Ynamides as racemization-free coupling reagents for amide and peptide synthesis

Transcription

1 Ynamides as racemization-free coupling reagents for amide and peptide synthesis Long Hu, Silin Xu, Zhenguang Zhao, Yang Yang, Zhiyuan Peng, Ming Yang, Changliu Wang, Junfeng Zhao* Key Laboratory of Chemical Biology of Jiangxi Province, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang, , P. R. China Supporting Information Table of 1. General Remarks.....S2 2. General Procedure for the Preparation of Ynamides....S3-S5 3. Amide Bond Formation with α-acyloxyenamide as Active Ester..S6 4. Reaction Condition Optimization for Hydroacyloxylation of Ynamides...S7-S9 5. General Procedure for Hydroacyloxylation of Ynamides with Various Carboxylic Acids...S10-S12 6. Control Experiments of Two-step Strategy and One-pot, Two-step Strategy for Amide Bond Formation...S13-S15 7. Ynamide MYTsA Mediated Amide Bond Formation from Carboxylic Acids and Amines S16-S18 8. Comparative Study of Epimerization/Racemization During Dipeptide Synthesis S19-S31 9. Ynamide MYTsA Mediated Peptide Bond Formation.S32-S Synthesis of Protected Leu-enkephalin with MYMsA as Coupling Reagent..S39-S NMR Spectra...S42-S119 S1

2 General Remarks: 1 H and 13 C NMR spectra were recorded on a Bruker (400 and 100 MHz for 1 H and 13 C respectively) instrument, and are internally referenced to residual solvent signals, CDCl 3 referenced at δ 7.26 and ppm and DMSO-d 6 referenced at δ 2.50 and ppm. Data for 1 H is reported as follows: chemical shift (δ ppm), integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), broad peaks (br), coupling constant (Hz) and assignment. Data for 13 C NMR are reported in terms of chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant (Hz) and no special nomenclature is used for equivalent carbons. HRMS (ESI) spectra were obtained by the electrospray ionization time-of-flight (ESI-TOF) mass spectrometry. The diastereomeric ratio (dr) was determined by chiral HPLC with chiral IC column with hexane and i-proh as eluent. Flash column chromatography purification of compound was carried out by gradient elution using ethyl acetate (EA) in light petroleum ether (PE).Unless otherwise noted, materials obtained from commercial suppliers were used without further purification. S2

3 General Procedure for the Preparation of Ynamides In a 500 ml three-neck round-bottom flask equipped with a stir-bar, CuCl 2 (2.0 mmol, 270 mg), nitrogen nucleophile (50.0 mmol) and Na 2CO 3 (20.0 mmol, 2.12 g) were combined. The reaction flask was purged with oxygen gas for 15 minutes. A solution of pyridine (20.0 mmol) in 40.0 ml of toluene was added to the reaction flask via a syringe. Two balloons filled with oxygen gas were connected to the reaction flask via needles. The flask was placed in an oil-bath and heated to 70 C. A solution of terminal alkyne (10.0 mmol) in 20.0 ml of toluene was added slowly to the flask over 4 h by using a syringe pump. After addition of terminal alkyne/toluene solution, the reaction mixture was allowed to stir at 70 C for another 16 hours and then cooled to room temperature. The crude mixture was concentrated under vacuumand then purified by flash chromatography on silica gel with PE/ethyl acetate to afford the ynamide. The unreacted nitrogen nucleophile was recovered. In a 10 ml round-bottom flask equipped with TMS-ynamide (1.0 equiv), CH 3OH and K 2CO 3 (2.0 equiv) were combined. The reaction mixture was stirred at room temperature under air until TMS-ynamide was fully consumed. The reaction mixture was treated with H 2O at room temperature and aqueous layer was extracted with Et 2O for three times. The combined organic layers were washed with brine, dried over Na 2SO 4, filtered and evaporated in vacuo. Crude product was purified by flash chromatography to afford the terminal ynamide in quantitative yield. S3

4 4-methyl-N-phenyl-N-(phenylethynyl)benzenesulfonamide 1 H NMR (400 MHz, CDCl 3) δ 7.65 (d, J = 8.3 Hz, 2H), (m, 2H), (m, 10H), 2.45 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 145.0, 139.0, 133.0, 131.4, 129.5, 129.0, 128.2, 128.2, 127.9, 126.2, 122.6, 83.0, 70.5, 21.6 ppm; HRMS m/z (ESI) calcd for C 21H 18NO 2S (M+H) + : , found N-benzyl-4-methyl-N-(phenylethynyl)benzenesulfonamide 1 H NMR (400 MHz, CDCl 3) δ 7.76 (t, J = 6.5 Hz, 2H), (m, 7H), (m, 5H), 4.54 (s, 2H), 2.39 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 144.6, 134.8, 134.5, 131.1, 129.7, 128.9, 128.5, 128.3, 128.2, 127.8, 127.6, 122.8, 82.7, 71.4, 55.7, 21.6 ppm; HRMS m/z (ESI) calcd for C 22H 20NO 2S (M+H) + : , found N,4-dimethyl-N-(phenylethynyl)benzenesulfonamide 1 H NMR (400 MHz, CDCl 3) δ 7.85 (d, J = 8.3 Hz, 2H), (m, 4H), (m, 3H), 3.15 (s, 3H), 2.46 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 144.8, 133.3, 131.4, 129.8, 128.2, 127.8, 127.5, 122.7, 84.0, 69.0, 39.3, 21.6 ppm; HRMS m/z (ESI) calcd for C 16H 16NO 2S (M+H) + : , found N-methyl-N-(phenylethynyl)methanesulfonamide 1 H NMR (400 MHz, CDCl 3) δ (m, 2H), (m, 3H), 3.29 (s, 3H), 3.12 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 131.4, 128.3, 128.0, 122.3, 83.0, 69.4, 39.1, 36.7 ppm; HRMS m/z (ESI) calcd for C 10H 12NO 2S (M+H) + : , found N-(hex-1-yn-1-yl)-N-methylmethanesulfonamide 1 H NMR (400 MHz, CDCl 3) δ 3.15 (s, 3H), 3.04 (s, 3H), 2.28 (t, J = 7.0 Hz, 2H), (m, 2H), 1.40 (dq, J = 14.1, 7.2 Hz, 2H), 0.92 (t, J = 7.2 Hz, 3H), 13 C NMR (100 MHz, CDCl 3) δ74.0, 68.9, 39.0, 35.6, 30.8, 21.7, 17.8, 13.4 ppm; HRMS m/z (ESI) calcd for C 8H 16NO 2S (M+H) + : , found N,4-dimethyl-N-((trimethylsilyl)ethynyl)benzenesulfonamide 1 H NMR (400 MHz, CDCl 3) δ 7.78 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 3.04 (s, 3H), 2.46 (s, 3H), 0.15 (s, 9H); 13 C NMR (100 MHz, CDCl 3) δ 144.7, 133.1, 129.6, 127.9, 96.6, 71.3, 39.0, 21.7, 0.1 ppm; HRMS m/z (ESI) calcd for C 13H 20NO 2SSi (M+H) + : , found N-benzyl-N-((trimethylsilyl)ethynyl)methanesulfonamide 1 H NMR (400 MHz, CDCl 3) δ (m, 2H), (m, 3H), 4.60 (s, 2H), 2.86 (s, 3H), 0.15 (s, 9H); 13 C NMR (100 MHz, CDCl 3) δ 134.4, 129.0, 128.7, 128.7, 94.6, 74.3, 55.6, 38.7, -0.1 ppm; HRMS m/z (ESI) calcd for C 13H 20NO 2SSi (M+H) + : , found ((trimethylsilyl)ethynyl)isoindoline-1,3-dione S4

5 1 H NMR (400 MHz, CDCl 3) δ 7.87 (dd, J = 5.5, 3.1 Hz, 2H), 7.78 (dd, J = 5.6, 3.0 Hz, 2H), 0.23 (s, 9H); 13 C NMR (100 MHz, CDCl 3) δ 164.8, 135.1, 130.9, 124.2, 84.5, 82.9, -0.2 ppm; HRMS m/z (ESI) calcd for C 13H 14NO 2Si (M+H) + : , found ((trimethylsilyl)ethynyl)oxazolidin-2-one 1 H NMR (400 MHz, CDCl 3) δ 4.40 (dd, J = 8.3, 7.7 Hz, 2H), 3.91 (dd, J = 8.6, 7.4 Hz, 2H), 0.16 (s, 9H); 13 C NMR (100 MHz, CDCl 3) δ 155.8, 91.3, 73.6, 63.0, 46.8, -0.1 ppm; HRMS m/z (ESI) calcd for C 8H 14NO 2Si (M+H) + : , found N-ethynyl-N,4-dimethylbenzenesulfonamide 1 H NMR (400 MHz, CDCl 3) δ 7.80 (d, J = 8.3 Hz, 2H), 7.36 (t, J = 7.4 Hz, 2H), 3.06 (s, 3H), 2.68 (s, 1H), 2.46 (s, 3H). 13 C NMR (100 MHz, CDCl 3) δ 144.9, 133.2, 129.8, 127.8, 77.6, 57.4, 38.8, 21.6 ppm; HRMS m/z (ESI) calcd for C 10H 11NO 2S (M+H) + : , found N-benzyl-N-ethynylmethanesulfonamide 1 H NMR (400 MHz, CDCl 3) δ (m, 5H), 4.62 (s, 2H), 2.89 (s, 3H), 2.84 (s, 1H); 13 C NMR (100 MHz, CDCl 3) δ 134.3, 128.8, 128.7, 75.8, 60.1, 55.3, 38.8 ppm; HRMS m/z (ESI) calcd for C 10H 11NO 2S (M+H) + : , found N-ethynyl-N-methylmethanesulfonamide 1 H NMR (400 MHz, CDCl 3) δ 3.20 (s, 3H), 3.08 (s, 3H), 2.78 (s, 1H); 13 C NMR (100 MHz, CDCl 3) δ76.8, 58.1, 38.7, 36.6 ppm; HRMS m/z (ESI) calcd for C 4H 8NO 2S (M+H) + : , found ethynylisoindoline-1,3-dione 1 H NMR (400 MHz, CDCl 3) δ 7.93 (dd, J = 5.4, 3.1 Hz, 2H), (m, 2H), 3.33 (s, 1H); 13 C NMR (100 MHz, CDCl 3) δ 164.9, 135.3, 130.9, 124.4, 67.9, 66.2 ppm; HRMS m/z (ESI) calcd for C 10H 6NO 2 (M+H) + : , found S5

6 Amide Bond Formation with α-acyloxyenamide as Active Ester A 5 ml round-bottomed flask was charged with α-acyloxyenamide 1 (0.2 mmol), CH 2Cl 2 (1 ml) and amine (0.2 mmol). The reaction mixture was stirred at room temperature under air until starting material was fully consumed. The reaction mixture was concentrated and purified by silica gel chromatography to afford the amides 2 and 3 in quantitative yields. (E)-1-((N,4-dimethylphenyl)sulfonamido)-2-phenylvinyl benzoate (1) White solid. 1 H NMR (400 MHz, CDCl 3) δ 7.82 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 7.8 Hz, 2H), (m, 3H), (m, 4H), (m, 1H), 7.02 (d, J = 8.0 Hz, 2H), 6.35 (s, 1H), 3.14 (d, J = 0.8 Hz, 3H), 2.24 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 164.6, 143.6, 139.9, 136.0, 133.7, 131.9, 130.0, 129.4, 128.6, 128.6, 128.4, 128.3, 127.5, 119.3, 36.6, 21.3 ppm; HRMS m/z (ESI) calcd for C 23H 21NNaO 4S (M+Na) + : , found N-phenethylbenzamide (2) White solid, 99% yield (44.6 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.69 (d, J = 7.5 Hz, 2H), 7.46 (t, J = 7.3 Hz, 1H), 7.38 (t, J = 7.3 Hz, 2H), 7.31 (t, J = 7.2 Hz, 2H), 7.23 (t, J = 8.0 Hz, 3H), 6.36 (s, 1H), 3.70 (q, J = 6.3 Hz, 2H), 2.92 (t, J = 6.8 Hz, 2H); 13 C NMR (100 MHz, CDCl 3) δ 167.5, 138.9, 134.6, 131.3, 128.7, 128.6, 128.5, 126.8, 126.5, 41.1, 35.6 ppm; HRMS m/z (ESI) calcd for C 15H 15NNaO (M+Na) + : , found N-methyl-2-phenyl-N-tosylacetamide (3) White solid, 99% yield (60.0 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.71 (d, J = 8.3 Hz, 2H), (m, 5H), 7.14 (d, J = 6.5 Hz, 2H), 4.06 (s, 2H), 3.29 (s, 3H), 2.45 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 171.2, 144.9, 136.0, 133.4, 129.8, 129.3, 128.5, 127.5, 127.1, 43.0, 33.2, 21.5 ppm; HRMS m/z (ESI) calcd for C 16H 17NNaO3S (M+Na) + : , found S6

7 Reaction Condition Optimization for Hydroacyloxylation of Ynamides Table 1. Hydroacyloxylation of Ynamides with Benzoic Acids To a 5 ml round-bottomed flask was added ynamide (0.2 mmol), CH 2Cl 2 (1 ml) and benzoic acid (0.2 mmol). The reaction mixture was stirred at room temperature under air until starting material was fully consumed. The reaction mixture was concentrated and purified by silica gel chromatography to afford the α-acyloxyenamides. S7

8 (E)-1-((4-methyl-N-phenylphenyl)sulfonamido)-2-phenylvinyl benzoate (5a) White solid, 38% yield (35.7 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.88 (d, J = 7.5 Hz, 2H), (m, 3H), 7.52 (d, J = 8.2 Hz, 2H), 7.44 (t, J = 7.7 Hz, 2H), (m, 5H), (m, 3H), 7.04 (d, J = 8.1 Hz, 2H), 6.65 (s, 1H), 2.31 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 163.5, 143.9, 139.2, 138.7, 136.5, 133.7, 132.2, 130.1, 129.2, 129.0, 129.0, 128.5, 128.4, 128.2, 128.2, 127.8, 127.0, 122.1, 21.5 ppm; HRMS m/z (ESI) calcd for C 28H 23NNaO 4S (M+Na) + : , found (E)-1-((N-benzyl-4-methylphenyl)sulfonamido)-2-phenylvinyl benzoate (5b) White solid, 90% yield (87 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 4H), 7.59 (t, J = 7.5 Hz, 1H), 7.39 (t, J = 7.8 Hz, 2H), (m, 2H), (m, 10H), 6.51 (s, 1H), 4.52 (s, 2H), 2.34 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 163.9, 143.8, 137.0, 136.8, 134.5, , 130.0, 129.7, 129.5, 129.1, 128.8, 128.3, 128.2, 128.1, 128.0, 127.8, 123.4, 52.8, 21.4 ppm; HRMS m/z (ESI) calcd for C 29H 25NNaO 4S (M+Na) + : , found (E)-1-((N,4-dimethylphenyl)sulfonamido)-2-phenylvinyl benzoate (5c) White solid, 93% yield (80.6 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.82 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 7.8 Hz, 2H), (m, 3H), (m, 4H), (m, 1H), 7.02 (d, J = 8.0 Hz, 2H), 6.35 (s, 1H), 3.14 (d, J = 0.8 Hz, 3H), 2.24 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 164.6, 143.6, 139.9, 136.0, 133.7, 131.9, 130.0, 129.4, 128.6, 128.5, 128.4, 128.3, 127.5, 119.3, 36.6, 21.3 ppm; HRMS m/z (ESI) calcd for C 23H 21NNaO 4S (M+Na) + : , found (E)-1-((N,4-dimethylphenyl)sulfonamido)-2-(trimethylsilyl)vinyl benzoate (5d) White solid, 92% yield (74.3 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.74 (d, J = 8.2 Hz, 2H), (m, 3H), 7.31 (t, J = 7.8 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H), 5.50 (s, 1H), 3.04 (s, 3H), 2.31 (s, 3H), 0.30 (s, 9H); 13 C NMR (100 MHz, CDCl 3) δ 163.9, 146.2, 143.6, 135.5, 133.4, 129.8, 129.4, 128.8, 128.2, 128.0, 118.5, 36.9, 21.3, -0.8 ppm; HRMS m/z (ESI) calcd for C 20H 25NNaO 4SSi (M+Na) + : , found ((N,4-dimethylphenyl)sulfonamido)vinyl benzoate (5e) White solid, 99% yield (66.3 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.86 (dd, J = 8.2, 1.0 Hz, 2H), 7.74 (d, J = 8.3 Hz, 2H), (m, 1H), 7.41 (t, J = 7.8 Hz, 2H), (m, 2H), 5.03 (d, J = 2.5 Hz, 1H), 4.88 (d, J = 2.5 Hz, 1H), 3.10 (s, 3H), 2.38 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 164.0, 146.8, 144.0, 134.3, 133.7, 130.1, 129.5, 128.6, 128.4, 127.9, 101.7, 37.1, 21.5 ppm; HRMS m/z (ESI) calcd for C 17H 17NNaO 4S (M+Na) + : , found (N-methylmethylsulfonamido)vinyl benzoate (5f) White solid, 99% yield (51.1 mg). 1 H NMR (400 MHz, CDCl 3) δ 8.09 (dd, J = 8.3, 1.2 Hz, 2H), (m, 1H), 7.50 (t, J = 7.8 Hz, 2H), 5.15 (d, J = 2.6 Hz, 1H), 4.99 (d, J = 2.6 Hz, 1H), 3.18 (s, 3H), 3.03 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 164.2, 146.1, 134.1, 130.1, 128.8, 128.3, 100.3, 37.7, 36.0 ppm; HRMS m/z (ESI) calcd for C 11H 13NNaO 4S (M+Na) + : , found (2-oxooxazolidin-3-yl)vinyl benzoate (5g) S8

9 White solid, 53% yield (23.8 mg). 1 H NMR (400 MHz, CDCl 3) δ 8.16 (dd, J = 8.4, 1.3 Hz, 2H), (m, 1H), 7.60 (t, J = 7.9 Hz, 2H), 5.05 (d, J = 2.5 Hz, 1H), 4.81 (d, J = 2.5 Hz, 1H), (m, 2H), (m, 2H); 13 C NMR (CDCl 3, 100 MHz) δ 165.2, 155.4, 137.6, 134.1, 132.1, 128.8, 128.6, 116.3, 63.1, 44.7 ppm; HRMS (ESI) m/z calculated for C 12H 12NO 4 (M+H) + : found: (E)-1-(N-methylmethylsulfonamido)-2-(trimethylsilyl)vinyl benzoate White solid, 89% yield (58.8 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 2H), (m, 1H), 7.49 (t, J = 7.7 Hz, 2H), 5.46 (s, 1H), 3.13 (s, 3H), 2.96 (s, 3H), 0.24 (s, 9H); 13 C NMR (100 MHz, CDCl 3) δ 164.4, 146.0, 134.0, 129.9, 128.8, 128.6, 119.0, 37.8, 36.9, -0.9 ppm; HRMS m/z (ESI) calcd for C 14H 22NO 4SSi (M+H) + : , found (E)-1-(N-methylmethylsulfonamido)-2-phenylvinyl benzoate White solid, 90% yield (59.6 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 2H), 7.67 (dd, J = 10.6, 4.3 Hz, 1H), (m, 4H), (m, 1H), (m, 2H), 6.37 (s, 1H), 3.16 (s, 3H), 2.90 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 165.1, 139.7, 131.8, 131.6, 130.3, 130.2, 129.0, 128.8, 128.6, 128.5, 119.7, 39.4, 36.3 ppm; HRMS m/z (ESI) calcd for C 17H 18NO 4S (M+H) + : , found (E)-1-(N-benzylmethylsulfonamido)-2-(trimethylsilyl)vinyl benzoate White solid, 88% yield (70.9 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 2H), 7.65 (t, J = 7.4 Hz, 1H), (m, 4H), (m, 3H), 5.54 (s, 1H), 4.53 (s, 2H), 2.98 (s, 3H), (s, 9H); 13 C NMR (100 MHz, CDCl 3) δ 164.3, 143.2, 134.7, 133.9, 130.2, 129.8, 129.0, 128.8, 128.5, 128.5, 120.9, 52.8, 40.0, -1.1 ppm; HRMS m/z (ESI) calcd for C 20H 26NO 4SSi (M+H) + : , found (N-benzylmethylsulfonamido)vinyl benzoate White solid, 92% yield (60.7 mg). 1 H NMR (400 MHz, CDCl 3) δ 8.05 (dd, J = 8.2, 1.0 Hz, 2H), (m, 1H), 7.50 (t, J = 7.8 Hz, 2H), 7.42 (d, J = 6.9 Hz, 2H), (m, 3H), 5.07 (d, J = 2.4 Hz, 1H), 5.00 (d, J = 2.4 Hz, 1H), 4.67 (s, 2H), 3.09 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 164.0, 143.7, 135.6, 134.1, 130.1, 128.7, 128.6, 128.6, 128.3, 128.0, 103.4, 51.7, 40.0 ppm; HRMS m/z (ESI) calcd for C 17H 18NO 4S (M+H) + : , found (E)-1-(N-methylmethylsulfonamido)hex-1-en-1-yl benzoate 1 H NMR (400 MHz, CDCl 3) δ (m, 2H), 7.63 (t, J = 7.5 Hz, 1H), 7.48 (t, J = 7.8 Hz, 2H), 5.48 (t, J = 7.6 Hz, 1H), 3.16 (s, 3H), 2.94 (s, 3H), (m, 7.3 Hz, 2H), (m, 4H), 0.93 (t, J = 7.1 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ 165.1, 138.9, 133.9, 130.0, 129.1, 128.9, 128.7, 128.6, 122.9, 38.5, 36.5, 30.8, 29.0, 28.3, 26.6, 22.3, 13.8 ppm; HRMS m/z (ESI) calcd for C 15H 22NO 4S (M+H) + : , found S9

10 General Procedure for Hydroacyloxylation of Ynamide with Various Carboxylic Acids A 5 ml round-bottomed flask was charged with N-methyl ynetoluenesulfonamide (MYTsA) (0.2 mmol), CH 2Cl 2 (1 ml) and carboxylic acid (0.2 mmol). The reaction mixture was stirred at room temperature under air until starting material was fully consumed. The reaction mixture was concentrated and purified by silica gel chromatography to afford the α-acyloxyenamides. 1-((N,4-dimethylphenyl)sulfonamido)vinyl formate (7a) White solid, 99% yield (50.5 mg). 1 H NMR (400 MHz, CDCl 3) δ 8.06 (s, 1H), 7.73 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 4.83 (d, J = 2.9 Hz, 1H), 4.73 (d, J = 2.8 Hz, 1H), 3.01 (s, 3H), 2.43 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 158.2, 146.9, 144.4, 134.1, 129.6, 127.8, 99.7, 36.8, 21.5 ppm; HRMS m/z (ESI) calcd for C 11H 13NNaO 4S (M+Na) + : , found ((N,4-dimethylphenyl)sulfonamido)vinyl pent-4-enoate (7b) White solid, 92% yield (56.9 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.70 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.1 Hz, 2H), (m, 1H), (m, 2H), 4.79 (d, J = 2.4 Hz, 1H), 4.62 (d, J = 2.4 Hz, 1H), 2.99 (s, 3H), 2.42 (s, 3H), 2.40 (d, J = 7.7 Hz, 2H), 2.32 (t, J = 6.7 Hz, 2H); 13 C NMR (100 MHz, CDCl 3) δ 170.4, 146.9, 144.0, 136.0, 133.9, 129.4, 127.9, 115.7, 100.4, 37.2, 33.1, 28.3, 21.5 ppm; HRMS m/z (ESI) calcd for C 15H 19NNaO 4S (M+Na) + : , found ((N,4-dimethylphenyl)sulfonamido)vinyl adamantane-1-carboxylate (7c) White solid, 98% yield (76.3 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.73 (d, J = 8.3 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 4.83 (d, J = 2.2 Hz, 1H), 4.79 (d, J = 2.2 Hz, 1H), 2.95 (s, 3H), 2.42 (s, 3H), 1.97 (s, 3H), 1.75 (d, J = 2.5 Hz, 6H), 1.70 (d, J = 12.4 Hz, 3H), 1.63 (d, J = 12.1 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ 174.8, 146.5, 143.9, 134.1, 129.5, 128.1, 102.0, 41.0, 38.3, 36.6, 36.2, 27.7, 21.5 ppm; HRMS m/z (ESI) calcd for C 21H 27NNaO 4S (M+Na) + : , found ((N,4-dimethylphenyl)sulfonamido)vinyl 4-chlorobenzoate (7d) White solid, 98% yield (71.5 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 2H), 7.72 (d, J = 8.3 Hz, 2H), (m, 2H), (m, 2H), 5.01 (d, J = 2.6 Hz, 1H), 4.82 (d, J = 2.6 Hz, 1H), 3.09 (s, 3H), 2.39 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 163.1, 146.9, 144.0, 140.3, 134.1, 131.5, 129.5, 128.8, 127.9, 127.1, 101.4, 37.3, 21.5 ppm; HRMS m/z (ESI) calcd for C 17H 17ClNO 4S (M+H) + : , found ((N,4-dimethylphenyl)sulfonamido)vinyl furan-2-carboxylate (7e) S10

11 White solid, 98% yield (63.0 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.66 (d, J = 8.0 Hz, 2H), 7.54 (s, 1H), 7.20 (d, J = 8.2 Hz, 2H), 7.02 (d, J = 3.2 Hz, 1H), 6.44 (s, 1H), 4.94 (d, J = 2.1 Hz, 1H), 4.75 (d, J = 2.1 Hz, 1H), 2.99 (s, 3H), 2.32 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 155.6, 147.4, 146.3, 144.0, 143.1, 134.0, 129.4, 127.9, 119.8, 112.1, 101.7, 37.2, 21.5 ppm; HRMS m/z (ESI) calcd for C 15H 15NNaO 5S (M+Na) + : , found ((N,4-dimethylphenyl)sulfonamido)vinyl 1-methyl-1H-indazole-3-carboxylate (7f) White solid, 99% yield (76.2 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.96 (d, J = 8.2 Hz, 1H), 7.78 (d, J = 8.2 Hz, 2H), 7.47 (d, J = 2.7 Hz, 2H), (m, 1H), 7.21 (d, J = 8.1 Hz, 2H), 5.08 (d, J = 2.4 Hz, 1H), 4.90 (d, J = 2.4 Hz, 1H), 4.18 (s, 3H), 3.15 (s, 3H), 2.33 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 159.8, 146.5, 143.8, 141.0, 134.4, 133.1, 129.4, 128.0, 127.0, 123.7, 123.5, 122.0, 109.6, 101.6, 37.2, 36.5, 21.5 ppm; HRMS m/z (ESI) calcd for C 19H 19N 3NaO 4S (M+Na) + : , found ((N,4-dimethylphenyl)sulfonamido)vinyl benzo[b]thiophene-3-carboxylate (7g) White solid, 99% yield (77.4 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.92 (s, 1H), 7.86 (d, J = 8.1 Hz, 2H), 7.76 (d, J = 8.2 Hz, 2H), (m, 2H), (m, 2H), 5.09 (d, J = 2.5 Hz, 1H), 4.92 (d, J = 2.5 Hz, 1H), 3.12 (s, 3H), 2.33 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 160.0, 146.5, 144.0, 142.7, 138.4, 134.3, 132.1, 131.6, 129.6, 127.9, , 125.7, 125.1, 122.7, 101.9, 37.1, 21.4 ppm; HRMS m/z (ESI) calcd for C 19H 17NNaO 4S 2 (M+Na) + : , found ((N,4-dimethylphenyl)sulfonamido)vinyl cinnamate (7h) White solid, 99% yield (70.7 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.75 (d, J = 8.3 Hz, 2H), 7.59 (d, J = 16.0 Hz, 1H), (m, 2H), (m, 3H), 7.30 (d, J = 8.0 Hz, 2H), 6.31 (d, J = 16.0 Hz, 1H), 4.95 (d, J = 2.4 Hz, 1H), 4.78 (d, J = 2.5 Hz, 1H), 3.08 (s, 3H), 2.36 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 164.1, 147.0, 146.9, 144.0, 134.5, 133.9, 130.8, 129.5, 129.0, 128.3, 128.0, 116.3, 101.1, 37.2, 21.5 ppm; HRMS m/z (ESI) calcd for C 19H 19NNaO 4S (M+Na) + : , found ((N,4-dimethylphenyl)sulfonamido)vinyl 3-phenylpropiolate (7i) White solid, 99% yield (71.0 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.76 (d, J = 8.2 Hz, 2H), (m, 2H), 7.49 (t, J = 7.5 Hz, 1H), 7.40 (t, J = 7.5 Hz, 2H), 7.31 (d, J = 8.1 Hz, 2H), 4.99 (d, J = 2.7 Hz, 1H), 4.81 (d, J = 2.7 Hz, 1H), 3.07 (s, 3H), 2.39 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 151.0, 146.4, 144.1, 134.1, 133.1, 131.1, 129.6, 128.6, 128.0, 119.0, 101.8, 89.4, 79.5, 37.2, 21.5 ppm; HRMS m/z (ESI) calcd for C 19H 18NO 4S (M+H) + : , found ((N,4-dimethylphenyl)sulfonamido)vinyl (tert-butoxycarbonyl)-l-phenylalaninate (7j) White solid, 99% yield (94.1 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.72 (d, J = 8.2 Hz, 2H), (m, 5H), 7.15 (d, J = 7.0 Hz, 2H), (m, 2H), 4.70 (s, 1H), 4.53 (dd, J = 13.4, 6.9 Hz, 1H), 3.12 (dd, J = 14.0, 5.4 Hz, 1H), (m, 4H), 2.42 (s, 3H), 1.40 (s, 9H); 13 C NMR (100 MHz, CDCl 3) δ 169.6, 155.0, 146.6, 144.3, 135.7, 133.7, 129.6, 129.4, 128.6, 128.1, 127.2, 101.8, 80.1, 54.4, 37.6, 37.0, 28.3, 21.6 ppm; HRMS m/z (ESI) calcd for C 24H 31N 2O 6S (M+H) + : , found S11

12 1-((N,4-dimethylphenyl)sulfonamido)vinyl ((benzyloxy)carbonyl)-l-serinate (7k) White solid, 99% yield (88.7 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.71 (d, J = 8.2 Hz, 2H), (m, 7H), 5.84 (d, J = 8.4 Hz, 1H), (m, 2H), 4.87 (d, J = 2.5 Hz, 1H), 4.46 (dd, J = 15.5, 5.5 Hz, 2H), (m, 1H), 3.85 (dd, J = 11.8, 3.2 Hz, 1H), 2.98 (s, 3H), 2.43 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 168.2, 156.2, 147.0, 144.6, 136.1, 132.0, 129.6, 128.5, 128.1, 128.0, 127.3, 100.5, 67.1, 62.6, 56.3, 38.0, 21.6 ppm; HRMS m/z (ESI) calcd for C 21H 25N 2O 7S (M+H) + : , found ((N,4-dimethylphenyl)sulfonamido)vinyl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-threoninate (7l) White solid, 99% yield (108.9 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 4H), 7.64 (t, J = 7.5 Hz, 2H), (m, 2H), 7.32 (d, J = 7.9 Hz, 4H), 5.84 (d, J = 9.4 Hz, 1H), 4.89 (d, J = 2.8 Hz, 1H), (m, 1H), (m, 4H), 4.26 (t, J = 7.2 Hz, 1H), 2.99 (s, 3H), 2.42 (s, 3H), 1.30 (d, J = 6.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ 168.5, 156.7, 147.0, 144.5, 141.2, 141.2, 132.0, 129.5, 128.1, 127.6, 127.0, 125.1, 119.9, 100.7, 67.3, 67.0, 59.6, 47.0, 38.0, 21.5, 19.5 ppm; HRMS m/z (ESI) calcd for C 29H 30N 2NaO 7S (M+Na) + : , found S12

13 Control Experiments for Two Steps Strategy and One-pot, Two-step Strategy for Amide Bond Formation 1. Two-step Strategy (eq. 1 & 2) and Two-step, One-pot Strategy (eq. 3) for Amide Bond Formation Two steps strategy: (1) A 5 ml round-bottomed flask was charged with N-methyl ynetoluenesulfonamide (MYTsA) (0.2 mmol), CH 2Cl 2 (1 ml) and Fmoc-Gly-OH (0.2 mmol). The reaction mixture was stirred at room temperature under air until starting material was fully consumed. The reaction mixture was concentrated and purified by silica gel chromatography to afford the α- acyloxyenamide in 99% yield. (2) To a 5 ml round-bottomed flask was added α-acyloxyenamide (0.2 mmol), CH 2Cl 2 (1 ml) and 2-phenylethan-1-amine (0.2 mmol). The reaction mixture was stirred at room temperature under air until starting material was fully consumed. The reaction mixture was concentrated and purified by silica gel chromatography to afford the respective amide in 99% yield. Two-step, One-pot strategy: (3) A 5 ml round-bottomed flask was charged with N-methyl ynetoluenesulfonamide (MYTsA) (0.2 mmol), CH 2Cl 2 (1 ml) and Fmoc-Gly-OH (0.2 mmol). The reaction mixture was stirred at room temperature under air until MYTsA was fully consumed. 2- phenylethan-1-amine (0.2 mmol) was added to the above solution and reaction mixture was stirred at room temperature under air until α-acyloxyenamide was fully consumed. The reaction mixture was concentrated and purified by silica gel chromatography to afford the respective amide in 99% yield. S13

14 2. Solvent effect (DMF) of the Aminolysis Step A 5 ml round-bottomed flask was charged with N-methyl ynetoluenesulfonamide (MYTsA) (0.2 mmol), CH 2Cl 2 (1 ml) and Fmoc-L-Trp-OH (0.2 mmol), or Cbz-L-Trp-OH (0.2 mmol), or Boc-L-Trp-OH (0.2 mmol). The reaction mixture was stirred at room temperature under air until starting material was fully consumed. The solvent was removed under reduced pressure to afford α-acyloxyenamide, which was charged with DMF (1 ml) and H-L-Thr(tBu)-OtBu (0.24 mmol). The reaction mixture was stirred at room temperature under air until α-acyloxyenamide was fully consumed. The reaction mixture was concentrated and purified by silica gel chromatography to afford the respective peptide. S14

15 (9H-fluoren-9-yl)methyl (2-oxo-2-(phenethylamino)ethyl)carbamate White solid, 99% yield (79.2 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.77 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 7.3 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), (m, 4H), (m, 3H), 6.01 (s, 1H), 5.45 (s, 1H), 4.41 (d, J = 6.9 Hz, 2H), 4.20 (t, J = 6.8 Hz, 1H), 3.80 (d, J = 5.3 Hz, 2H), 3.53 (dd, J = 13.0, 6.7 Hz, 2H), 2.80 (t, J = 6.9 Hz, 2H); 13 C NMR (100 MHz, CDCl 3) δ 168.9, 156.6, 143.6, 141.2, 138.5, 128.6, 128.5, 127.7, 127.0, 126.5, 124.9, 119.9, 67.0, 47.0, 44.4, 40.6, 35.5 ppm; HRMS m/z (ESI) calcd for C 25H 25N 2O 3 (M+H) + : , found N-methyl-N-tosylacetamide White solid, 99% yield (45.0 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.74 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 3.25 (s, 3H), 2.42 (s, 3H), 2.36 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 170.3, 144.9, 136.1, 129.9, 127.2, 32.9, 24.9, 21.5 ppm; HRMS m/z (ESI) calcd for C 10H 14NO 3S (M+H) + : , found Fmoc-L-Trp-L-Thr(tBu)-OtBu Glassy solid, 72% yield (92.2 mg). 1 H NMR (400 MHz, CDCl 3) δ 8.46 (s, 1H), 7.72 (d, J = 7.5 Hz, 3H), 7.51 (t, J = 7.5 Hz, 2H), (m, 3H), 7.25 (t, J = 7.3 Hz, 2H), (m, 3H), 6.59 (d, J = 6.5 Hz, 1H), 5.65 (s, 1H), 4.59 (t, J = 20.0 Hz, 1H), (m, 3H), (m, 2H), (m, 2H), 1.45 (s, 9H), 1.09 (s, 9H), 1.02 (d, J = 6.0 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ 171.5, 169.4, 155.8, 143.8, 143.7, 141.1, 136.2, 127.5, 127.5, 126.9, 125.1, 125.0, 123.5, 121.9, 119.8, 119.7, 119.5, 118.7, 111.2, 81.8, 73.7, 67.0, 60.3, 58.5, 55.2, 47.0, 28.8, 28.5, 27.9, 20.5 ppm; HRMS m/z (ESI) calcd for C 38H 46N 3O 6 (M+H) + : , found Cbz-L-Trp-L-Thr(tBu)-OtBu Glassy solid, 97% yield (106.9 mg). 1 H NMR (400 MHz, CDCl 3) δ 8.33 (s, 1H), 7.71 (d, J = 6.4 Hz, 1H), 7.32 (t, J = 5.1 Hz, 6H), 7.17 (t, J = 7.5 Hz, 1H), 7.10 (d, J = 8.3 Hz, 2H), 6.55 (d, J = 8.6 Hz, 1H), 5.55 (d, J = 5.8 Hz, 1H), 5.10 (s, 2H), 4.62 (s, 1H), 4.33 (dd, J = 8.7, 2.3 Hz, 1H), 4.13 (dd, J = 13.4, 7.8 Hz, 1H), (m, 2H), 1.46 (s, 9H), 1.11 (s, 9H), 1.00 (d, J = 5.9 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ 171.6, 169.5, 156.0, 136.4, 136.3, 128.5, 128.1, 128.0, 127.6, 123.5, 122.1, 119.7, 118.9, 111.2, 110.4, 81.9, 73.9, 67.1, 66.9, 58.6, 55.3, 28.8, 28.6, 28.1, 20.6 ppm; HRMS m/z (ESI) calcd for C 31H 42N 3O 6 (M+H) + : , found Boc-L-Trp-L-Thr(tBu)-OtBu Glassy solid, 99% yield (102.4 mg). 1 H NMR (400 MHz, CDCl 3) δ 8.38 (s, 1H), 7.67 (d, J = 7.7 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.17 (t, J = 7.5 Hz, 1H), (m, 2H), 6.61 (d, J = 8.6 Hz, 1H), 5.22 (d, J = 46.6 Hz, 1H), 4.54 (s, 1H), 4.31 (dd, J = 8.7, 2.4 Hz, 1H), 4.12 (dd, J = 6.1, 1.9 Hz, 1H), (m, 2H), 1.44 (s, 9H), 1.41 (s, 9H), 1.12 (s, 9H), 0.99 (d, J = 6.1 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ 171.9, 169.5, 155.3, 136.2, 127.7, 123.3, 122.0, 119.5, 118.9, 111.1, 110.5, 81.8, 79.7, 73.8, 67.1, 58.4, 54.8, 28.5, 28.2, 28.0, 20.4 ppm; HRMS m/z (ESI) calcd for C 28H 44N 3O 6 (M+H) + : , found S15

16 Ynamide MYTsA Mediated Amide Bond Formation from Carboxylic Acids and Amines A 5 ml round-bottomed flask was charged with N-methyl ynetoluenesulfonamide (MYTsA) (0.2 mmol), CH 2Cl 2 (1 ml) and carboxylic acid (0.2 mmol). The reaction mixture was stirred at room temperature under air until MYTsA was fully consumed. Amine (0.22 mmol) was added and the reaction mixture was stirred at room temperature under air until α-acyloxyenamide was fully consumed. The reaction mixture was concentrated and purified by silica gel chromatography to afford the respective amide. N-phenethylacetamide (9a) White solid, 98% yield (31.9 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.27 (t, J = 7.3 Hz, 2H), (m, 3H), 6.50 (s, 1H), 3.45 (dd, J = 13.3, 7.0 Hz, 2H), 2.79 (t, J = 7.2 Hz, 2H), 1.90 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 170.2, 138.7, 128.4, 128.3, 126.1, 40.5, 35.3, 22.8 ppm; HRMS m/z (ESI) calcd for C 10H 14NO (M+H) + : , found N-phenethylcinnamamide (9b) White solid, 80% yield (40.2 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.62 (d, J = 15.6 Hz, 1H), (m, 2H), (m, 5H), (m, 3H), 6.35 (d, J = 15.6 Hz, 1H), 5.85 (s, 1H), 3.66 (dd, J = 13.0, 6.8 Hz, 2H), 2.89 (t, J = 6.9 Hz, 2H); 13 C NMR (100 MHz, CDCl 3) δ 165.9, 141.0, 138.9, 134.8, 129.6, 128.8, 128.7, 128.6, 127.7, 126.5, 120.7, 40.8, 35.6 ppm; HRMS m/z (ESI) calcd for C 17H 18NO (M+H) + : , found N-phenethylpropiolamide (9c) White solid, 99% yield (34.3 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.33 (t, J = 7.3 Hz, 2H), (m, 3H), 5.94 (s, 1H), 3.57 (q, J = 6.9 Hz, 2H), 2.85 (t, J = 7.0 Hz, 2H), 2.75 (s, 1H); 13 C NMR (100 MHz, CDCl 3) δ 13 C NMR (100 MHz, CDCl 3) δ 152.1, 138.2, 128.7, 128.7, 126.7, 79.2, 73.1, 40.9, 35.2 ppm; HRMS m/z (ESI) calcd for C 11H 12NO (M+H) + : , found N-phenethylfuran-2-carboxamide (9d) White solid, 99% yield (42.6 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 1H), 7.32 (t, J = 7.5 Hz, 2H), 7.24 (t, J = 7.4 Hz, 3H), 7.09 (d, J = 3.4 Hz, 1H), (m, 2H), 3.68 (dd, J = 13.4, 6.8 Hz, 2H), 2.91 (t, J = 7.1 Hz, 2H); 13 C NMR (100 MHz, CDCl 3) δ 158.3, 148.0, 143.7, 138.7, 128.7, 128.6, 126.5, 114.0, 112.0, 40.24, ppm; HRMS m/z (ESI) calcd for C 13H 14NO 2 (M+H) + : , found N-phenethylbenzo[b]thiophene-3-carboxamide (9e) S16

17 White solid, 98% yield (55.1 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 2H), 7.68 (s, 1H), (m, 2H), (m, 2H), (m, 3H), 6.26 (s, 1H), 3.71 (dd, J = 13.0, 6.8 Hz, 2H), 2.94 (t, J = 6.9 Hz, 2H); 13 C NMR (100 MHz, CDCl 3) δ 162.2, 140.7, 139.0, 138.7, 138.4, 128.8, 128.7, 126.6, 126.3, 125.1, 125.0, 124.9, 122.7, 41.3, 35.7 ppm; HRMS m/z (ESI) calcd for C 17H 16NOS (M+H) + : , found N-(2-(1H-indol-3-yl)ethyl)-3-phenylpropiolamide (9f) White solid, 99% yield (57.0 mg). 1 H NMR (400 MHz, CDCl 3) δ 8.51 (s, 1H), 7.59 (d, J = 7.8 Hz, 1H), (m, 2H), (m, 2H), (m, 2H), (m, 1H), (m, 1H), 7.01 (d, J = 2.1 Hz, 1H), 6.23 (s, 1H), 3.66 (dd, J = 12.8, 6.7 Hz, 2H), 3.00 (t, J = 6.7 Hz, 2H); 13 C NMR (100 MHz, CDCl 3) δ 153.5, 136.4, 132.4, 129.9, 128.4, 127.1, 122.3, 122.0, 120.0, 119.3, 118.5, 112.1, 111.4, 84.7, 83.0, 40.1, 25.0 ppm; HRMS m/z (ESI) calcd for C 19H 17N 2O (M+H) + : , found chloro-N-(2-morpholinoethyl)benzamide (9g) White solid, 98% yield (57.0 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 2H), (m, 2H), (m, 4H), 3.52 (dd, J = 11.2, 5.8 Hz, 2H), 2.58 (t, J = 6.0 Hz, 2H), (m, 4H); 13 C NMR (100 MHz, CDCl 3) δ 166.3, 137.6, 132.9, 128.8, 128.3, 66.9, 56.8, 53.3, 36.1 ppm; HRMS m/z (ESI) calcd for C 13H 18ClN 2O 2 (M+H) + : , found (3r,5r,7r)-N-(tert-butyl)adamantane-1-carboxamide (9h) White solid, 98% yield (46.1 mg). 1 H NMR (400 MHz, CDCl3) δ 5.35 (s, 1H), 2.01 (s, 3H), (m, 6H), (m, 6H), 1.31 (s, 9H); 13 C NMR (100 MHz, CDCl3) δ 177.6, 50.7, 41.1, 39.6, 36.7, 29.0, ppm; HRMS m/z (ESI) calcd for C 15H 26NO (M+H) + : , found N-cycloheptyl-3-phenylpropiolamide (9i) White solid, 92% yield (44.3 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 2H), (m, 3H), 6.06 (d, J = 6.8 Hz, 1H), (d, J = 4.0 Hz, 1H), 1.97 (t, J = 11.6 Hz, 2H), (m, 10H); 13 C NMR (100 MHz, CDCl 3) δ 152.4, 132.5, 130.0, 128.6, 120.5, 84.1, 83.6, 51.2, 35.0, 28.1, 24.1 ppm; HRMS m/z (ESI) calcd for C 16H 20NO (M+H) + : , found N-(benzyloxy)-3-phenylpropiolamide (9j) White solid, 90% yield (45.2 mg). 1 H NMR (400 MHz, CDCl 3) δ 9.14 (s, 1H), 7.49 (d, J = 6.1 Hz, 2H), (m, 8H), 4.97 (s, 2H); 13 C NMR (100 MHz, CDCl 3) δ 151.9, 134.8, 132.5, 130.3, 129.2, 128.7, 128.5, 128.4, 119.6, 78.0, 78.4, 29.6 ppm; HRMS m/z (ESI) calcd for C 16H 13NNaO 2 (M+Na) + : , found phenyl-1-(pyrrolidin-1-yl)prop-2-yn-1-one (9k) S17

18 White solid, 99% yield (39.4 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.53 (d, J = 6.8 Hz, 2H), (m, 3H), 3.72 (t, J = 6.4 Hz, 2H), 3.52 (t, J = 6.6 Hz, 2H), (m, 4H); 13 C NMR (100 MHz, CDCl 3) δ 152.8, 132.5, 130.0, 128.6, 120.8, 88.8, 82.8, 48.2, 45.5, 25.5, 24.8 ppm; HRMS m/z (ESI) calcd for C 13H 14NO (M+H) + : , found phenyl-1-(piperidin-1-yl)prop-2-yn-1-one (9l) White solid, 99% yield (42.2 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 2H), (m, 3H), (m, 2H), (m, 2H), 1.65 (dt, J = 9.9, 6.3 Hz, 4H), 1.56 (dt, J = 11.0, 5.6 Hz, 2H); 13 C NMR (100 MHz, CDCl 3) δ 152.8, 132.2, 129.7, 128.4, 120.7, 90.1, 81.4, 48.1, 42.3, 26.4, 25.3, 24.4 ppm; HRMS m/z (ESI) calcd for C 14H 16NO (M+H) + : , found N,N-diisopropyl-3-phenylpropiolamide (9m) White solid, 76% yield (34.8 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 2H), (m, 3H), (m, 1H), (m, 1H), 1.40 (d, J = 6.8 Hz, 6H), 1.29 (d, J = 6.8 Hz, 6H); 13 C NMR (100 MHz, CDCl 3) δ 153.5, 132.1, 129.6, 128.4, 121.0, 88.4, 83.1, 50.3, 45.7, 21.0, 20.1 ppm; HRMS m/z (ESI) calcd for C 15H 20NO (M+H) + : , found N-(2-hydroxyethyl)-3-phenylpropiolamide (9n) White solid, 95% yield (35.9 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 2H), 7.37 (t, J = 4.9 Hz, 1H), 7.30 (t, J = 7.7 Hz, 2H), 6.92 (s, 1H), (m, 2H), 3.49 (dd, J = 10.5, 5.5 Hz, 2H); 13 C NMR (100 MHz, CDCl 3) δ 154.5, 132.6, 130.2, 128.6, 120.2, 85.6, 82.9, 61.4, 42.7 ppm; HRMS m/z (ESI) calcd for C 11H 12NO 2 (M+H) + : , found S18

19 Table 2. Comparative Study of Epimerization/Racemization During Dipeptide Synthesis with Fmoc-L- Ser(tBu)-L-Leu-OtBu as the Model a Reaction conditions: 11a (0.2 mmol), 12a (0.22 mmol), coupling reagent (0.22 mmol), DIEA (0.4 mmol). b Isolated yield. c Determined by HPLC analysis. HBTU, HATU, PyBop, DEPBT: A 5 ml round-bottomed flask was charged with the respective coupling reagent (0.22 mmol), CH 2Cl 2 (1 ml), Fmoc-L-Ser(tBu)-OH (0.2 mmol), H-L-Leu-OtBu (0.22 mmol) and DIEA (0.4 mmol). The reaction mixture was stirred at room temperature under air until starting material was fully consumed. The diastereomeric ratio (dr) was determined by chiral HPLC with chiral IC column with hexane and i-proh as eluent. The reaction mixture was concentrated and purified by silica gel chromatography to afford the respective peptide. DCC: A 5 ml round-bottomed flask was charged with coupling reagent (0.22 mmol), CH 2Cl 2 (1 ml), Fmoc-L-Ser(tBu)-OH (0.2 mmol) and H-L-Leu-OtBu(0.22 mmol). The reaction mixture was stirred at room temperature under air until starting material was fully consumed by TLC. The diastereomeric ratio (dr) was determined by chiral HPLC with chiral IC column with hexane and i-proh as eluent. The reaction mixture was concentrated and purified by silica gel chromatography to afford the respective peptide. MYMsA, MYTsA: A 5 ml round-bottomed flask was charged with coupling reagent (0.22 mmol), CH 2Cl 2 (1 ml) and Fmoc-L-Ser(tBu)-OH (0.2 mmol). The reaction mixture was stirred at room temperature under air until starting material was fully consumed. H-L-Leu-OtBu(0.22 mmol) was added to the above solution and the reaction mixture was stirred at room temperature under air until α- acyloxyenamide was fully consumed. The diastereomeric ratio (dr) was determined by chiral HPLC with chiral IC column with hexane and i-proh as eluent. The reaction mixture was concentrated and purified by silica gel chromatography to afford the respective peptide. S19

20 Copies of HPLC Chromatograms 1:1 mixture of Fmoc-L-Ser(tBu)-L-Leu-OtBu and Fmoc-D-Ser(tBu)-L-Leu-OtBu Peak Info Peak No % Area Area RT (min) Height (mav) Total: HBTU: mixture of Fmoc-D-Ser(tBu)-L-Leu-OtBu (minor) and Fmoc-L-Ser(tBu)-L-Leu-OtBu (major) Peak Info Peak No % Area Area RT (min) Height (mav) Total: S20

21 HATU: mixture of Fmoc-D-Ser(tBu)-L-Leu-OtBu (minor) and Fmoc-L-Ser(tBu)-L-Leu-OtBu (major) Peak Info Peak No % Area Area RT (min) Height (mav) Total: PyBop: mixture of Fmoc-D-Ser(tBu)-L-Leu-OtBu (minor) and Fmoc-L-Ser(tBu)-L-Leu-OtBu (major) Peak Info Peak No % Area Area RT (min) Height (mav) Total: S21

22 DEPBT: mixture of Fmoc-D-Ser(tBu)-L-Leu-OtBu (minor) and Fmoc-L-Ser(tBu)-L-Leu-OtBu (major) Peak Info Peak No % Area Area RT (min) Height (mav) Total: DCC: mixture of Fmoc-D-Ser(tBu)-L-Leu-OtBu (minor) and Fmoc-L-Ser(tBu)-L-Leu-OtBu (major) Peak Info Peak No % Area Area RT (min) Height (mav) Total: S22

23 MYMsA: mixture of Fmoc-D-Ser(tBu)-L-Leu-OtBu (minor) and Fmoc-L-Ser(tBu)-L-Leu-OtBu (major) Peak Info Peak No % Area Area RT (min) Height (mav) Total: MYTsA: mixture of Fmoc-D-Ser(tBu)-L-Leu-OtBu (minor) and Fmoc-L-Ser(tBu)-L-Leu-OtBu(major) Peak Info Peak No % Area Area RT (min) Height (mav) Total: S23

24 Comparative Study of Epimerization/Racemization During Dipeptide Synthesis with Fmoc-L-Phg-L-Leu-OtBu as the Model The control experiment with H-L-phenylglycine, a non-natural amino acid which is extremely prone to epimerize/racemize during peptide synthesis, was used as a model to test the limit of ynamide coupling reagent were performed. According to the experimental results (Table 3), the ynamide coupling reagent MYMsA still be the best one among the tested coupling reagents albeit small amount of epimerization was observed. To further probe the origin of the epimerization/racemization of phenylglycine, a control experiment regarding the racemization of phenylglycine during hydroacyloxylation was performed. As shown in Scheme 1, no racemization was observed during hydroacyloxylation of phenylglycine. Such result clearly demonstrated that the epimerization/racemization of phenylglycine in peptide synthesis was not occurred in the activation step but in the subsequent aminolysis of α-acyloxyenamide. We believed that such epimerization/racemization could be suppressed by careful optimization the reaction conditions of the aminolysis step. Table 3. Comparative Study of Epimerization/Racemization During Dipeptide Synthesis with Fmoc-L- Phg-L-Leu-OtBu as the Model a Reaction conditions: 11x (0.2 mmol), 12a (0.22 mmol), coupling reagent (0.22 mmol), DIEA (0.4 mmol). b Isolated yield. c Determined by HPLC analysis. Scheme 1. Study on the racemization of phenylglycine during hydroacyloxylation with MYMsA S24

25 HBTU, HATU, PyBop, DEPBT: A 5 ml round-bottomed flask was charged with the respective coupling reagent (0.22 mmol), CH 2Cl 2 (1 ml), Fmoc-L-Phg-OH (0.2 mmol), H-L-Leu-OtBu (0.22 mmol) and DIEA (0.4 mmol). The reaction mixture was stirred at room temperature under air until starting material was fully consumed. The diastereomeric ratio (dr) was determined by chiral HPLC with chiral IC columncolumn with hexane and i-proh as eluent. The reaction mixture was concentrated and purified by silica gel chromatography to afford the respective peptide. MYMsA, MYTsA: A 5 ml round-bottomed flask was charged with coupling reagent (0.22 mmol), CH 2Cl 2 (1 ml) and Fmoc-L-Phg-OH (0.2 mmol). The reaction mixture was stirred at room temperature under air until starting material was fully consumed. H-L-Leu-OtBu(0.22 mmol) was added to the above solution and the reaction mixture was stirred at room temperature under air until α-acyloxyenamide was fully consumed. The diastereomeric ratio (dr) was determined by chiral HPLC with chiral IC column with hexane and i-proh as eluent. The reaction mixture was concentrated and purified by silica gel chromatography to afford the respective peptide. S25

26 Fmoc-L-Phg-L-Leu-OtBu (13x) White solid, 97% yield (105.3 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.75 (d, J = 7.6 Hz, 2H), 7.58 (s, 2H), (m, 9H), 6.20 (d, J = 21.8 Hz, 2H), 5.29 (s, 1H), 4.47 (dd, J = 13.8, 8.0 Hz, 1H), 4.36 (d, J = 6.9 Hz, 2H), 4.19 (t, J = 6.7 Hz, 1H), (m, 3H), 1.35 (s, 9H), 0.94 (d, J = 6.3 Hz, 6H); 13 C NMR (100 MHz, CDCl 3) δ 171.1, 169.3, 155.7, 143.9, 143.8, 141.3, 137.9, 129.1, 128.6, 127.7, 127.3, 127.1, 125.2, 112.0, 82.0, 67.2, 58.8, 52.0, 47.2, 41.8, 27.9, 24.9, 22.7, 22.2 ppm; HRMS m/z (ESI) calcd for C 33H 38N 2NaO 5 (M+Na) + : , found (N-methylmethylsulfonamido)vinyl (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2- phenylacetate White solid, 95% yield (96.2 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.76 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.3 Hz, 2H), (m, 7H), 7.30 (t, J = 6.9 Hz, 2H), 5.86 (d, J = 6.7 Hz, 1H), 5.44 (d, J = 6.7 Hz, 1H), 5.09 (s, 1H), 4.88 (s, 1H), 4.42 (d, J = 6.5 Hz, 2H), 4.21 (t, J = 6.7 Hz, 1H), 2.89 (s, 3H), 2.72 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 168.7, 155.4, 145.2, 143.6, 143.5, 141.2, 135.1, 129.2, 127.7, 127.4, 127.0, 124.9, 119.9, 101.5, 67.3, 58.2, 47.0, 37.6, 35.3 ppm; HRMS m/z (ESI) calcd for C 27H 26N 2NaO 6S (M+Na) + : , found S26

27 1:1 mixture of Fmoc-L-Phg -L-Leu-OtBu and Fmoc-D-Phg -L-Leu-OtBu Peak Info Peak No % Area Area RT (min) Height (mav) Total: HBTU: mixture of Fmoc-L-Phg -L-Leu-OtBu (major) and Fmoc-D-Phg -L-Leu-OtBu (minor) Peak Info Peak No % Area Area RT (min) Height (mav) Total: S27

28 HATU: mixture of Fmoc-L-Phg -L-Leu-OtBu (major) and Fmoc-D-Phg -L-Leu-OtBu (minor) Peak Info Peak No % Area Area RT (min) Height (mav) Total: PyBop: mixture of Fmoc-L-Phg -L-Leu-OtBu (major) and Fmoc-D-Phg -L-Leu-OtBu (minor) Peak Info Peak No % Area Area RT (min) Height (mav) Total: S28

29 DEPBT: mixture of Fmoc-L-Phg -L-Leu-OtBu (major) and Fmoc-D-Phg -L-Leu-OtBu (minor) Peak Info Peak No % Area Area RT (min) Height (mav) Total: MYMsA: mixture of Fmoc-L-Phg -L-Leu-OtBu (major) and Fmoc-D-Phg -L-Leu-OtBu (minor) Peak Info Peak No % Area Area RT (min) Height (mav) Total: S29

30 MYTsA: mixture of Fmoc-L-Phg -L-Leu-OtBu (major) and Fmoc-D-Phg -L-Leu-OtBu (minor) Peak Info Peak No % Area Area RT (min) Height (mav) Total: Study on the racemization of phenylglycine during hydroacyloxylation with MYMsA Racemate: Peak Info Peak No % Area Area RT (min) Height (mav) Total: S30

31 Peak Info Peak No % Area Area RT (min) Height (mav) Total: S31

32 Ynamide MYTsA Mediated Peptide Bond Formation A 5 ml round-bottomed flask was charged with N-methyl ynetoluenesulfonamide (MYTsA) (0.2 mmol), CH 2Cl 2 (1 ml) and acids (0.2 mmol). The reaction mixture was stirred at room temperature under air until starting material was fully consumed. Amine (0.24 mmol) was added and the reaction mixture was stirred at 35 under air until α-acyloxyenamide was fully consumed. The reaction mixture was concentrated and purified by silica gel chromatography to afford the respective peptide. Larger-scale reactions Larger-scale reactions (10 & 20 mmol scale) to provide 5 or 11 gram dipeptide were carried out with Fmoc-L-Ser(OtBu)-L-Leu-(OtBu) as the model substrate. As shown below, excellent efficiency were retained for 10 & 20 mmol reactions. S32

33 Copies of HPLC Chromatograms 1:1 mixture of Fmoc-L-Ser(tBu)-L-Leu-OtBu and Fmoc-D-Ser(tBu)-L-Leu-OtBu Peak Info Peak No % Area Area RT (min) Height (mav) Total: MYTsA: mixture of Fmoc-D-Ser(tBu)-L-Leu-OtBu (minor) and Fmoc-L-Ser(tBu)-L-Leu- OtBu(major)) (0.2 mmol scale) Peak Info Peak No % Area Area RT (min) Height (mav) Total: S33

34 MYTsA: mixture of Fmoc-D-Ser(tBu)-L-Leu-OtBu (minor) and Fmoc-L-Ser(tBu)-L-Leu- OtBu(major)) (10 mmol scale) Peak Info Peak No % Area Area RT (min) Height (mav) Total: MYTsA: mixture of Fmoc-D-Ser(tBu)-L-Leu-OtBu (minor) and Fmoc-L-Ser(tBu)-L-Leu- OtBu(major)) (20 mmol scale) Peak Info Peak No % Area Area RT (min) Height (mav) Total: S34

35 Fmoc-L-Ser(tBu)-L-Leu-OtBu (13a) White solid, 99% yield (109.3 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.75 (d, J = 7.5 Hz, 2H), (m, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 7.25 (d, J = 8.8 Hz, 1H), 5.81 (d, 1H), 4.50 (d, J = 4.9 Hz, 1H), (m, 2H), (m, 2H), 3.84 (dd, J = 8.4, 3.7 Hz, 1H), 3.41 (t, J = 8.2 Hz, 1H), (m, 2H), (m, 1H), 1.46 (s, 9H), 1.23 (s, 9H), 0.96 (d, J = 6.3 Hz, 6H); 13 C NMR (100 MHz, CDCl 3) δ 171.5, 169.8, 155.9, 143.7, 141.2, 127.6, 127.0, 125.0, 119.9, 81.6, 74.1, 67.0, 61.7, 54.2, 51.6, 47.1, 41.8, 27.9, 27.3, 24.8, 22.7, 22.1 ppm; HRMS m/z (ESI) calcd for C 32H 44N 2NaO 6 (M+Na) + : , found Fmoc-L-Asp-L-Tyr(tBu)-OtBu (13b) White solid, 97% yield (131.7 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.76 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.3 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.2 Hz, 2H), 7.07 (t, J = 8.3 Hz, 3H), 6.89 (d, J = 8.3 Hz, 2H), 5.99 (d, J = 8.1 Hz, 1H), 4.66 (dd, J = 13.4, 6.3 Hz, 1H), (m, 1H), 4.38 (p, J = 10.4 Hz, 2H), 4.23 (t, J = 7.1 Hz, 1H), (m, 2H), 2.90 (dd, J = 17.0, 3.9 Hz, 1H), 2.63 (dd, J = 16.7, 5.9 Hz, 1H), 1.45 (s, 9H), 1.36 (s, 9H), 1.29 (s, 9H); 13 C NMR (100 MHz, CDCl 3) δ 171.0, 169.9, 169.9, 155.9, 154.3, 143.6, 141.2, 130.9, 129.9, 127.7, 127.0, 125.0, 124.0, 119.9, 82.2, 81.8, 78.2, 67.3, 54.0, 51.0, 47.0, 37.4, 37.4, 28.7, 28.0, 27.8 ppm; HRMS m/z (ESI) calcd for C 40H 50N 2NaO 8 (M+Na) + : , found Fmoc-L-Trp(Boc)-L-Tyr(tBu)-OtBu (13c) Glassy solid, 96% yield (153.8 mg). 1 H NMR (400 MHz, CDCl 3) δ 8.14 (d, J = 7.3 Hz, 1H), 7.75 (d, J = 7.5 Hz, 2H), 7.62 (d, J = 7.0 Hz, 1H), (m, 3H), (m, 5H), (m, 1H), 6.85 (d, J = 7.3 Hz, 2H), 6.77 (d, J = 8.3 Hz, 2H), 6.36 (d, J = 6.2 Hz, 1H), 5.51 (d, J = 4.9 Hz, 1H), 4.55 (dd, J = 12.8, 6.0 Hz, 2H), (m, 1H), (m, 1H), 4.20 (t, J = 6.7 Hz, 1H), (m, 1H), 3.14 (dd, J = 14.2, 7.0 Hz, 1H), 2.94 (d, J = 5.4 Hz, 2H), 1.60 (s, 9H), 1.30 (s, 9H), 1.26 (s, 9H); 13 C NMR (100 MHz, CDCl 3) δ 170.1, 169.7, 155.9, 154.3, 149.4, 143.7, 143.7, 141.2, 135.5, 130.7, 130.2, 129.8, 127.7, 127.1, 125.1, 124.7, 124.6, 123.9, 122.8, 119.9, 118.9, 115.4, 115.1, 83.6, 82.3, 78.3, 67.3, 55.0, 53.9, 47.0, 37.3, 28.8, 28.2, 28.1, 27.8 ppm; HRMS m/z (ESI) calcd for C 48H 55N 3NaO 8 (M+Na) + : , found Fmoc-L-Ser-L-Tyr(tBu)-OtBu (13d) Glassy solid, 98% yield (118.0 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.76 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.3 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), (m, 2H), 7.04 (d, J = 8.4 Hz, 2H), 6.95 (s, 1H), 6.88 (d, J = 8.4 Hz, 2H), 5.84 (s, 1H), 4.70 (dd, J = 14.1, 6.4 Hz, 1H), (m, 2H), 4.22 (t, J = 7.0 Hz, 2H), 4.01 (d, J = 10.1 Hz, 1H), 3.64 (d, J = 5.0 Hz, 1H), 3.29 (s, 1H), 3.03 (qd, J = 14.1, 6.4 Hz, 2H), 1.39 (s, 9H), 1.29 (s, 9H); 13 C NMR (100 MHz, CDCl 3) δ 170.6, 170.4, 156.4, 154.4, 143.7, 143.6, 141.3, 130.7, 129.8, 127.7, S35

36 127.1, 125.1, 124.1, 120.0, 82.7, 78.4, 67.3, 62.9, 55.3, 54.0, 47.0, 37.1, 28.8, 27.9 ppm; HRMS m/z (ESI) calcd for C 35H 42N 2NaO 7 (M+Na) + : , found Cbz-L-Ser-L-Tyr(tBu)-OtBu (13e) Glassy solid, 98% yield (100.7 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 5H), 7.04 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 7.3 Hz, 1H), (m, 2H), 5.79 (d, J = 7.3 Hz, 1H), (m, 2H), 4.69 (dd, J = 14.2, 6.4 Hz, 1H), 4.23 (s, 1H), (m, 1H), 3.61 (dt, J = 11.5, 6.9 Hz, 1H), 3.24 (s, 1H), 3.02 (qd, J = 14.0, 6.4 Hz, 2H), 1.39 (s, 9H), 1.31 (s, 9H); 13 C NMR (100 MHz, CDCl 3) δ 170.6, 170.5, 156.4, 154.4, 136.1, 130.8, 129.9, 128.6, 128.3, 128.1, 124.2, 82.7, 78.5, 67.3, 62.9, 55.4, 54.0, 37.2, 28.8, 28.0 ppm; HRMS m/z (ESI) calcd for C 28H 38N 2NaO 7 (M+Na) + : , found Boc-L-Thr-L-Tyr(tBu)-OtBu (13f) Glassy solid, 96% yield (94.8 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.05 (d, J = 8.4 Hz, 3H), 6.88 (d, J = 8.4 Hz, 2H), 5.45 (d, J = 7.5 Hz, 1H), 4.67 (dd, J = 13.7, 6.5 Hz, 1H), 4.28 (d, J = 4.7 Hz, 1H), 4.07 (d, J = 6.9 Hz, 1H), 2.99 (t, J = 6.1 Hz, 2H), 1.43 (s, 9H), 1.36 (s, 9H), 1.30 (s, 9H), 1.13 (d, J = 6.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ 170.8, 170.3, 156.2, 154.3, 130.9, 129.8, 124.0, 82.3, 80.2, 78.3, 66.9, 58.3, 53.8, 37.5, 28.8, 28.2, 27.9, 18.2 ppm; HRMS m/z (ESI) calcd for C 26H 42N 2NaO 7 (M+Na) + : , found Fmoc-L-Thr-L-Thr-OtBu (13g) Glassy solid, 97% yield (96.6 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.73 (d, J = 7.5 Hz, 2H), 7.57 (d, J = 6.6 Hz, 2H), 7.36 (t, J = 7.5 Hz, 3H), 7.27 (t, J = 7.5 Hz, 2H), 6.15 (s, 1H), (m, 2H), (m, 4H), 4.19 (t, J = 7.0 Hz, 1H), 1.45 (s, 9H), 1.19 (d, J = 6.4 Hz, 6H); 13 C NMR (100 MHz, CDCl 3) δ 171.2, 169.9, 156.7, 143.6, 141.2, 127.7, 127.0, 125.0, 119.9, 82.7, 68.1, 67.4, 67.3, 59.0, 58.5, 47.0, 27.9, 20.1, 18.0 ppm; HRMS m/z (ESI) calcd for C 27H 34N 2NaO 7 (M+Na) + : , found Cbz-L-Thr-L-Thr-OtBu (13h) Glassy solid, 98% yield (80.4 mg). 1 H NMR (400 MHz, CDCl 3) δ (m, 5H), 6.09 (s, 1H), 5.09 (s, 2H), 4.43 (dd, J = 8.9, 3.2 Hz, 1H), (m, 3H), 3.99 (s, 1H), 3.50 (s, 1H), 2.59 (s, 1H), 1.45 (s, 9H), (m, 6H); 13 C NMR (100 MHz, CDCl 3) δ 171.2, 169.9, 156.7, 136.1, 128.5, 128.1, 127.9, 82.7, 68.1, 67.3, 67.2, 59.1, 58.4, 27.9, 20.0, 18.1ppm; HRMS m/z (ESI) calcd for C 20H 30N 2NaO 7 (M+Na) + : , found Boc-L-Thr-L-Thr-OtBu (13i) Glassy solid, 97% yield (72.2 mg). 1 H NMR (400 MHz, CDCl 3) δ 7.35 (d, J = 7.7 Hz, 1H), 5.73 (s, 1H), 4.42 (dd, J = 8.7, 2.4 Hz, 1H), (m, 3H), 1.43 (d, J = 10.9 Hz, 18H), 1.18 (t, J = 6.2 Hz, 6H); 13 C NMR (100 MHz, CDCl 3) δ 171.7, 169.8, 156.2, 82.6, 80.3, 68.2, 67.3, 58.7, 58.4, 28.2, 27.9, 20.0, 18.2 ppm; HRMS m/z (ESI) calcd for C 17H 32N 2NaO 7 (M+Na) + : , found Fmoc-L-Trp-L-Thr-OtBu (13j) S36