H, H8), 8.09 (s, 1H, H2), 8.05 (s, 1H, H2), 6.14 (d, J = 3.0 Hz, 1H, H1′), 5.75 (d, J = 5.0 Hz, 1H, H1′), 5.22 (dd, J = 6.5, 3.0 Hz, 1H, H2′), 5.20 (s, 1H, OCH₂O), 5.17 (s, 1H, OCH₂O), 5.04 (dd, J = 6.5, 4.0 Hz, 1H, H3′), 4.56–4.52 (m, 3H, H2′, H3′& H4′), 4.20 (dt, J = 11.5, 3.0 Hz, 1H, H5′), 4.15–4.12 (m, 2H, H4′, H5′′), 3.65 (d, J = 3.0, 2H, H5′, H5′′). ¹³C NMR (126 MHz, D₂O) δ: 154.8 (C6), 154.5 (C6), 151.5 (C2), 151.4 (C2), 148.3 (C4), 148.0 (C4), 140.7 (C8), 140.4 (C8), 119.1 (C5), 118.5 (C5), 95.7 (OCH₂O), 88.7 (C1′), 88.6 (C1′), 84.2 (d, J = 3.9 Hz, C4′), 83.4 (d, J = 9.2 Hz, C4′), 83.2 (C2′), 80.1 (C3′), 74.4 (d, J = 5.5 Hz, C3′), 73.1 (d, J = 5.5 Hz, C2′), 65.7 (d, J = 5.6 Hz, C5′), 60.9 (C5′). ³¹P NMR (202 MHz, D₂O): δ = -0.93. HRMS (ESI) m/z: [M − H]⁻ calcd for C₂₁H₂₃N₁₀O₁₀P⁻ 607.1420; found 607.1424.

3.7. Adenylyl-3′,5′-(2′,3′-O-methyleneadenosine) phosphoromonothioate (2a)

Compound 5 (70 mg, 0,068 mmol) and triethylamine trihydrofluoride (44.0 µL, 0.270 mmol) were dissolved in anhydrous CH₂Cl₂ (2.0 mL). After stirring for 72 h, the precipitation formed was recovered and washed with CH₂Cl₂ (3 × 5 mL). The crude product was purified by reverse phase chromatography on a ThermoHypersil Hyperprep column (10 × 250 mm, 8 µm) eluting with a mixture of MeCN and 50 mM aqueous ammonium formate (15:85, v/v). Compound 2a was obtained as a solid ammonium salt in a 27% overall yield (6.5 and 4.5 mg of the faster and slower eluting diastereomers, respectively). The faster and slower eluting diastereomers were assigned as the S_p and R_p diastereomers based on their ³¹P chemical shifts, as described in the literature [20]. ¹H NMR (500 MHz, (CD₃)₂SO, R_p diastereomer) δ: 8.53 (s, 1H, H8), 8.45 (s, 1H, H8), 8.26 (s, 1H, H2), 8.22 (s, 1H, H2), 7.81 (br, 4H, NH₂), 6.20 (d, J = 3.0 Hz, 1H, H1′), 5.91 (d, J = 7.0 Hz, 1H, H1′), 5.31 (dd, J = 6.5, 3.0 Hz, 1H, H2′), 5.19 (s, 1H, OCH₂O), 5.15 (s, 1H, OCH₂O), 5.03 (dd, J = 6.5, 3.0 Hz, 1H, H3′), 4.83 (m, 1H, H3′), 4.70 (m, 1H, H2′), 4.41 (m, 1H, H4′), 4.14 (m, 1H, H4′), 4.07 (m, 1H, H5′), 4.02 (m, 1H, H5′′), 3.65 (dd, J = 12.5, 3.0 Hz, 1H, H5′), 3.56 (dd, J = 12.5, 3.0 Hz, 1H, H5′′). ¹H NMR (500 MHz, (CD₃)₂SO, S_p diastereomer) δ: 8.55 (s, 1H, H8), 8.43 (s, 1H, H8), 8.26 (s, 1H, H2), 8.21 (s, 1H, H2), 7.78 (br, 4H, NH₂), 6.19 (d, J = 3.0 Hz, 1H, H1′), 5.91 (d, J = 6.5 Hz, 1H, H1′), 5.29 (dd, J = 6.3, 3.0 Hz, 1H, H2′), 5.19 (s, 1H, OCH₂O), 5.14 (s, 1H, OCH₂O), 5.02 (dd, J = 6.3, 3.3 Hz, 1H, H3′), 4.74–4.70 (m, 2H, H2′ & H3′), 4.41 (m, 1H, H4′), 4.15 (m, 1H, H4′), 4.07 (m, 1H, H5′), 3.99 (m, 1H, H5′′), 3.60 (dd, J = 12.0, 3.0 Hz, 3.51 (dd, J = 12.0, 3.0 Hz, 1H, H5′′). ¹³C NMR (126 MHz, (CD₃)₂SO, R_p diastereomer) δ: 155.4 (C6), 155.2 (C6), 151.5 (C2), 151.3 (C2), 149.31 (C4), 149.26 (C4), 140.8 (C8), 140.7 (C8), 119.7 (C5), 119.3 (C5), 95.7 (OCH₂O), 88.6 (C1′), 88.4 (C1′), 85.4 (d, J = 6.8 Hz, C4′), 83.6 (d, J = 8.1 Hz, C4′), 83.2 (C2′), 81.4 (C3′), 75.2 (d, J = 8.1 Hz, C3′), 73.6 (C2′), 65.8 (d, J = 5.5 Hz, C5′), 61.9 (C5′). ¹³C NMR (126 MHz, (CD₃)₂SO, S_p diastereomer) δ: 155.4 (C6), 155.1 (C6), 151.5 (C2), 151.3 (C2), 149.33 (C4), 149.26 (C4), 140.8 (2 × C8), 119.7 (C5), 119.2 (C5), 95.7 (OCH₂O), 88.5 (C1′), 88.3 (C1′), 85.7 (d, J = 4.5 Hz, C4′), 83.5 (d, J = 8.7 Hz, C4′), 83.3 (C2′), 81.4 (C3′), 75.8 (d, J = 5.9 Hz, C3′), 73.3 (d, J = 3.3 Hz, C2′), 65.8 (d, J = 5.6 Hz, C5′), 62.0 (C5′). ³¹P NMR (202 MHz, (CD₃)₂SO, R_p diastereomer): δ = 58.7. ³¹P NMR (202 MHz, (CD₃)₂SO, S_p diastereomer): δ = 57.2. HRMS (ESI) m/z: [M − H]⁻ calcd for C₂₁H₂₃N₁₀O₉PS⁻ 623.1192, found 623.1221.

3.8. N⁶,N⁶-dibenzoyl-2′,3′-O-methyleneadenosine (3)

Compound 3 was prepared from 2′,3′-O-methyleneadenosine [36] as described previously for N⁶,N⁶-dibenzoyladenosine [37]. ¹H NMR (500 MHz, CDCl₃) δ: 8.76 (s, 1H, H2), 8.21 (s, 1H, H8), 8.09 (m, 2H, Bz), 7.83 (m, 2H, Bz), 7.60 (m, 1H, Bz), 7.52 (m, 2H, Bz), 7.45 (m, 3H, Bz), 6.03 (d, J = 4.3 Hz, 1H, H1′), 5.34 (s, 1H, OCH₂O), 5.25 (d, J = 6.0, 4.3 Hz, 1H, H2′), 5.12 (s, 1H, OCH₂O), 5.01 (dd, J = 6.1, 1.9 Hz, 1H, H3′), 4.53 (m, 1H, H4′), 4.00 (dd, J = 12.7, 1.9 Hz, 1H, H5′), 3.84 (dd, J = 12.6, 2.5 Hz, 1H, H5′′). ¹³C NMR (126 MHz, CDCl₃) δ: 170.5 (C=O), 170.2 (C=O), 152.2 (C2), 150.9 (C6), 150.5 (C4), 142.6 (C8), 133.3 (Bz), 133.2 (Bz), 132.9 (Bz), 132.1 (Bz), 130.2 (Bz), 130.0 (Bz), 128.8 (Bz), 128.6 (Bz), 128.4 (Bz), 128.2 (Bz), 127.4 (Bz), 124.3 (C5), 97.0 (OCH₂O), 92.2 (C1′), 85.7 (C4′), 82.9 (C2′), 81.8 (C3′), 63.0 (C5′). HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₅H₂₁N₅O₆Na⁺ 510.1384, found 510.1373.

4. 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.