## Materials and Methods
### Chemical Syntheses  
5.1 P1-(N1-Propargylguanosin-5'-yl) P3-(N7-methylguanosin-5'-yl) triphosphate (m\({}^{7}\)Gp\({}_{3}\)G-N1-propagaryl) (**1**)  
The m\({}^{7}\)GDP (TEAH\({}^{+}\) salt, 50.0 mg, 0.066 mmol, 1.0 equivalent) **(19)** and compound **18** (Na\({}^{+}\) salt, 46.7 mg, 0.098 mmol, 1.5 equivalent) were suspended in anhydrous DMF (1.0 mL) followed by addition of anhydrous ZnCl\({}_{2}\) (89.7 mg, 0.66 mmol, 10 equivalent). The mixture was vigorously shaken until the reagents dissolved. The reaction progress was monitored by RP-HPLC. After completion (24 h), an appropriate amount of EDTA solution (Na\({}_{2}\)EDTA, 246.8 mg, 0.66 mmol, 10 equivalent) was added to the mixture to disassociate the nucleotide-metal complex, then the pH of the mixture was adjusted to 6 with solid NaHCO\({}_{3}\), followed by purification with DEAE-Sephadex and isolated as TEAH\({}^{+}\) salts (43.6 mg, 862 mmol, 0.038 mmol, 58%).Triethylammonium salts were then re-purified by semipreparative RP-HPLC and after repeated freeze-drying, were isolated as ammonium salts. HPLC method A t\({}_{\text{R}}\) = 5.539 min. \({}^{1}\)H NMR (deuterium oxide, 400 MHz) 6 7.98 (1H, s), 5.89 (1H, d, \(J\) = 3.7 Hz), 5.80 (1H, d, \(J\) = 6.2 Hz), 4.86 (1H, d, \(J\) = 7.0, 2.4 Hz), 4.72 (1H, t, \(J\) = 6.2, 5.2 Hz), 4.55 (1H, t, \(J\) = 5.0, 3.7 Hz), 4.49 (2H, dd, \(J\) = 5.2, 3.3 Hz), 4.43 (1H, t, \(J\) = 5.0 Hz), 4.38-4.32 (3H, m), 4.29-4.22 (3H, m), 4.05 (3H, s), 2.76 (1H, t, \(J\) = 2.4 Hz). \({}^{31}\)P NMR (deuterium oxide, 162 MHz) \(\delta\) -10.62 (2P, dq, \(J\) = 19.6, 6.5 Hz), \(-\)22.24 (1P, t, \(J\) = 19.6 Hz). HRMS ESI (-) _m/z_ Calc. for C\({}_{24}\)H\({}_{30}\)N\({}_{10}\)O\({}_{18}\)P\({}_{3}\)\({}^{-}\) [M \(-\) H]\({}^{-}\): 839.0958 found: 839.0965.  
5.2 P1-(N7-Methylguanosin-5'-yl) P3-(N1-propargylguanosin-5'-yl) 1,2-methylenetriphosphate (m\({}^{7}\)GpCH\({}_{2}\)ppG-N1-propargyl) **(2**)  
The m\({}^{7}\)GpCH\({}_{2}\)p (28.4 mg, 60.0 \(\upmu\)mol) **(20)**, compound **18** (39.5 mg, 60.0 \(\upmu\)mol, 1 equivalent), and ZnCl\({}_{2}\) (65.3 mg, 480.1 \(\upmu\)mol, 8 equivalent) were suspended in anhydrous DMF (601 \(\upmu\)L). The mixture was stirred overnight at RT and quenched by the addition of 8.9 mL of Na\({}_{2}\)EDTA/NaHCO\({}_{3}\) solution (20 g/L and 10 g/L, respectively). Side product propargyl-N1-GppG-N1-propargyl was observed. The product was purified with ion exchange chromatography on DEAE-Sephadex resin using TEAB buffer gradient (0.0-1.2 M) and isolated as TEAH\({}^{+}\) salt (703 mmol, 31.1 \(\upmu\)mol, 52%). The product was re-purified using semi-preparative RP-HPLC. HPLC method A t\({}_{\text{R}}\) = 5.641 min. NMR: \({}^{1}\)H NMR(400 MHz, deuterium oxide) 5 9.28 (1H, s), 8.10 (1H, s), 5.95 (1H, d, \(J\) = 4.2 Hz), 5.84 (1H, d, \(J\) = 5.5 Hz), 4.86 (2H, dd, \(J\) = 6.1, 2.5 Hz), 4.70 (1H, t, \(J\) = 5.5 Hz), 4.63 (1H, t, \(J\) = 4.2 Hz), 4.51-4.48 (2H, m), 4.38-4.31 (2H, m), 4.31-4.16 (4H, m), 4.07 (3H, s), 2.76 (1H, s), 2.40 (2H, t, \(J\) = 20.3 Hz), \({}^{31}\)P NMR (162 MHz, deuterium oxide) 8 17.49-16.82 (1P, m), 7.63-6.82 (1P, m), \(-\)11.14 (1P, ddt, \(J\) = 1026.2, 25.6, 5.5 Hz), HRMS ESI (\(-\)) _m/z_ Calc. for C\({}_{25}\)H\({}_{32}\)N\({}_{10}\)O\({}_{17}\)P\({}_{3}\)\({}^{-}\) [M \(-\) H]\({}^{-}\): 837.1165 found: 837.1177.