Cleavage of an RNA Model Compound by an Arylmercury Complex

Abstract A water‐soluble arylmercury complex has been synthesized, and its ability to catalyze the cleavage of the phosphodiester linkage of the RNA model compound adenylyl‐3′,5′‐(2′,3′‐O‐methyleneadenosine) has been assessed over a pH range of 3–8.5 and a catalyst concentration range of 0–7 mM. In the presence of 1 mM catalyst, the observed pH–rate profile featured a new pH‐independent region between pH 6 and 7, the catalyzed reaction being as much as eight times faster than the background reaction. At pH 7, the acceleration increased linearly from three‐ to 17‐fold upon increasing the catalyst concentration from 1 to 7 mM. The linear dependence indicates a relatively low affinity of the catalyst for the substrate and, hence, the potential for considerable improvement on tethering to an appropriate targeting group, such as an oligonucleotide.


General methods
Dichloromethane and N,N-dimethylformamide were dried over 4 Å molecular sieves. NMR spectra were measured on a Bruker Biospin 500 MHz NMR spectrometer 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 using electrospray ionization.

Kinetic measurements
The hydrolytic reactions of adenylyl-3´,5´-(2´,3´-O-methyleneadenosine) (4) were carried out in the absence and presence of the organometallic catalyst 1-Hg in sealed tubes immersed in a water bath, the temperature of which was kept at 90 °C (± 0.1 °C). The pH of the reaction solutions was adjusted with 125 μL samples were withdrawn from the reaction solutions at appropriate time intervals and cooled to 0 °C to quench the hydrolysis. Composition of the samples was determined by RP-HPLC on a Thermo Scientific Aquasil column (4 × 150 mm, 5 µm) eluting with a linear gradient of acetonitrtile (3-15% over 30 min) in 50 mM aqueous ammonium acetate. The flow rate was 1.0 mL min -1 and the detection wavelength 260 nm. The observed retention times (min) were 9.8 (cAMP), 13.7 (adenosine), 19.4 (5), 24.4 (6) and 26.6 (4). The products were identified by spiking with authentic samples as well as by HPLC/MS analysis. A representative chromatogram has been included as Figure S8.
Irrespective of the presence of 1-Hg, the pH-rate profile for the cleavage of 4 and 5 features a pHindependent minimum flanked by an acid-catalyzed (first-order in [H + ]) region under more acidic conditions and a base-catalyzed (first-order in [OH -]) region under more basic conditions. The kinetics of these partial reaction are described by the rate equations (S1), (S2) and (S3), respectively.
kcl H2O is the first-order rate constant for the pH-independent reaction and kcl H and kcl OH the second-order rate constants for the hydronium and hydroxide ion catalyzed reactions. KW is the ion product of water under S3 the experimental conditions (6.2 × 10 -13 M 2 ) and [4+5] the total concentration of compounds 4 and 5. In the presence of 1-Hg, an additional plateau at pH 6-7 and a second-order dependence on [OH -] at pH 5-6 are observed. The second-order dependence on [OH -] indicates the loss of two protons on going from the dominant ionic form of the complex formed by 4 or 5 with 1-Hg to the transition state of the 1-Hg catalyzed reaction. The concentration of this doubly deprotonated species is given by equation (S4) and the kinetics of the 1-Hg catalyzed partial reaction by equation (S5).
Ka1 and Ka2 are acid dissociation constants related to the two deprotonation events to give the reactive complex, kcl cat is the second-order rate constant for the 1-Hg catalyzed reaction and [1-Hg] the total concentration of 1-Hg. Finally, the observed rate constant at any pH is given by equation (S6) as the sum of the pseudo first-order rate constants (including the concentration of hydronium ion, hydroxide ion and 1-Hg in case of the respective catalyzed reactions) of the various partial reactions.
Non-linear least-squares fitting of the experimental data to equation (S6) was carried out using Origin 2016 software. The initial guesses for the fitted parameters were kcl H = 1 × 10 -3 , kcl H2O = 1 × 10 -7 , kcl OH = 0.1, kcl cat = 1 × 10 -6 and Ka1 = Ka2 = 3 × 10 -6 . KW was locked to 6.2 × 10 -13 . ). The samples (125 μL) were withdrawn from the reaction solution at appropriate time intervals (Table   S1) and were cooled to 0 °C to quench the reactions. Composition of the samples was analyzed by RP-HPLC and the observed peak areas were converted to mole fractions by dividing with the combined peak area of all reaction components (Table S1). Molar absorptivity of the dimeric compounds 4 and 5 was assumed to be twice as high as that of the monomeric compounds 2´,3´-cAMP and 6. Pseudo first-order rate constants for the decomposition of 4 and its 2´,5´-isomer 5 were obtained by applying the integrated first-order rate equation to the time-dependent combined concentration of these compounds. Accordingly,

S4
ln(x(4+5)t / x(4+5)0) was plotted as a function of t and the rate constant was obtained as the opposite number of the slope of a straight line fitted to the experimental data points ( Figure S1).

Pentaethylene glycol monotosylate (2)
Pentaethylene glycol (1.91 g, 8.00 mmol) was dissolved in dry dichloromethane (50 mL). The solution was cooled on an ice-water bath and silver oxide (2.78 g, 12 mmol) and sodium iodide (1.27 g, 8.80 mmol) were added. Finally, 4-toluenesulfonyl chloride (1.6 g, 8.40 mmol) was added in small portions over 15 min. The ice-water bath was removed and the reaction mixture was stirred for 10 min at room temperature. The mixture was filtrated and the filtrate washed with a 10% aqueous solution of sodium bicarbonate (50 ml). The organic layer was dried with sodium sulfate, filtrated and evaporated to dryness.
The filtrate was fractioned by reverse phase chromatography on a Sunfire Hyperprep column (10 × 250 mm, 5 μm) using a gradient elution with a mixture of acetonitrile (30-50% over 30 min) and 50 mM