Factors Influencing the Activity of Nanozymes in the Cleavage of an RNA Model Substrate

A series of 2-nm gold nanoparticles passivated with different thiols all featuring at least one triazacyclonanone-Zn(II) complex and different flanking units (a second Zn(II) complex, a triethyleneoxymethyl derivative or a guanidinium of arginine of a peptide) were prepared and studied for their efficiency in the cleavage of the RNA-model substrate 2-hydroxypropyl-p-nitrophenyl phosphate. The source of catalysis for each of them was elucidated from the kinetic analysis (Michaelis–Menten profiles, pH dependence and kinetic isotope effect). The data indicated that two different mechanisms were operative: One involving two Zn(II) complexes and the other one involving a single Zn(II) complex and a flanking guanidinium cation. The mechanism based on a dinuclear catalytic site appeared more efficient than the one based on the cooperativity between a metal complex and a guanidinium.


The synthesis of thiol (1)
2-(Hept-6-en-1-yl)isoindoline-1,3-dione (18): In a round-bottom flask, tetrabutylammonium iodide (0.02 eq.) and potassium phthalimide (2.0 eq.) were suspended in acetonitrile. 17 (1.0 eq.) was added dropwise and the resulting mixture was heated up to 80˚C for exactly 24 hours. Once the mixture reached room temperature, it was diluted with diethyl ether and passed through a celite plug. The filtrate was concentrated in vacuo and the yellow residue was purified by column chromatography (hexane/ethyl acetate 95/5) giving the desired product as a white solid (83% yield).
Then sodium carbonate (3.0 eq.) and methyl bromoacetate (1.0 eq.) were added. The mixture was stirred overnight at room temperature under nitrogen. Acetonitrile was evaporated and the oily crude was purified by column chromatography (petroleum ether/ ethyl acetate 80/20-70/30) yielding the desired product as a colorless oil (83% yield).

[M + H] +
2-(4,7-Bis(tert-butoxycarbonyl)-1,4,7-triazonan-1-yl)acetic acid (7): In a round-bottom flask placed in an ice bath, 21 (4.7 mmol) was dissolved in methanol (40 ml). 1M sodium hydroxide (40 mL) was added dropwise. After the addition, the reaction was stirred for 1 hour at room temperature. Next, methanol was evaporated and the aqueous phase was acidified with 10% aqueous s olution of potassium hydrogen sulfate until pH 3 was reached. The product was extracted with ethyl acetate and the collected organic phases were dried over sodium sulfate. After evaporation of the solvents, the desired product was isolated as a white solid (85% yield).

S5
Obtained product was used in situ without any additional purification in the synthesis of gold nanoparticles (AuNP1).
Potassium thioacetate (1.0 eq.) together with an additional portion of acetone was added. This resulting mixture was stirred overnight at room temperature. Afterwards the solution was concentrated, extracted with ethyl acetate and dried over a Na2SO4. After solvent evaporation, the remaining orange oil was purified by column chromatography (petroleum ether/ethyl acetate 98/2-96/4). The desired product was obtained as a white solid (46% yield).  : 196.51, 34.49, 33.19, 31.07, 29.87, 29.72, 29.52, 29.44, 29.16, 29.11, 28.53 Di-tert-butyl-7-(10-(acetylthio)decyl)-1,4,7-triazonane-1,4-dicarboxylate (10): In a round-bottom flask previously purged with nitrogen, potassium carbonate (3.0 eq.) and sodium bicarbonate (3.0 eq.) were suspended in dry MeCN. Then 24 (1.2 eq.) and 6 (1.0 eq.) were added. The mixture was stirred at 60˚C for 4 hours. Afterwards, the s olvent was removed and resulting oily crude was purified by column chromatography (dichloromethane/methanol 97/3). The desired product was isolated as a yellow oil (40% yield). (0.1 mmol) was dissolved in a small amount of ethanol. An equal amount of 6M hydrochloric acid was added dropwise. After the addition, the resulting mixture was refluxed for 2 hours. Afterwards, the solvents were evaporated and the crude was dried under high vacuum. Obtained product was used in situ without any additional purification in the synthesis of gold nanoparticles (AuNP2).

The synthesis of thiols (30, 4, 5)
Ethyl 3,5-diaminobenzoate (11): In a two-neck round bottom flask, thionyl chloride (SOCl2; 1.0 eq.) was added dropwise to dry ethanol under nitrogen at 0°C. Afterwards, 25 (3.0 eq.) was added in 3 portions and the resulting mixture was stirred at reflux overnight. The ethanol and unreacted SOCl2 were evaporated, remaining residue was dissolved in water and alkalized with sat. aqueous solution of NaHCO3. The cloudy mixture was extracted with ethyl acetate, collected organic phases were washed with water and dried over a MgSO4. Brown crystals were obtained (91% yield).
The obtained product, being a precursor of thiol 3, was used in situ without any additional purification in the synthesis of gold nanoparticles.
A total volume was adjusted using dichloromethane (up to 15 mL) and then everything was stirred for 1 hour. Afterwards the solvents were filtrated, resin washed with NMP, MeOH and DCM. The Fmoc group of the last coupled amino acid was deprotected by 20% solution of piperidine in DMF.
The liquids were again filtered off and the resin washed with NMP, MeOH and DCM. Described steps were repeated until desired oligopeptide was built. After the last coupling, the cocktail made from 95% (v) of TFA, 2.5% (v) of triisopropylsilane and 2.5% (v) of water was poured into dry resin, in order to cleave the oligopeptide from the solid platform. The concoction was stirred for 2 hours.
Next the crude was quenched and washed several times with cold tert-butylmethyl ether. The protocol yielded a formation of desired compounds with high purity.

The synthesis of 2-hydroxypropyl p-nitrophenyl phosphate (34)
The synthesis of HPNP was performed basing on the method published by Brown and Usher. 1 33 (5.0 mmol) was dissolved in 10 mL of distilled water. The solution was passed through an ionexchange column (IR-120 (H+) resin). The acid effluent was alkalized to pH 8.0 with an aqueous solution of ammonia. Following this, 1,2-epoxypropane (17.0 mmol) was added and the reaction was stirred at 40°C for 2 days and any unreacted epoxide was removed in vacuo. The remaining liquid was diluted with a small amount of water and passed through an ion-exchange column ((IR-120 (H+) resin). The aqueous solution was carefully neutralized to pH 6.5-7.0 with an aqueous solution of barium hydroxide (carbonate-free) and then concentrated in vacuo at room temperature until the volume was reduced to 70%. Ethanol (2 vol.) was added and the white precipitate was filtered off.

The synthesis of gold nanoparticles
The synthesis of AuNPs was performed using the method published by Scrimin. 2 All the glassware was washed with Aqua Regia and rinsed with distilled water. The aqueous solution of HauCl4·3H2O (1.0 eq.) was extracted with tetraoctylammonium bromide (3.0 eq.) previously dissolved in degassed toluene. To the red-orange organic phase, dioctylamine (20 eq.) was added.
The mixture was vigorously stirred at room temperature under nitrogen for 30 minutes. During that time the colour changed from an orange to colourless. The flask was placed in an ice-bath and then aqueous solution of NaBH4 (10 eq.) was rapidly added. The colour changed immediately to brownishblack due to nanoparticles' formation. After 2 hr stirring, a small amount of left water was removed.
Keeping the reaction under an ice-bath, desired thiol (2.0 eq.) dissolved in methanol was added to the solution and then the reaction was left stirring for additional 2 hours. AuNPs were purified by their sonication and washing with different solvents (hexane, toluene, petroleum ether, ethyl acetate and methanol). Eventually, their concentrated solution was applied on the gel permeation