**2. Materials and Methods**

#### *2.1. Chemistry*

All nucleoside analogue ProTides, including McGuigan aryloxy phosphoramidite ProTides and 2-(methylthio)ethyl tryptamine ProTides, were synthesized using established phosphorus chemistry [25,26]. The 2- -*C*-ethenyluridine ProTide was synthesized using a known ethynyl ribose precursor **1**, which was obtained by following a previously reported synthetic route (Scheme 1) [27]. The ethenyl group of compound **2** was obtained by hydrogenation of the alkyne using 5% Pd/BaSO4 in (1:1) quinoline/benzene [28]. Ribosylation of uracil to compound **2** using *N*,*O*-bis(trimethylsilyl)acetamide (BSA) and SnCl4 in ACN afforded benzoyl-protected 2- -*C*-ethenyluridine **3**. The deprotection of compound **3** was performed using NaOMe in MeOH to afford 2- -*C*-ethenyluridine **4**, which was then treated with *t*BuMgCl in THF followed by *N*-[(*S*)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphinyl]-l-alanine 1-methylethyl ester and to afford 2- -*C*-ethenyluridine aryloxy phosphoramidate **5**.

#### 2.1.1. General Synthetic Experimental

All reagents, chemicals, and nucleoside analogues precursors were purchased from Acros Organics, Carbosynth, Chem-Impex International, AK Scientific, and Fisher Chemical at ACS reagent grade or higher purity and used as received. The (3R,4R,5R)-5-(benzoyloxy)methyl)-3-ethynyltetrahydrofuran-2, 3,4-triyl tribenzoate **1** was synthesized by following a previously reported procedure [27]. Synthetic details for aryloxy phosphoramidate ProTides other than for 2- -*C*-ethynyluridine and 2- -*C*-ethenyluridine were published previously by our group [24]. All reactions were carried out in either an oven-dried round bottom flask or a Schlenk tube under a nitrogen atmosphere using commercially available anhydrous solvents and monitored by thin-layer chromatography with detection by UV light. The 1H NMR and the 31P NMR spectra were acquired on a Varian 400-MHz spectrometer and recorded at 298 K. Chemical shifts were referenced to the residual protio solvent peak and are given in parts per million (ppm). Splitting patterns are denoted as s (singlet), d (doublet), dd (doublet of doublet), dq (doublet of quartet), ddd (doublet of doublet of doublet), ddt (doublet of doublet of triplet), t (triplet), td (triplet of doublet), tdd (triplet of doublet of doublet), q (quartet), and m (multiplet).

#### 2- -*C*-Ethynyluridine Aryloxy Phosphoramidate

To a stirred suspension of 2- -*C*-ethynyluridine (0.09 mmol, dried under vacuum at 50 ◦C overnight) in dry THF (1 mL) was added a 2.0 M solution of tert-butyl magnesium chloride in THF (96 μL, 0.19 mmol). The mixture was stirred at 0 ◦C for 30 min and then allowed to warm to room temperature and stirred for an additional 30 min. The reaction mixture was then cooled to 0 ◦C and *N*-[(*S*)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphinyl]-L-alanine 1-methylethyl ester (46 mg, 0.10 mmol) was added. The reaction mixture was stirred for 18 h as the temperature was allowed to warm to room temperature. The solvent was removed by rotary evaporation. The reaction mixture was purified first using flash chromatography (a gradient from 0 to 30% methanol in dichloromethane) and then using preparative, normal-phase HPLC (10 to 40% MeOH in dichloromethane gradient) to afford 25.5 mg of the product (54.3%) in <sup>≥</sup> 95% purity. 1H NMR (400 MHz, CD3OD): <sup>δ</sup>= 7.65 (d, 1H, *J* = 8.1 Hz), 7.40–7.34 (m, 2H), 7.27 (dq, 2H, *J* = 7.7, 1.2 Hz), 7.20 (ddd, 1H, *J* = 8.2, 7.1, 1.1 Hz), 6.04 (s, 1H), 5.61 (d, 1H, *J* = 8.1 Hz), 4.97 (m, 1H), 4.49 (ddd, 1H, *J* = 11.8, 6.0, 2.1 Hz), 4.36 (ddd, 1H, *J* = 11.8, 6.1, 3.7 Hz), 4.16 (d, 1H, *J* = 9.1 Hz), 4.08 (ddt, 1H, *J* = 9.0, 3.9, 2.1 Hz), 3.92 (dq, 1H, *J* = 9.9, 7.1 Hz), 3.08 (s, 1H), 1.35 (dd, 3H, *J* = 7.2, 1.0 Hz), 1.22 (dd, 6H, *J* = 6.3, 1.8 Hz). 31P NMR (162 MHz, CD3OD) δ= 3.78.

#### 5-((Benzoyloxy)methyl)-3-vinyltetrahydrofuran-2,3,4-triyl tribenzoate **2**

To a stirring solution of **1** (208.7 mg, 0.362 mmol, 1 equivalent) in benzene (2 mL) and ethanol (2 mL) under H2 (g), we added 5% palladium on barium sulfate (20.8 mg, 10 wt%) followed by quinolone (22 μL) and stirred at room temperature for 2 h. The mixture was diluted in ethyl acetate, washed 3 times with water, and dried over anhydrous sodium sulfate. The reaction mixture was purified using flash chromatography (0 to 30% ethyl acetate in hexane gradient) to afford purified product; (162.7 mg, 0.281 mmol, 77.7%) 1H NMR (400 MHz, CDCl3): δ = 8.23–8.09 (m, 4H), 8.07–8.03 (m, 2H), 7.92–7.88 (m, 2H), 7.69–7.39 (m, 10H), 7.18–7.12 (m, 2H), 6.46 (dd, 1H, *J* = 17.6, 11.2 Hz), 6.25 (d, 1H, *J* = 8.3 Hz), 4.54 (dd, 2H, *J* = 12.2, 4.8 Hz), 4.81 (ddd, 2H, *J* = 8.4, 4.7, 3.9 Hz), 4.73 (dd, 1H, *J* = 12.2, 3.9 Hz), 4.54 (dd, 1H, *J* = 12.2, 4.8 Hz)

5-((Benzoyloxy)Methyl)-2-(2,4-Dioxo-3,4-Hihydropyrimidin-1(2H)-yl)-3-Vinyltetrahydrofuran-3,4-diyl Benzoate **3**

Uracil (63.0 mg, 0.562 mmol, 2 equivalent) and **2** (162.7 mg, 0.281 mmol, 1 equivalent) were dried under high vacuum in separate round bottom flasks for 2 h. Under N2 (g) and stirring, we added dry acetonitrile (2 mL) to uracil followed by the addition of bis(trimethylsilyl)acetamide (550.1 μL, 2.250 mmol, 8 equivalent). The reaction mixture was refluxed at 80 ◦C for 1 h then cooled to 0 ◦C. Then, compound 2 in dry acetonitrile (2 mL) was added to the reaction mixture followed by tin (IV) chloride (229.9 μL, 1.968 mmol, 7 equivalent) and heated to 60 ◦C for 3 h. The reaction mixture was poured into a separatory funnel containing ice cold water, extracted 3 times with ethyl acetate, and the combined organic layer was dried over anhydrous sodium sulfate. The reaction mixture was purified using flash chromatography (0 to 100% ethyl acetate in hexane gradient) to afford purified product; (89.4 mg, 0.153 mmol, 54.6%) 1H NMR (400 MHz, CDCl3): δ= 9.22 (s, 1H), 8.09 (m, 4H), 7.86–7.82 (m, 2H), 7.63–7.56 (m, 2H), 7.63–7.56 (m, 6H), 7.29–7.21 (m, 2H), 6.65 (s, 1H), 6.12 (dd, 1H, *J* = 17.5, 11.1 Hz), 6.04 (d, 1H, *J* = 5.2 Hz), 5.64 (dd, 1H, *J* = 8.2, 2.1 Hz), 5.46–5.40 (dd, 2H), 4.94 (dd, 1H, *J* = 12.3, 3.2 Hz), 4.81 (dd, 1H, *J* = 12.3, 5.7 Hz), 4.66 (td, 1H, *J* = 5.5, 3.2 Hz)

1-(3,4-dihydroxy-5-(hydromethyl)-3-vinyltetrahydrofuran2-yl)pyrimidine-2,4(1H,3H)-dione **4**

Compound **3** (90.5 g, 0.155 mmol, 1 equivalent) was dried overnight on high vacuum. Under N2 (g), methanol (1.5 mL) was added, then the reaction mixture was cooled to 0 ◦C followed by the dropwise addition of sodium methoxide (86.5 μL, 1.553 mmol, 10 equivalent). The reaction mixture was raised to room temperature and stirred for 1.5 h. The reaction mixture was cooled to 0 ◦C followed by the addition of formic acid until pH = 4. The reaction mixture was dried in vacuo then purified using flash chromatography (0 to 40% methanol in dicholoromethane gradient) to afford a purified product; (30.2 mg, 0.146 mmol, 93.8%) 1H NMR (400 MHz, CDCl3): δ= 8.13 (d, 1H, *J* = 8.1 Hz), 5.95 (s, 1H), 5.74–5.65 (m, 2H), 5.44 (dd, 1H, *J* = 17.3, 1.3 Hz), 5.26 (dd, 1H, *J* = 10.8, 1.3 Hz), 4.22 (d, 1H, *J* = 9.2 Hz), 4.03–3.97 (m, 2H), 3.84–3.79 (m, 1H)

isopropyl(((5-(2,4-dioxo-3,4-dihydropyridimin-1(2H)-yl)-3,4-dihydroxy-3-inyltetrahydrofuran-2 yl)methoxy)(phenoxy)phosphoryl)-L-alaninate **5**

To a stirring solution of **4** (36.4 mg, 0.176 mmol, 1 equivalent) in dry tetrahydrofuran (1 mL) at 0 ◦C was added tert-butyl magnesium chloride (184.5 μL, 0.369 mmol, 2.1 equivalent). The reaction mixture was raised to room temperature and allowed to react for 30 min. The reaction mixture was cooled to 0 ◦C, then *N*-[(*S*)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphinyl]-L-alanine 1-methylethyl ester (95.5 mg, 0.211 mmol, 1.2 equivalent) was added and gradually warmed to room temperature overnight. The reaction mixture was purified using flash chromatography (0 to 30% methanol [MeOH] in dichloromethane gradient) to afford the purified product; (42.5 mg, 0.0788 mmol, 44.8%) 1H NMR (400 MHz, CD3OD): δ= 7.76 (d, 1H, *J* = 8.1 Hz), 7.38 (dd, 2H, *J* = 8.6, 7.2 Hz), 7.31–7.25 (m, 2H), 7.25–7.16 (m, 2H), 5.94 (s, 1H), 5.68 (dd, 1H, *J* = 17.3, 10.8 Hz), 5.60 (d, 1H, *J* = 8.1 Hz), 5.48 (d, 1H, *J* = 1.4 Hz), 5.44 (d, 1, *J* = 1.4 Hz), 5.27 (dd, 1H, *J* = 10.8, 1.4 Hz), 4.96 (1H, m), 4.58–4.49 (m, 1H), 4.45–4.38 (m, 1H), 4.17 (s, 2H), 4.00–3.87 (m, 1H), 1.38 (dd, 3H, *J* = 7.1, 1.0 Hz), 1.21 (d, 6H, *J* = 6.3 Hz). 31P NMR (162 MHz, CD3OD) δ = 3.78.

(5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methyl (2-(methylthio)ethyl) (2-(1H-indol-3-yl)ethyl)phosphoramidate (2- -*C*-methyluridine methylthioethyl tryptamine ProTide)

The 2- -*C*-methyluridine (45.3 mg, 0.1790 mmol, 1 equivalent) and the triethylammonium 2-(methylthio)ethyl phosphonate (92.1 mg, 0.3579 mmol, 1.5 equivalent) were added to an oven-dried schlenk tube and dried overnight on the high vacuum. To the flask under N2 (g) was added dry pyridine (2 mL) followed by the dropwise addition of trimethylacetyl chloride (60.6 μL, 0.4923 mmol, 2.75 equivalent), and the reaction was stirred for 3 h at room temperature. The reaction was quenched by saturated sodium bicarbonate and extracted 3 times with dicholormethane. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo at room temperature and dried for 2 h on high vacuum. The dried residue was dissolved in pyridine (2 mL) under N2 (g). To the reaction mixture was simultaneously added trimethylamine (49.9 μL, 0.3579 mmol, 2 equivalent), tryptamine (114.7 mg, 0.7158 mmol, 4 equivalent, dissolved in dry pyridine 2 mL), and carbon tetrachloride (26.0 μL, 0.2685 mmol, 1.5 equivalent) and stirred for 25 min at room temperature. The reaction mixture was diluted with methanol and purified using flash chromatography (0 to 30% methanol in dichloromethane gradient) to afford a purified product as a mixture of two epimers at phosphorus; (8.0 mg, 0.01443 mmol, 7.3%) 1H NMR (400 MHz, CD3OD): δ= 7.72 (dd, 1H, *J* = 21.4, 8.2 Hz), 7.55–7.50 (m, 1H), 7.33–7.29 (m, 1H), 7.06 (t, 2H, *J* = 7.3 Hz), 6.97 (tdd, 1H, *J* = 7.5, 4.6, 2.4 Hz), 5.95 (d, 1H, *J* = 5.2 Hz), 5.64 (dd, 1H, *J* = 8.1, 3.8 Hz), 4.37–4.29 (m, 1H), 4.17 (ddd, 1H, *J* = 11.7, 5.0, 3.1 Hz), 4.04 (dt, 1H, *J* = 10.4, 6.8 Hz), 4.00–3.90 (m, 1H), 3.77 (dd, 1H, *J* = 12.4, 9.3 Hz), 3.21 (td, 2H, *J* = 10.9, 5.6 Hz), 2.96 (dt, 2H, *J* = 7.3, 4.4 Hz), 2.66 (td, 2H, *J* = 6.7, 2.3 Hz), 2.07 (d, 3H, *J* = 1.4 Hz), 1.13 (s, 3H). 31P NMR (162 MHz, CD3OD) δ= 10.52 and 10.41.

**Scheme 1.** Synthesis of 2- -*C*-ethenyluridine ProTide. (i) H2 (g), Pd/BaSO4, quinoline, benzene, 78%; (ii) bovine serum albumin (BSA), uracil, SnCl4, ACN, 60 ◦C to 80 ◦C, 55%; (iii) NaOMe, MeOH, 0 ◦C to reflux, 94%; (iv) tBuMgCl, *N*-[(*S*)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphynyl]-*L*-alanine 1-methylethyl ester, THF, −70 ◦C to room temperature (RT), 45%.
