## Materials and Methods
### Chemical Syntheses
#### 4.5.3 P1-(N7-Methylguanosin-5'-yl) P3-(N1-propargylguanosin-5'-yl) 1,2-imidotriphosphate  
(m\({}^{7}\)GpNHfppG-N1-propargyl) (**3**)  
The m\({}^{7}\)GpNHfp (TEAH\({}^{+}\) salt, 50 mg, 0.066 mmol, 1.0 equivalent) (**21**) 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. 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 disassociate the nucleotide-metal complex, adjusted to pH 6 with solid NaHCO\({}_{3}\). The product was purified with ion exchange chromatography on DEAE-Sephadex in a TEAB buffer gradient (0.0-1.2 M) and isolated as TEAH\({}^{+}\) salt (33.2 mg, 657 mODU, 0.029 mmol, 44%). The product was re-purified with semi-preparative RP-HPLC. \({}^{1}\)H NMR (deuterium oxide, 400 MHz) 8 9.23 (1H, s), 8.11 (1H, d, \(J\) = 0.8 Hz), 5.94 (1H, d, \(J\) = 3.7 Hz), 5.84 (1H, d, \(J\) = 5.7 Hz), 4.87 (2H, dd, \(J\) = 5.9, 2.5 Hz), 4.72 (1H, t, \(J\) = 5.7 Hz), 4.60 (1H, t, \(J\) = 4.7, 3.8 Hz), 4.51-4.46 (2H, m), 4.37 (1H, dd, \(J\) = 5.2, 2.5 Hz), 4.35-4.32 (1H, m), 4.31-4.13 (4H, m), 4.10-4.07 (3H, m), 3.21 (1H, q, \(J\) = 7.3 Hz), 2.76 (1H, t, \(J\) = 2.5 Hz), 1.29 (1H, t, \(J\) = 7.3 Hz). \({}^{31}\)P NMR (deuterium oxide, 162 MHz) 8 1.47-1.24 (1P, m), \(-\)8.90 (1P, dt, \(J\) = 20.0, 5.5 Hz), \(-\)9.37 (1P, dd, \(J\) = 20.0, 5.9 Hz). HRMS ESI (\(-\)) _m/z_ Calc. for C\({}_{24}\)H\({}_{31}\)N\({}_{11}\)O\({}_{17}\)P\({}_{3}\)\({}^{-}\) [M \(-\) H]\({}^{-}\): 838.1112 found: 838.1126. 5.4 P1-(N7-Methylguanosin-5'-yl) P3-(N1-propargylguanosin-5'-yl) 2,3-methylenetriphosphate (m\({}^{7}\)GppCH\({}_{2}\)PG-N1-propargyl) (**4**)  
Compound **22** (37.3 mg, 54.8 \(\upmu\)mol), m\({}^{7}\)GMP-Im (Na\({}^{+}\) salt, 25.0 mg, 55.7 \(\upmu\)mol, 1.0 equivalent) (**24**), and ZnCl\({}_{2}\) (62.1 mg, 456.6 \(\upmu\)mol, 8.3 equivalent) were suspended in anhydrous DMF (547 \(\upmu\)L). The reaction mixture was stirred overnight at RT and quenched by the addition of 8.5 mL of Na\({}_{2}\)EDTA/NaHCO\({}_{3}\) aqueous solution (20 g/L and 10 g/L, respectively). Traces of the side product m\({}^{7}\)G-pp-m\({}^{7}\)G were observed. The product was purified using ion exchange chromatography on DEAE-Sephadex resin in TEAB buffer gradient (0.0-1.2 M) and isolated as TEAH\({}^{+}\) salt (515 mODU, 22.8 \(\upmu\)mol, 42%). The product was repurified with semi-preparative RP-HPLC. HPLC method A t\({}_{\rm R}\) = 5.741 min. \({}^{1}\)H NMR (400 MHz, deuterium oxide) 8 9.15 (1H, s), 8.30 (1H, s), 5.95 (1H, d, \(J\) = 3.5 Hz), 5.86 (1H, d, \(J\) = 5.4 Hz), 4.86 (2H, dd, \(J\) = 4.8, 2.5 Hz), 4.72 (1H, t, \(J\) = 5.4 Hz), 4.60 (1H, dd, \(J\) = 4.8, 3.5 Hz), 4.50 (1H, dd, \(J\) = 4.8, 4.3 Hz), 4.47 (1H, t, \(J\) = 5.4 Hz), 4.37 (1H, t, \(J\) = 5.4, 2.5 Hz), 4.35-4.33 (1H, m), 4.21 (4H, ddd, \(J\) = 22.3, 11.2, 6.2, 3.8 Hz), 4.08 (3H, s), 2.76 (1H, t, \(J\) = 2.5 Hz), 2.40 (2H, t, \(J\) = 20.4 Hz); \({}^{31}\)P NMR (162 MHz, deuterium oxide) 8 17.26-16.77 (1P, m), 7.76-7.10 (1P, m), \(-\)11.17--11.50 (1P, m), HRMS ESI (\(-\)) _m/z_ Calc. for C\({}_{25}\)H\({}_{32}\)N\({}_{10}\)O\({}_{17}\)P\({}_{3}\)\({}^{-}\) [M \(-\) H]\({}^{-}\): 837.1165 found: 837.1174. 5.5 P1-(N7-Methylguanosin-5'-yl) P3-(N1-propargylguanosin-5'-yl) 2,3-imidotriphosphate (m\({}^{7}\)GppNHfpG-N1-propargyl) (**5**)  
Compound **23** (TEAH\({}^{+}\) salt, 50 mg, 0.064 mmol, 1.0 equivalent) and m\({}^{7}\)GMP-Im (Na\({}^{+}\) salt, 43.2 mg, 0.096 mmol, 1.5 equivalent) (**24**) were suspended in anhydrous DMF (1.0 mL) followed by the addition of anhydrous ZnCl\({}_{2}\) (87.0 mg, 10 equivalent, 0.64 mmol). The mixture was vigorously shaken until the reagents dissolved. The reaction progress was monitored by RP-HPLC.