## Materials and Methods
### Chemical Syntheses
#### 4.5.16 N1-Propargylguanosine-5\({}^{\prime}\)-O-monophosphate (propargyl-N1-GMP) (**17**)  
Compound **16** (217.5 mg, 0.678 mmol) was suspended in trimethyl phosphate (6.8 mL) and luttidine (470.3 \(\
icefrac{{\rm{\mu}}}{{\rm{L}}}\), 435.0 mg, 4.07 mmol, 6 equivalent) was added and the reaction mixture was stirred at 0 \({}^{\circ}\)C for approx. 20 min. Then, freshly distilled phosphoryl chloride (189.4 \(\
icefrac{{\rm{\mu}}}{{\rm{L}}}\), 311.6 mg, 2.03 mmol, 3.0 equivalent) was added and the reaction was stirred at 0 \({}^{\circ}\)C until the starting material disappeared, as determined by RP-HPLC. Reaction was quenched by the addition of water (70 mL) and pH was neutralized with solid NaHCO\({}_{3}\). The crude product was purified using ion exchange chromatography on DEAE-Sephadex (0.0-0.7 M TEAB buffer gradient) and isolated as a TEAH\({}^{+}\) salt (4395 mmol, 0.364 mmol, 54%). HPLC method A: t\({}_{\text{R}}\) = 5.998 min. \({}^{1}\)H NMR (400 MHz, deuterium oxide) \(\delta\) 8.14 (1H, s), 5.92 (1H, d, \(J\) = 6.1 Hz), 4.86 (2H, d, \(J\) = 2.3 Hz), 4.78 (1H, m, overlapped with HDO), 4.49 (1H, dd, \(J\) = 5.2, 3.5 Hz), 4.32 (1H, qd, \(J\) = 3.5, 1.5 Hz), 4.10-3.99 (2H, m), 2.73 (1H, t, \(J\) = 2.3 Hz), \({}^{31}\)P NMR (162 MHz, deuterium oxide) \(\delta\) 2.04 (1P, t, \(J\) = 4.9 Hz). HRMS ESI (-) _m_/_z_ Calc. for C\({}_{13}\)H\({}_{15}\)N\({}_{5}\)O\({}_{8}\)P\({}^{-}\) [M - H]\({}^{-}\):400.06583 found: 400.06630. 5.17 N1-Propargylguanosine 5\({}^{\prime}\)-O-monophosphate P-imidazolide (propargyl-N1-GMP-Im) (**18**)  
Compound **18** was prepared according to the Mukaiyama and Hashimoto procedure [29]. Compound **17** (1.0 g, 2.5 mmol, 1 equivalent, TEAH\({}^{+}\) salt), imidazole (1.70 g, 25.00 mmol, 10 equivalent), and 2,2\({}^{\prime}\)-dithiiodpyridine (1.65 g, 7.5 mmol, 3 equivalent) were suspended in dry DMF (-1.5 mL/100 mg of nucleotide) before adding triethylamine (757.00 mg, 1.04 mL, 7.5 mmol, 3 equivalent) and triphenylphosphine (1.96 g, 7.5 mmol, 3 equivalent). The mixture was stirred for 24 h at RT. Then, the product was precipitated by the addition of a solution of anhydrous NaClO\({}_{4}\) (1.22 g, 10 mmol, 4 equivalent) in dry acetone (15 mL). The precipitate was filtered off, washed repeatedly with cold, dry acetone, and dried under vacuum over P\({}_{4}\)O\({}_{10}\) (1.07 g, 95%). HPLC method A t\({}_{\text{R}}\) = 8.579 min. \({}^{1}\)H NMR (400 MHz, deuterium oxide) \(\delta\) 7.88 (1H, s), 7.81 (1H, t, \(J\) = 1.3 Hz), 7.10 (1H, q, \(J\) = 1.3 Hz), 6.88 (1H, m), 5.83 (1H, d, \(J\) = 5.3 Hz), 4.87 (2H, t, \(J\) = 2.2 Hz), 4.84 (1H, t, \(J\) = 5.3 Hz), 4.44 (1H, dd, \(J\) = 5.3, 4.1 Hz), 4.25 (1H, qd, \(J\) = 4.1, 1.8 Hz), 4.11 (2H, dd, \(J\) = 5.5, 4.1 Hz), 2.76 (1H, t, \(J\) = 2.2 Hz). \({}^{31}\)P NMR (162 MHz, deuterium oxide) \(\delta\) -8.00 (1P, qd, \(J\) = 5.5, 2.3 Hz). HRMS ESI (-) _m_/_z_ Calc. for C\({}_{16}\)H\({}_{17}\)N\({}_{7}\)O\({}_{7}\)P\({}^{-}\) [M - H]\({}^{-}\): 450.0932 found: 450.0933. 4.5.18. N1-Propargylguanosine 5'-O-(1,2-methylened diphosphate) (propagyl-N1-GpCH\({}_{2}\)p) (**22**)  
Compound **16** (100 mg, 0.31 mmol) was suspended in trimethyl phosphate (1.5 mL) and stirred at 0 \({}^{\circ}\)C for approx. 15 min. Then methylene bis(phosphonic dichloride) (CH\({}_{2}\)(POCI\({}_{2}\))\({}_{2}\), 3 equivalent, 0.93 mmol, 230 mg) was added and the reaction was stirred at 0 \({}^{\circ}\)C until the starting material disappeared as determined by RP-HPLC (usually 4-5 h). Then, the reaction was quenched by the addition of 0.7 M TEAB (pH 7) or 1 M NaHCO\({}_{3}\) until neutral pH was reached. Crude product was purified by DEAE-Sephadex and isolated as TEAH\({}^{+}\) salts (111.70 mg, 1705 mOD, 75%). HPLC method A t\({}_{\text{R}}\) = 4.801 min.