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Supporting Information Liu et al. 10.1073/pnas.1111561108 SI Text General Methods for Synthesis. All solvents and reagents were ob- tained from commercial suppliers and were used without further purification. NMR spectra were recorded on a Varian Oxford AS600 600 MHz NMR instrument. NMR chemical shifts were expressed in ppm relative to internal solvent peaks, and coupling constants were measured in Hertz (br ¼ broad). Mass spectra were determined on a Waters Micromass ZQ instrument, using an electrospray ionization (ESI) source coupled to a Waters 2525 HPLC system operating in reverse mode, with an Waters Sunfir- eTM C18 5 μM 4.6 × 50 mm column. Flash chromatography was performed using a Biotage Isolera One flash purification system. Synthesis of O-Propargyl-Puromycin (OP-puro). NHBoc O O OMe NHBoc OH O OMe Br K 2 CO 3 , DMF, 25 C O-Propargyl Boc-Tyr-OMe (1). A solution of Boc-Tyr-OMe (2.01 g, 6.0 mmol), propargyl bromide (80 % wt solution in toluene, 910 μL, 8.16 mmol), K 2 CO 3 (2.82 g, 20.4 mmol) in dry dimethyl- formamide (DMF) (19 mL) was stirred for 17 h at room tempera- ture. After dilution with water (200 mL), the resulting mixture was extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with saturated NaHCO 3 and brine followed by drying over Na 2 SO 4 . After removal of the solvent in vacuo, the crude product was obtained as a yellow liquid and was used in the next step without further purification. NHBoc O O OMe NHBoc O O OH Me 3 SnOH 1,2-dichloroethane, 80 C O-Propargyl Boc-Tyr-OH (2). A solution of O-Propargyl Boc-Tyr- OMe 1 (2.27 g, 6.80 mmol) was dissolved in 27 mL 1,2-dichlor- oethane and, after addition of trimethyltin hydroxide (3.69 g, 20.4 mmol), the mixture was heated to 80 °C until TLC analysis indicated a complete reaction. This hydrolysis method (1) was used to avoid the possible racemization of the tyrosine derivative. The mixture was then concentrated in vacuo, and the residue was dissolved in EtOAc (100 mL). The organic layer was washed with aqueous HCl (5%) (3 × 60 mL), then washed with brine, and dried over Na 2 SO 4 . After removal of the solvent in vacuo, the residue was purified by flash column chromatography (SiO 2 , step- wise gradient from 2–20 % MeOH in CH 2 Cl 2 ) to give 2 (2.08 g, 96%) as a clear oil. NHBoc O O O N O O NHBoc O O OH DSC Pyridine, MeCN, 25 C O-Propargyl Boc-Tyrosine N-Hydroxysuccinimide Ester (3). Disuccini- midyl carbonate (2.46 g, 9.60 mmol) was added to a solution of 2 (2.04 g, 6.40 mmol) and pyridine (1.04 mL, 12.80 mmol) in acet- onitrile (16 mL). The reaction was stirred at room temperature for 15 h, during which the solution became clear and evolved gas. The reaction mixture was added to EtOAc, washed twice with 1 N HCl and twice with saturated NaHCO 3 , dried over Na 2 SO 4 , and concentrated in vacuo to yield a white solid (1.90 g, 71%), which was used in the next step without further purification. O HO NH OH N N N N N NHBoc O O O HO NH 2 OH N N N N N NHBoc O O O N O O Et 3 N, CH 2 Cl 2 25 C + tert-Butyl ((S)-1-(((2S,3S,4R,5R)-5-(6-(dimethylamino)-9H-purin-9-yl)-4- hydroxy-2- (hydroxymethyl)tetrahydrofuran-3-yl)amino)-1-oxo-3-(4- (prop-2-yn-1-yloxy)phenyl)propan-2- yl)carbamate (O-propargyl Boc- puromycin) (4). O-Propargyl Boc-Tyrosine N-hydroxysuccinimide ester 3 (1.42 g, 3.40 mmol) and triethylamine (0.47 mL, 3.40 mmol) were added to a solution of puromycin aminoglyco- side (500.0 mg, 1.70 mmol) in CH 2 Cl 2 (15 mL). The solution was stirred at room temperature for 1.5 h and then directly purified by flash chromatography (SiO 2 , stepwise gradient from 2–10% MeOH in CH 2 Cl 2 ), to yield the product as a white solid (480 mg, 47%). 1 H NMR (600 MHz, DMSO-d 6 ): δ 8.44 (s, 1H), 8.24 (s, 1H), 8.00 (d, J ¼ 7.8 Hz, 1H), 7.21 (d, J ¼ 8.4 Hz, 2H), 6.84–6.92 (m, 3H), 6.05 (d, J ¼ 4.8 Hz, 1H), 5.99 (d, J ¼ 3.0 Hz, 1H), 5.15 (t, J ¼ 5.4 Hz, 1H), 4.74 (d, J ¼ 1.8 Hz, 2H), 4.43–4.53 (m, 2H), 4.17–4.24 (m, 1H), 3.91–3.96 (m, 1H), 3.64–3.72 (m, 1H), 3.10–3.62 (m, 8H), 2.91 (dd, J ¼ 13.8, 4.2 Hz, 1H), 2.70 (dd, J ¼ 13.8, 10.2 Hz, 1H), 1.30 (s, 9H); liquid chromatography / MS (ESI, m∕z): calcd for C 29 H 37 N 7 O 7 ½M þ H þ 596, found 596. (Definitions are as follows: singlet, s; doublet, d; triplet, t; multiplet, m; doublet of doublets, dd.) Liu et al. www.pnas.org/cgi/doi/10.1073/pnas.1111561108 1 of 4
