A novel route for preparing 5′ cap mimics and capped RNAs: phosphate-modified cap analogues obtained via click chemistry

A different approach for synthesizing 5′ cap mimics to yield a novel class of dinucleotide cap analogues containing a triazole ring within the oligophosphate chain.

Tables: Table S1. Yields of syntheses of triazole-modified dinucleotide cap analogues.   GpppG is average from 5 repetitions as for practical reasons the experiment was performed independently for groups of no more than 8 analogues.    Figure S1.

1.2.2
Analytical and preparative reverse-phase (RP) HPLC Both analytical and semi-preparative HPLC were performed on Agilent Technologies Series 1200 with UV-detection at 254 nm and fluorescence detection (Ex: 260 nm, Em: 370 nm). For chemical and enzymatic reactions, monitoring analytical HPLC was performed using Supelcosil LC-18-T column (4.6 x 250 mm, 5 μm, flow rate 1.3 mL/min) with one of three different linear gradients of methanol in 0.05 M ammonium acetate buffer (pH 5.9): program A -gradient 0-25% of methanol in 15 min, program B -gradient 0-50% of methanol in 15 min, program C -gradient 0-50% of methanol in 7.5 min and then isocratic elution (50% of methanol) until 15 min. For pH-dependent degradation studies and reactions monitoring of different steps of ARCA analogues synthesis analytical HPLC was performed using Grace VisionHT C18-HL column (4.6 x 250 mm, 5μm, flow rate 1.3 mL/min) with linear gradient 0-25% of methanol in 0.05 M ammonium acetate buffer (pH 5.9) in 15 min. Semi-preparative RP HPLC was performed using Discovery RP Amide C-16 HPLC column (25 cm x 21.2 mm, 5 μm, flow rate 5.0 mL/min) with linear gradients of acetonitrile in 0.05 M ammonium acetate buffer (pH 5.9). The products, after at least triple lyophilisation, were isolated as ammonium salts.

Yields and concentrations determination
The yields of mononucleotide analogues after ion-exchange purification and the concentrations of mono-and dinucleotide analogues solutions used for biophysical and biological experiments were determined on the basis of absorbance measurements performed at 260 nm in 0.1 M phosphate buffer pH 6.0 for 7-methylguanine mononucleotide analogues and in 0.1 M phosphate buffer pH 7.0 for dinucleotide analogues and guanine mononucleotide analogues. The quantities of obtained ion-exchange purified products were expressed as optical density miliunits (opt. mu = absorbance of the solution by volume in mL). For calculations of yields and concentrations following molar extinction coefficients [M -1 cm -1 ] were employed: ε = 22600 (dinucleotides), ε = 11400 (m 7 G mononucleotides), ε = 12080 (G mononucleotides). Concentrations of transcripts were determined using NanoDrop 2000c Spectrofotometer (Thermo Scientific).

NMR spectroscopy and mass spectrometry
The structure and purity of each final product were confirmed by high resolution mass spectrometry using negative or positive electrospray ionization (HRMS (-) ESI or HRMS (+) ESI) and 1 H NMR, 31  P NMR chemical shifts were reported in ppm and referenced to respective internal standards: sodium 3-(trimethylsilyl)-2,2',3,3' tetradeuteropropionate (TSP) and 20% phosphorus acid in D 2 O. Signals in 1 H NMR spectra of dinucleotides were assigned according to 2D NMR spectra (gDQCOSY, gHSQCAD). In 31 P signal assignment of dinucleotide cap analogues the phosphates were denoted analogously to m 7 Gp γ p β p α G.

S19
Phosphoramidate cap analogues (6a-d, 7a-9b) hydrolyzed in D 2 O gradually. Although pure compounds (see HPLC profiles, Supporting Information 2) were dissolved in D 2 O just before measurements, the spectra indicated some level of hydrolysis. % of hydrolysis is provided along with compound characterization data, if higher than 5%.

Preparation of triethylammonium (TEA) salts
The commercially available guanosine 5'-monophosphate (GMP) disodium salt and tetrasodium pyrophosphate were converted into triethylammonium forms by passing their aqueous solutions (ca. 1 g/20 mL) through Dowex 50 W x 8 cationite. The collected eluates were evaporated under reduced pressure with repeated additions of ethanol and acetonitrile to dryness yielding the nucleotide triethylammonium salt as a white solid and triethylammonium pyrophosphate as colorless oil.
Triethylammonium phosphate was prepared by slowly adding triethylamine to the low-concentrated solution of H 2 PO 4 in water until pH 7 was obtained which was followed by evaporation to afford oily, colorless residue.

N7-methylation of guanine nucleotides (GMP, GDP and GTP)
An appropriate analogue (GMP, GDP or GTP, TEA salt) was dissolved in dry DMSO to obtain ca. 0.1 M solution followed by addition of CH 3 I (8 equiv.). The mixture was stirred at room temperature for several hours until HPLC analysis indicated more than 90% conversion of the substrate and the presence of N 7 -methylated nucleotide as the major product. The reaction was stopped by 10-fold dilution with water and organic-soluble compounds were removed by 3-time washing with diethyl ether. The aqueous phase was then treated with a pinch of Na 2 S 2 O 5 to reduce the residual iodine and the pH of solution was set to 7 by addition of solid NaHCO 3 . The following ionexchange purification afforded triethylammonium salt of m 7 GMP (63%), m 7 GDP (68%) or m 7 GTP (58%).

General procedure A (GP A): Coupling of nucleotide imidazolides with phosphoester subunit (18d)
Compound 18d (3 equiv.) was stirred in DMF until complete dissolution (to ̴ 0.3 M concentration). Then, 19a or 19b (1 equiv.) along with ZnCl 2 (8 equiv.) was added and the mixture was stirred for 1-2 h at room temperature. The reaction was stopped by 10-fold dilution with water and addition of EDTA (8 equiv.) and NaHCO 3 (ca. 17.6 equiv.). The product was purified by ion-exchange chromatography on DEAE Sephadex A-25 and evaporated to dryness as described in General Information. Prior to NMR characterization the product was additionally purified by semi-preparative HPLC as described in General Information.
General procedure B (GP B): N 7 -methylation of guanine nucleotides using CH 3 I An appropriate nucleotide (TEA salt) was dissolved in dry DMSO to obtain ca. 0.1 M solution followed by addition of CH 3 I (8 equiv.). The mixture was stirred at room temperature for several hours until HPLC analysis indicated more than 90% conversion of the substrate and the presence of N 7 -methylated nucleotide as the major product. The reaction was stopped by 10-fold dilution with water and organic-soluble compounds were removed by 3-time washing with diethyl ether. The aqueous phase was then treated with a pinch of Na 2 S 2 O 5 to reduce the residual iodine and the pH of solution was set to 7 by addition of solid NaHCO 3. The product was purified by ionexchange chromatography on DEAE Sephadex A-25 and evaporated to dryness as described in General Information. Prior to NMR characterization the product was additionally purified by semi-preparative HPLC as described in General Information.

General procedure C (GP C): N 7 -methylation of guanine nucleotides using (CH 3 ) 2 SO 4
An appropriate nucleotide (TEA salt) was dissolved at ̴ 0.2 M concentration in ca. 0.5 mM aqueous CH 3 COOH (pH 4) to obtain ca. 0.2 M solution. Then, 5 portions of (CH 3 ) 2 SO 4 (2 equiv. each) were added every 10 min to the mixture under vigorous stirring and the pH was maintained at 4 by adding 10% KOH if necessary. The stirring was continued at room temperature for several hours until HPLC analysis indicated more than 90% conversion of the substrate and the presence of N 7 -methylated nucleotide as the major product. The reaction was stopped by 10-fold dilution with water and organic-soluble compounds were removed by 3-time washing with diethyl ether. The pH of aqueous phase was then set to 7 by addition of solid NaHCO 3. The product was purified by ion-exchange chromatography on DEAE Sephadex A-25 and evaporated to dryness as described in General Information. Prior to NMR characterization the product was additionally purified by semi-preparative HPLC as described in General Information.

(12e) γ-C-(2-ethynyl) 2'-O-methylguanosine triphosphate triethylammonium salt
Analogue 12e was obtained analogously to compounds 12a-b. Triethylammonium  stopped by 10-fold dilution with water. The product was purified by ion-exchange chromatography on DEAE Sephadex A-25 and evaporated to dryness as described in General Information to afford 6385 mOD (0.528 mmol, 93%) of 12e triethylammonium salt. Additional HPLC purification of a fraction of obtained product gave 12e as ammonium salt.

Synthesis of phosphoramidate nucleotide analogues General procedure D (GP D): Coupling of nucleotide imidazolides with amine linker
Analogues 15a-d were synthesized analogously as described in Guranowski et al. for the reaction of diamine linkers with guanosine 5'phosphorimidazolide. 7 An appropriate nucleotide imidazolide (19a-f) was dissolved in 0.1 M Tris-HCl buffer pH 8.0 (approx. 1 mL per 100 mg nucleotide) and propargylamine or 2-azidoethyloamine (8 equiv.) was added. The mixture was stirred at room temperature for 24 h. The reaction was diluted with ten volumes of water and extracted with diethyl ether. After setting pH to 7 with 5% HCl, the mixture was either subjected to ion-exchange chromatography purification as described in General Information to afford the desired product as triethylammonium salt or directly purified by semi-preparative HPLC to afford the desired product as ammonium salt.

General procedure E (GP E): S-alkylation of thiophosphate nucleotide analogues
An appropriate nucleotide (20a-d) was dissolved in DMSO to a concentration of ca. 0.1 M and propargyl bromide (1 equiv.) was added. The mixture was stirred at room temperature for approx. 15 min. Then the reaction was diluted with ten volumes of water and extracted with diethyl ether. After setting pH to 7 (if needed), the mixture was subjected to ion-exchange chromatography purification as described in General Information to afford the desired product as triethylammonium salt.  (16b) 5'-azido-5'deoxy-7-methylguanosine 5'-azido-5'deoxyguanosine (16a) (350 mg, 1.14 mmol) was dissolved in DMF (4 mL) and CH 3 I (566 µl, 9.09 mmol) was added. The mixture was stirred at room temperature for 3h (until 95% conversion into the desired product as determined by HPLC). The excess CH 3  CuSO 4 or sodium ascorbate solutions or solvents (DMF or H 2 O) were added upon precipitation or slow kinetics. Final concentrations of reagents are given in the detailed procedures below. When completed, the reaction was quenched by 5-fold dilution with water and addition of Na 2 EDTA (ten equivalents of added CuSO 4 ) followed by direct semi-preparative RP HPLC purification.

General procedure G (GP G): Synthesis of dinucleotide cap analogues containing triazole located between P-subunits
Aqueous solutions of an alkyne-containing nucleotide (1 equiv., 0.2-1.0 M) and an azide-containing nucleotide (1 equiv., 0.2-1.0 M) were mixed together followed by addition of H 2 O (to the concentration of each analogue ca. 50-150 mM) and aqueous solutions of CuSO 4 •5H 2 O (0.2 equiv., 0.5-6.0 M) and sodium ascorbate (0.4 equiv., 1-12 M). The reaction was stirred at room temperature for several hours and monitored by RP HPLC. Additional portions of CuSO 4 or sodium ascorbate solutions were added upon slow kinetics. Final concentrations of reagents are given in the detailed procedures below. When completed, the reaction was quenched by 5-fold dilution with water and addition of Na 2 EDTA (ten equivalents of added CuSO 4 ) directly followed by semi-preparative RP HPLC purification.     The reference 5'-triphosphate RNA1 and 5' capped RNA1 were obtained analogously but with usage of 0.5 mM GTP and no cap analogue or compound 1b instead of 10b, respectively.
Relative bands intensity was determined using CLIQS v1.0 program. with NucleoSpin RNA Clean-up XS kit (Macherey-Nagel) to afford luciferase-coding RNA strands capped with appropriate cap analogue.

Translation
For each capped luciferase-coding RNA four diluted solutions were prepared -3.0 ng/µl, 1.5 ng/µl, 0.75 ng/µl, 0.375 ng/µl. Translation studies were performed using Rabbit Reticulocyte Lysate System (Promega). 9 µl of a mixture containing Rabbit Reticulocyte Lysate Translation efficiency was determined using Luciferase Reporter System (Promega). The samples were defrosted just before the experiment. To every sample 50 µl of Luciferase Assay Reagent was added just before measurement of luminescence on Synergy H1 Microplate Reader (Bio Tek). The measurement were performed for every four samples independently due to low stability of luminescence signal. The results are presented as proportions between regression coefficients of linear relationships between capped luciferase-coding RNA concentration in translation reaction (300 pg/µl, 150 pg/µl, 75 pg/µl, 37.5 pg/µl) and corresponding luminescence signal. Non-hydrolyzed fraction of the substrate (Y) at each time point was calculated as follows: Y = . •( + )+ , where S -absorbance of the substrate, P 1 , P 2 -absorbance of the hydrolysis product(s).

Studies of susceptibility to degradation by hDcpS
The analogue was considered as resistant to degradation by hDcpS if no products of hydrolysis were observed after 24 h of incubation in the presence of the enzyme and then subjected to analogous experiment but using different enzyme and analogue concentrations -200 nM and 10 µM, respectively. In this case, the reaction was stopped after 30, 120 and 240 min.