Comparison of diffusion coefficients for matched pairs of macrocyclic and linear molecules

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry Supporting Information for: Comparison of diffusion coefficients for matched pairs of macrocyclic and linear molecules over a drug-like molecular weight range Andrew R. Bogdan, ichola L. Davies, and Keith James, * Department of Chemistry, The Scripps Research Institute, 10550. Torrey Pines Rd., La Jolla, CA 92037; Pfizer armatherapeutics Research, Sandwich, Kent CT13 9J, UK. kjames@scripps.edu Table of Contents Page General Considerations S-2 MR Diffusion Coefficient Determination S-2 LC/MS Analysis S-4 Copper Reactor Diskette S-4 Synthesis of Azido Linkers S-5 Synthesis of Macrocycle Precursors S-6 Synthesis of Macrocycles S-28 Synthesis of Acyclic Analog Precursors S-35 Synthesis of Acyclic Analogs S-45 MR Spectra S-54 Example of arrayed MR spectra used to calculate diffusion coefficients S-70 Example DSY Report S-70 References S-77

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry General Considerations. All reagents and solvents were used as received. THF was distilled from sodium. MR spectra were recorded on Bruker DRX-600, Bruker DRX-500, or Bruker AMX-400 instruments using residual solvent peak as a reference. Data are reported as s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. LC/MS analyses were carried out using an Agilent Technologies HPLC (Agilent Technologies 1100 Series diode array detector, Agilent Technologies 1100 Series column heater, Agilent 1100 Series pump, and Agilent 1100 Series degasser) interfaced with an Agilent Technologies 6110 Quadrupole LC/MS. Column chromatography was performed using a Biotage Horizon automated flash chromatography system equipped with a Biotage Horizon detector, fraction collector and pump where noted. MR Diffusion Coefficient Determination. All the MR experiments were acquired on a Bruker Avance 600 MHz spectrometer, equipped with a 5 mm TCI cryoprobe with z-gradients capable of generating 54 G/cm field strengths. The temperature controller was set to 298K with an air flow of 535 l/h in order to avoid any temperature fluctuations due to sample heating during acquisition and to avoid sample vibrations from a high air-flow. Samples were made up to 10mM solutions in DMS-d 6 or CDCl3 with some TMS vapour, and 180uL of this solution was added to a 3 mm MR tube to avoid problems of convection. In the case of samples in 10% DMS in D 2, samples were made up at 2.5 mm and 600 ul of this solution was added to a 5mm MR tube. The lower concentration was required due to the limited solubility in this solvent. To maintain good signal to noise, the use of 5mm MR tubes is possible for more viscous solvents, as the onset of Rayleigh-Benard convection is ablated at the temperatures used during this investigation. S-2

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry Diffusion coefficients were determined with a high degree of reproducibility on the MR system used. Using robust statistical analysis, we have determined that the relative differences in diffusion between the linear and macrocyclic analogues are significant. However, the absolute accuracy of the diffusion coefficient as measured by MR is limited by the accuracy of the calibration of the gradient and temperature, and often prone to errors introduced during the calculation method. Steps were taken to minimise these errors as detailed, however, it would be difficult to accurately compare diffusion coefficients between different MR systems. The gradient strength was calibrated using the diffusion coefficient of water in a standard solution of 0.1 mg/ml GdCl 3, 0.1% DSS, 1% H 2 in D 2. The values of the measured diffusion coefficient (D) of water, the known diffusion coefficient of water and the current gradient calibration value (gc(old)) were used to obtain the new gradient calibration value (gc(new)) using the following equation: 1 gc(new) = gc(old) x sqrt.( D(measured)/ D(known)) The temperature was calibrated with a sample of methanol-d4 (99.8 at%), sealed under atmospheric pressure, using details described elsewhere. 2 All DSY experiments used the ledbpgp2s sequence (available in standard Bruker pulse sequence library). A gradient duration (d) of 2msec and an eddy current delay of 5 ms was used in all cases. The diffusion time (D) was 100 ms in the case of CDCl 3, and 200 ms in the case of d 6 -DMS and 10% d 6 -DMS in D 2. In each PFG MR experiment, a series of 16-32 spectra on 16K data points were collected, using a linear gradient ramp from 5-95% of the maximum gradient strength. After acquisition, the data was zero filled to 32k, Fourier transformed and baseline corrected in f2. The diffusion coefficients were calculated with the T1/T2 relaxation module using mono- S-3

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry exponential fitting, rather than the 2D processing protocol. This is available in Bruker Topspin v.2. For each sample, several well resolved signals were used to extract individual diffusion coefficients (an example of the raw data and the fitting report is included in this supporting information). These signals have been averaged for each run to give the diffusion coefficient and a standard deviation. Statistical analysis confirms that the variation in diffusion coefficient within each run is the same as the comparison between runs. The analysis also demonstrates that a difference in diffusion coefficients between macrocycles and linear controls of 1% would be statistically significant. The observed difference of 5% is highly statistically significant. TMS was included in the CDCl 3 and DMS datasets, as a control to check temperature was consistent in all experiments. As can be seen in the table of diffusion data, this is very reproducible and allows confidence in the comparison of data between macrocycles and linear analogues. LC/MS Analysis. HPLC analyses was performed using a water (formic acid 0.1% w/v / ammonium formate 0.05% w/v) and C (water 5% v/v, formic acid 0.1% v/v, ammonium formate 0.05% w/v) based gradient from 0-100% C over 4 minutes. A Waters XBridge C18 2.5 µm (3.0 x 30 mm) column was used at 80 C with a flow rate of 2.4 ml min -1. Injections were made from diluted reaction mixtures and ionization monitored in positive or negative mode. Copper Reactor Diskette. The copper tubing used in the Conjure flow reactor comes in the form of a reactor diskette. Copper tubing (0.75 mm inner diameter, 3.0 m length, 1500 µl internal volume) is housed between two metal plates (Figure 1a). Figure 1b shows the fully S-4

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry assembled reactor diskette (145 mm x 165 mm x 5 mm). Figure S1. The copper reactor diskette. Synthesis of Azido Linkers. 4-azidobutyric acid and 6-azidohexanoic acid were synthesized according to literature precedence. [3] 3 H 3 Cl 9. CAUTI: Extreme care should be taken with preparing low molecular weight organic azides. Bis(2-bromoethyl) ether (11.5 g, 50.0 mmol, 1.0 eq) and a 3 (8.13 g, 125.0 mmol, 2.5 eq) were suspended in DMF at 55 C until the reaction was deemed complete by TLC (~3 hrs). DI H 2 (50 ml) was added to the reaction flask, and the solution was transferred to a separatory funnel. The aqueous phase was extracted with Et 2 (3x 100 ml), the organic extracts were washed with brine, dried over a 2 S 4 and filtered. The solvent was removed under a stream of S-5

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 2 until the total volume was ~50 ml. Aqueous HCl (2.5 M, 45 ml, 112.5 mmol, 2.25 eq) was added to the organic phase and stirred at 0 C. P 3 (12.5 g, 47.6 mmol, 0.95 eq) in EtAc (50 ml) was added to the ether/hcl mixture drop wise over 2 hours at 0 C, and the reaction was allowed to warm to room temperature and stir overnight. Excess Et 2 (50 ml) was added to the reaction mixture and the phases were allowed to separate. The organic phase was extracted with aqueous HCl (1 M, 50 ml). The combined aqueous extracts were washed with Et 2 (2x 50 ml), concentrated at 60 C under a stream of 2, and dried under vacuum to yield 9 as a light brown solid (7.6 g, 96% yield): 1 H MR (600 MHz, CD 3 D): δ 3.76 (d, J = 4.8 Hz, 2 H), 3.71 (t, J = 4.2 Hz, 2 H), 3.46 (t, J = 5.4 Hz, 2 H), 3.15 (t, J = 4.8 Hz, 2 H); 13 C MR (150 MHz, CD 3 D): δ 71.0, 67.8, 51.6, 40.6. Synthesis of Macrocycle Precursors. H H 3 10: General Procedure A. (1R,2S)-(-)-norephedrine (1.0 g, 6.6 mmol, 1.0 eq) and PyBP (3.43 g, 6.6 mmol, 1.0 eq) were dissolved in dry CH 2 Cl 2 (50 ml) under Ar. Diisopropylethylamine (3.43 ml, 19.8 mmol, 3.0 eq) was added to the reaction mixture and the reaction was cooled to 0 C using an ice bath. 4-azidobutyric acid (940.0 mg, 7.3 mmol, 1.1 eq) was dissolved in CH 2 Cl 2 (5 ml) and slowly added to the cooled reaction mixture. The reaction was allowed to warm to room temperature and continued to stir overnight. S-6

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry To the reaction mixture was added DI H 2 (50 ml). The mixture was stirred vigorously then transferred to a separatory funnel. The aqueous phase was extracted with CH 2 Cl 2 (3x 25 ml) and the combined organic extracts were dried over a 2 S 4, filtered and concentrated in vacuo. The residue was dissolved in Et 2 and the solids (PyBP impurities) were filtered off. The solution was concentrated in vacuo and the residue purified by column chromatography (silica gel, 1:2 hexanes/etac, R f = 0.33) to yield 10 as a yellow oil (1.06 g, 63% yield): 1 H MR (500 MHz, CDCl 3 ): δ 7.28-7.40 (m, 5 H), 5.60 (d, J = 7.3 Hz, 1 H), 4.87 (t, J = 3.3 Hz, 1 H), 4.35 (m, 1 H), 3.37 (t, J = 6.4 Hz, 2 H), 3.29 (d, J = 3.3 Hz, 1 H), 2.26-2.33 (m, 2 H), 1.88-2.01 (m, 2 H), 1.04 (d, J = 7.0 Hz, 3 H); 13 C MR (125 MHz, CDCl 3 ): δ 172.3, 140.5, 128.2, 127.7, 126.3, 66.2, 51.0, 50.7, 33.2, 24.8, 14.8. m/z = 263.1 [M+H] +. H 3 11: General Procedure B. 10 (1.06 g, 4.04 mmol, 1.0 eq) was dissolved in dry THF (40 ml) at room temperature. ah (dry, 120 mg, 4.8 mmol, 1.2 eq) was added to the reaction mixture and allowed to stir until gas evolution ceased. Propargyl bromide (80% w/w in toluene, 520 µl, 4.8 mmol, 1.2 eq) was added drop wise and allowed to stir overnight. The reaction was quenched carefully with DI H 2 and concentrated. The resulting residue was purified using column chromatography (silica gel, 1:1 hexanes/etac, R f = 0.45) to yield 11 as a yellow oil (1.00 g, 83% yield): 1 H MR (600 MHz, CDCl 3 ): δ 7.28-7.42 (m, 5 H), 5.90 (d, J=7.5 Hz, 1 H), 4.72 (br. s., 1 H), 4.30 (d, J = 15.8 Hz, 1 H), 4.24 (br. s., 1 H), 3.95 (d, J = 16.2 Hz, 1 H), 3.35 (t, J = 6.6 Hz, 2 H), 2.45 (s, 1 H), 2.29 (t, J = 7.2 Hz, 2 H), 1.94 (m, 2 S-7

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry H), 1.00 (d, J = 7.0 Hz, 3 H); 13 C MR (150 MHz, CDCl 3 ): δ 170.8, 137.6, 128.5, 127.9, 126.8, 81.9, 79.7, 74.6, 56.5, 50.7, 49.6, 33.4, 24.8, 13.7. m/z = 301.1 [M+H] +. H 3 12. Prepared according General Procedure A using (1R,2S)-(-)-ephedrine (495.0 mg, 3.0 mmol, 1.0 eq), PyBP (1.56 g, 3.0 mmol, 1.0 eq), diisopropylethylamine (1.6 ml, 9.2 mmol, 3.0 eq) and 6-azidohexanoic acid (520.0 mg, 3.3 mmol, 1.1 eq) in dry CH 2 Cl 2 (75 ml). The crude reaction mixture was purified using column chromatography (silica gel, 1:1 hexanes/etac, R f = 0.31) to yield 12 as a colorless oil (850.0 mg, 93% yield): 1 H MR (4.1:1 rotamer ratio, asterisks denote minor rotamer peaks, 500 MHz, CDCl 3 ): δ 7.28-7.34 (m, 5 H), 4.80 (t, 1 H), 4.60* (t, 1 H), 4.49 (m, 1 H), 4.03 (s, 1 H), 3.93* (m, 1 H), 3.24 (t, J = 7.0 Hz, 2 H), 3.17* (t, J = 7.0 Hz, 2 H), 2.79* (s, 3 H), 2.67 (s, 3 H), 1.56 (m, 4 H), 1.34 (m, 2 H), 3.17 (d, J = 7.5 Hz, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 125 MHz, CDCl 3 ): δ 174.5, 172.5*, 142.4*, 142.0, 128.7*, 128.3, 126.5, 126.2*, 77.5, 76.0*, 58.3, 57.9*, 51.4, 34.1, 33.1, 32.7*, 28.9, 26.6, 24.6, 15.2*, 12.4. m/z = 305.2 [M+H] +. 3 13. Prepared according General Procedure B using 12 (425.0 mg, 1.4 mmol, 1.0 eq), ah (60% w/w dispersion in mineral oil, 84 mg, 2.1 mmol, 1.5 eq), propargyl bromide (80% w/w in S-8

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry toluene, 230 µl, 2.1 mmol, 1.5 eq) in dry THF (25 ml). The resulting residue was purified using column chromatography (silica gel, 4:1 hexanes/etac, R f = 0.20) to yield 13 as a yellow oil (420.0 mg, 88% yield): 1 H MR (2:1 rotamer ratio, asterisks denote minor rotamer peaks, 500 MHz, CDCl 3 ): δ 7.25-7.32 (m, 5 H), 4.70 (m, 1 H), 4.65 (d, J = 5.5 Hz, 1 H), 4.46* (d, J = 8.0 Hz, 1 H), 4.14 (m, 1 H), 4.00* (m, 1 H), 3.82 (m, 1 H), 3.24 (m, 2 H), 2.86 (s, 3 H), 2.81* (s, 3 H), 2.41* (t, J = 2.5 Hz, 1 H), 2.36 (t, J = 2.5 Hz, 1 H), 2.15 (m, 2 H), 1.47-1.58 (m, 4 H), 1.36* (d, J = 6.0 Hz, 3 H), 1.29 (m, 2 H), 1.18 (d, J = 6.5 Hz, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 125 MHz, CDCl 3 ): δ 172.8, 138.7, 128.9*, 128.5, 128.1, 127.4, 127.2*, 83.2, 82.2*, 80.1, 79.5*, 77.4, 74.9*, 74.3, 57.5*, 56.3, 51.5, 33.9, 32.7, 29.0, 28.8*, 26.7, 24.7*, 24.6, 15.4, 12.1. m/z = 343.2 [M+H] +. Boc 14. Prepared according General Procedure B using tert-butyl 3-hydroxy-4-phenylpyrrolidine-1- carboxylate (racemic, 14.53 g, 55.2 mmol, 1.0 eq), ah (dry, 1.60 g, 66.7 mmol, 1.2 eq), propargyl bromide (80% w/w in toluene, 8.8 ml, 81.8 mmol, 1.5 eq) in dry THF (400 ml). The resulting residue was purified using column chromatography (silica gel, 33-100% EtAc in hexanes gradient, R f = 0.50 in 3:1 hexanes/etac) to yield racemic 14 as a brown oil (16.5 g, 99% yield). The racemic mixture was resolved by preparative chiral SFC using a Chiralpak AD- H column (4.6 x 250 mm) using a 10% H in C 2 solvent system (3.0 ml/min, 140 bar). Two peaks were collected into separate fractions, yielding 14 and the (3R,4S) enantiomer in a 1:1 ratio: 1 H MR (600 MHz, CDCl 3 ): δ 7.30 (m, 2 H), 7.22 (m, 3 H), 4.19 (m, 1 H), 4.10 (m, 2 H), 3.63-3.82 (m, 2 H), 3.46-3.57 (m, 1 H), 3.34-3.43 (m, 2 H), 2.40 (s, 1 H), 1.46 (m, 9 H); 13 C S-9

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry MR (asterisks denote minor rotamer peaks, 150 MHz, CDCl 3 ): δ 154.5, 140.0, 139.9*, 128.9, 127.35, 127.34*, 127.25, 127.20*, 83.2*, 82.3, 79.7, 79.4, 75.04, 75.00*, 57.1, 57.0*, 50.4, 50.2*, 49.63, 49.61*, 49.5, 48.5*, 28.6. m/z = 245.9 [M+H, - tbu] +. 15: General Procedure C. 14 (516.6 mg, 1.7 mmol, 1.0 eq) was dissolved in 4.0 M HCl in 1,4- dioxane (3.0 ml, 12.0 mmol, 7.0 eq) and stirred at room temperature until the deprotection was complete by TLC. The solvent was removed under a stream of 2 and the residue was dried under vacuum. The residue was dissolved in CH 2 Cl 2 (20 ml), and PyBP (890 mg, 1.71 mmol, 1.0 eq), diisopropylethylamine (1.48 ml, 8.5 mmol, 5.0 eq) and (R)-(+)-3-methylsuccinic acid 1- monomethyl ester (275 mg, 1.88 mmol, 1.1 eq) were added and the reaction allowed to stir overnight at room temperature. To the reaction mixture was added DI H 2 (50 ml). The mixture was stirred vigorously then transferred to a separatory funnel. The aqueous phase was extracted with CH 2 Cl 2 (3x 25 ml) and the combined organic extracts were dried over a 2 S 4, filtered and concentrated in vacuo. The residue was purified using a Biotage Horizon automated flash column chromatography system (silica gel, 10-100% EtAc in hexanes gradient, R f = 0.20 in 1:1 hexanes/etac) to yield 15 as a light brown oil (475.1 mg, 81% yield): 1 H MR (500 MHz, CDCl 3 ): δ 7.19-7.31 (m, 5 H), 4.12-4.29 (m, 3 H), 3.87-3.96 (m, 2 H), 3.78 (m, 1 H), 3.70 (m, 1 H), 3.63 (s, 3 H), 3.55 (m, 1 H), 2.95-3.08 (m, 1 H), 2.83-2.93 (m, 1 H), 2.40-2.42 (m, 1 H), 2.34 (m, 1 H), 1.15 (d, J = 7.0 Hz, 3 H); 13 C MR (asterisks denote minor rotamer peaks, S-10

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 125 MHz, CDCl 3 ): δ 174.3, 174.2*, 173.1, 173.0*, 139.6, 129.1, 129.0*, 127.5*, 127.41, 127.39*, 127.3, 83.8*, 82.0, 79.5*, 79.4, 75.2*, 75.1, 57.2*, 57.1, 51.9*, 51.8, 50.45, 50.42*, 49.8*, 49.6, 49.4*, 47.8, 37.91*, 37.88, 34.3*, 34.1, 17.5, 17.3*. m/z = 330.2 [M+H] +. H 16: General Procedure D. 15 (475.1 mg, 1.44 mmol, 1.0 eq) was dissolved in THF/H (25 ml, 3:2 v/v). LiH monohydrate (242 mg, 5.8 mg, 4.0 eq) was dissolved in DI H 2 (5 ml) and added drop wise to the solution of 15 in THF/H and allowed to stir overnight at RT. The reaction was concentrated and the resultant residue was acidified using 5 M HCl. The residue was partitioned between DI H 2 and CH 2 Cl 2, and the aqueous phase extracted with CH 2 Cl 2 (2x 25 ml). The organic phase was dried over a 2 S 4, concentrated and dried in vacuo to yield 16 as a light brown oil (454 mg, >99% yield): 1 H MR (500 MHz, CDCl 3 ): δ 7.29-7.32 (m, 2 H), 7.18-7.25 (m, 3 H), 4.14-4.30 (m, 3 H), 3.88-3.97 (m, 2 H), 3.66-3.81 (m, 2 H), 3.58 (m, 1 H), 2.95-3.09 (m, 1 H), 2.79-2.94 (m, 1 H), 2.40-2.54 (m, 2 H), 1.20 (d, J = 7.0 Hz, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 125 MHz, CDCl 3 ): δ 176.5, 176.5*, 175.1*, 175.0, 139.4, 139.3*, 129.2, 129.1*, 127.6, 127.5*, 127.28, 127.25*, 83.2*, 82.1, 79.4, 79.3*, 75.4*, 75.3, 57.2*, 57.1, 50.6*, 50.5, 49.8*, 49.7, 49.6, 47.6*, 38.13*, 38.06, 34.3*, 34.1, 17.0, 16.8*. m/z = 316.2 [M+H] +. S-11

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry H 3 17. Prepared according General Procedure A using 9 (100.0 mg, 0.60 mmol, 1.1 eq), PyBP (170 mg, 0.54 mmol, 1.0 eq), diisopropylethylamine (470 µl, 2.69 mmol, 5.0 eq) and 16 (170.0 mg, 0.54 mmol, 1.0 eq) in dry CH 2 Cl 2 (30 ml). The residue was purified (without filtering off PyBP impurities) using a Biotage Horizon automated flash column chromatography system (silica gel, 20-100% EtAc in hexanes gradient, R f = 0.41 in EtAc) to yield 17 as a light yellow oil (223.2 mg, 97% yield): 1 H MR (600 MHz, CD 3 D): δ 7.24-7.35 (m, 5 H), 4.26-4.39 (m, 1 H), 4.15-4.25 (m, 2 H), 3.82-4.04 (m, 2 H), 3.68-3.78 (m, 1 H), 3.51-3.68 (m, 5 H), 3.32-3.51 (m, 5 H), 3.08-3.20 (m, 1 H), 2.88 (m, 1 H), 2.59-2.66 (m, 1 H), 2.28-2.32 (m, 1 H), 1.20 (d, J = 4.2 Hz, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 150 MHz, CD 3 D): δ 176.9, 176.8*, 174.14, 174.07*, 140.9*, 140.8, 129.90*, 129.89*, 128.5, 128.3, 128.2*, 84.1*, 83.0, 80.47*, 80.46, 76.34*, 76.30, 70.93*, 70.92, 70.44*, 70.43, 57.76*, 57.75, 51.74*, 51.72, 51.64*, 51.52, 51.0, 50.6*, 50.4, 40.56, 40.55*, 40.36*, 40.33, 35.8*, 35.5, 17.4, 17.2*. m/z = 428.3 [M+H] +. H S-12

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 18. Prepared according General Procedure A using (1R,2S)-(-)-ephedrine (825 mg, 5.0 mmol, 1.0 eq), PyBP (2.6 g, 5.0 mmol, 1.0 eq), diisopropylethylamine (2.6 ml, 15.0 mmol, 3.0 eq) and 3-(4-thoxycarbonyl)propionic acid methyl ester (1.14 g, 5.5 mmol, 1.1 eq) in dry CH 2 Cl 2 (40 ml). The crude reaction mixture was purified using a Biotage Horizon automated flash column chromatography system (silica gel, 10-100% EtAc in hexanes gradient, R f = 0.16 in 1:1 hexanes/etac) to yield 18 as a colorless oil (1.07 g, 60% yield): 1 H MR (3.2:1 rotamer ratio, asterisks denote minor rotamer peaks, 500 MHz, CDCl 3 ) δ 7.93 (d, J = 9.5 Hz, 2 H), 7.87* (d, J = 8.0 Hz), 7.21-7.32 (m, 7 H), 7.05* (d, J = 8.0 Hz, 2 H), 4.81 (m, 1 H), 4.58* (m, 1 H), 4.49 (m, 1 H), 3.82-3.92 (m, 4 H), 2.94 (t, J = 7.5 Hz, 2 H), 2.79* (s, 3 H), 2.64 (s, 3 H), 2.52 (t, J = 8.5 Hz, 2 H), 1.29* (d, J = 6.0 Hz, 3 H), 1.16 (d, J = 7.5 Hz, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 125 MHz, CDCl 3 ) δ 173.5, 171.9*, 167.2, 147.2*, 147.0, 142.3*, 142.0, 130.0, 129.9*, 128.7, 128.6*, 128.5, 128.4*, 128.36, 128.32*, 128.1*, 127.7, 126.4, 126.2*, 77.4, 75.9*, 58.3, 58.0*, 52.20, 52.16*, 35.6, 34.2*, 33.1, 31.28*, 31.25, 28.5*, 15.3*, 12.3. m/z = 356.0 [M+H] +. 19. Prepared according General Procedure B using 18 (1.07 g, 3.01 mmol, 1.0 eq), ah (dry, 90 mg, 3.75 mmol, 1.3 eq), propargyl bromide (80% w/w in toluene, 480 µl, 4.5 mmol, 1.5 eq) in dry THF (50 ml). The crude reaction mixture was purified using a Biotage Horizon automated flash column chromatography system (silica gel, 8-100% EtAc in hexanes gradient, R f = 0.33 S-13

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry in 2:1 hexanes/etac) to yield 19 as a colorless oil (900 mg, 76% yield): 1 H MR (1.9:1 rotamer ratio, asterisks denote minor rotamer peaks, 500 MHz, CDCl 3 ): δ 7.90 (m, 2 H), 7.19-7.32 (m, 7H), 7.08* (d, J = 6.5 Hz, 2 H), 4.65 (m, 2 H), 4.30* (d, J = 8.5 Hz, 1 H), 4.13 (m, 1 H), 3.94* (m, 1 H), 3.86 (m, 3 H), 3.80 (m, 1 H), 2.88 (m, 2 H), 2.83 (s, 3 H), 2.79* (s, 3 H), 2.40-2.51 (m, 2 H), 2.39* (t, J = 2.5 Hz, 1 H), 2.33 (t, J = 2.5 Hz, 1 H), 1.29* (d, J = 7.0 Hz, 3 H), 1.15 (d, J = 6.5 Hz, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 125 MHz, CDCl 3 ): δ 171.8, 171.7*, 167.2, 147.3, 147.2*, 138.6, 138.2*, 129.9, 129.8*, 128.9, 128.8*, 128.6, 128.5*, 128.4, 128.2*, 128.08, 128.06*, 127.2, 127*, 83.1, 82.0*, 80.0, 79.4*, 74.9*, 74.3, 57.5, 56.26, 56.22*, 54.6*, 52.13, 52.12*, 35.4, 34.1*, 31.7*, 31.2*, 31.1, 28.4, 15.6, 12.2*. m/z = 394.0 [M+H] +. H 20. Prepared according to General Procedure D using 19 (900 mg, 2.3 mmol, 1.0 eq), LiH monohydrate (386 mg, 9.2 mg, 4.0 eq) and THF/H/DI H 2 (15 ml, 3:2:1 v/v) to yield 20 as a light brown solid (823 mg, 94% yield): 1 H MR (1.9:1 rotamer ratio, asterisks denote minor rotamer peaks, 500 MHz, CDCl 3 ): δ 8.00 (m, 2 H), 7.22-7.35 (m, 5 H), 7.15 (m, 2 H), 4.69 (m, 2 H), 4.46* (d, J = 7.5 Hz, 1 H), 4.16 (m, 1 H), 3.98* (m, 1 H), 3.82 (m, 1 H), 2.93 (m, 2 H), 2.87 (s, 3 H), 2.84* (s, 3 H), 2.70* (m, 2 H), 2.46-2.56 (m, 2 H), 2.42* (t, J = 2.5 Hz, 1 H), 2.36 (t, J = 2.5 H, 1 H), 2.07-2.13* (m, 2 H), 1.32 (d, J = 6.5 Hz, 3 H), 1.19 (d, J = 7.0 Hz, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 125 MHz, CDCl 3 ): δ 172.1. 172.0*, 171.6*, 171.5, S-14

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 148.0, 138.5, 138.2*, 130.6, 130.5*, 128.92, 128.88*, 128.7, 128.6*, 128.5, 128.4*, 127.7, 127.6*, 127.3, 127.2*, 83.1, 81.9*, 80.0, 79.4*, 74.9*, 74.4, 57.7, 56.3, 56.2*, 35.4, 34.1*, 31.8*, 31.4*, 31.2, 28.6, 15.6*, 12.2. m/z = 379.9 [M+H] +. 3 H 21. Prepared according General Procedure A using 9 (164.0 mg, 1.0 mmol, 1.0 eq), PyBP (513 mg, 1.0 mmol, 1.0 eq), diisopropylethylamine (864 µl, 5.0 mmol, 5.0 eq) and 20 (965.0 mg, 6.6 mmol, 1.1 eq) in dry CH 2 Cl 2 (30 ml). The crude reaction mixture was purified using a Biotage Horizon automated flash column chromatography system (silica gel, EtAc, R f = 0.41) to yield 21 as a colorless oil (207.8 mg, 43% yield): 1 H MR (1.9:1 rotamer ratio, asterisks denote minor rotamer peaks, 500 MHz, CDCl 3 ): δ 7.67 (m, 2 H), 7.05-7.34 (m, 7 H), 6.59 (br. s., 1 H), 4.69 (m, 1 H), 4.65 (d, J = 5.0 Hz, 1 H), 4.42* (d, J = 7.5 Hz, 1 H), 4.13 (m, 1 H), 3.94* (m, 1 H), 3.81 (m, 1 H), 3.58-3.70 (m, 6 H), 3.36 (m, 2 H), 2.74-2.90 (m, 5 H), 2.40-2.51 (m, 2 H), 2.38* (m, 1 H), 2.33 (m, 1 H), 1.29* (d, J = 6.5 Hz, 3 H), 1.14 (d, J = 6.5 Hz, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 125 MHz, CDCl 3 ): δ 171.9, 171.8*, 167.5*, 167.4, 145.63, 145.58*, 138.6, 138.3*, 132.3, 132.2*, 128.9, 128.8*, 128.7, 128.6*, 128.5, 128.1*, S-15

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 127.34, 127.26*, 127.24, 127.17*, 83.2, 82.0*, 80.0, 79.5*, 74.9*, 74.3, 70.3, 70.2*, 57.5, 56.30, 56.25*, 50.8, 39.8, 35.5, 34.3, 31.1*, 31.0, 28.5, 15.6*, 12.2. m/z = 492.3 [M+H] +. Boc H 22. To a 1 L-3 necked-rbf equipped with a stir bar and an internal thermometer was added 1- tert-butyl 2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (19.9 g, 81 mmol, 1.0 eq) and THF (300 ml). The clear solution was evacuated and back filled with nitrogen gas and cooled to -78 C. When the internal temperature was at about -50 C, ah (60 wt % dispersion in mineral oil, 5 g ca., 122 mmol, 1.5 eq) was added in small portions. The reaction flask was quickly capped and charged with nitrogen and stirred for 10 min. Propargyl bromide (80 wt % in toluene, 9.5 ml, 122 mmol, 1.5 eq) was added via a syringe over 5 min. During the addition the internal temperature was at about -60 C. Stirring continued and the reaction was allowed to warm to ambient temperature overnight. After 20 h at ambient temperature, LCMS analysis of the reaction mixture showed that starting material still remained. The reaction was cooled again to about -60 C. Additional ah (60 wt % dispersion in mineral oil, 5 g, 122 mmol, 1.5 mol eq) was added. At about -60 C, propargyl bromide (80 wt % in toluene, 10 ml, 122 mmol, 1.5 mol eq) was added via a syringe over 5 min. Stirring continued and the reaction was allowed to warm to ambient temperature for 4 h. LCMS analysis of the reaction mixture showed major and clean product presence. The reaction mixture was cooled to -65 C and was carefully quenched with water (few drops first, total 20 ml). The reaction mixture was allowed to warm to ambient temperature and was used in the next reaction without further purification. S-16

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry To the above reaction crude solution in THF-water mixture was added solid lithium hydroxide (3.9 g, 162 mmol, 2 mol eq in 40 ml water). The reaction mixture was stirred at ambient temperature for 1.5 h. LCMS analysis of the reaction mixture showed clean and major product presence. The volatiles were removed in vacuo, providing an oil, which was suspended in water. To remove mineral oil from ah dispersion, the reaction crude mixture was partitioned in heptane (100 ml). The organic layer was separated. The aqueous layer was cooled in an ice bath and was acidified to ph 4 using acetic acid. The aqueous phase was extracted with CH 2 Cl 2 (2 x 250 ml). The organic layer was separated. The aqueous phase was then saturated with solid acl and extracted with CH 2 Cl 2 (2 x 100 ml). The organic layer was separated. The combined organic layer was dried over a 2 S 4 and evaporated to give an oil (44 g). The above oil was dissolved in ethyl acetate (10 ml). Heptane (100 ml) was slowly added. The mixture was stirred at ambient temperature for 30 min. A light yellow gummy solid came out and was stirred for another 10 min. The light clear top liquid was decanted. The remaining light gummy solid was again dissolved in ethyl acetate (10 ml) and heptane (100 ml) was slowly added with stirring until lightly cloudy. The mixture was stirred for 30 min. The clear liquid was decanted. The remaining oil was evaporated. This oil upon cooling solidified to a light yellow solid, which was dried to give the title compound 22 (17.64 g). 1 H MR analysis showed acetic acid presence (10 mole %). This solid was then dissolved in acetonitrile (30 ml) and water (100 ml). The solution was lyophilized to a residue. This residue was dissolved in ethyl acetate (20 ml). To this solution was added heptane (total 220 ml) with stirring. The mixture was stirred at ambient temperature for 1 h. The clear liquid was decanted. The resulting material was dried in vacuo to compound 22 as a light yellow solid (16.61 g, 76% yield): 1 H MR (500 MHz, DMS-d 6 ): δ 4.21 (m, 1 H), 4.16 (d, J=2.45 Hz, 2 H), 4.04-4.09 (m, 1 H), S-17

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 3.38-3.49 (m, 3 H), 2.24-2.35 (m, 1 H), 1.93-2.02 (m, 1 H), 1.39 (s, 3 H) 1.33 (s, 6 H); 13 C MR (asterisks denote minor rotamer peaks, 125 MHz, DMS-d 6 ): δ 174.1, 173.6*, 153.5*, 153.1, 80.3, 78.9, 77.2, 76.2*, 75.4, 57.6, 57.4*, 55.7*, 55.6, 51.6*, 51.2, 35.5, 34.7*, 28.1*, 27.9. m/z = 268.1 [M-H] +. HBoc H 3 23. Prepared according General Procedure A using 9 (350.0 mg, 2.1 mmol, 1.1 eq), PyBP (990 mg, 1.9 mmol, 1.0 eq), diisopropylethylamine (1.66 ml, 10.5 mmol, 5.0 eq) and -Boc-2- aminomethyl-phenylacetic acid (500.0 mg, 1.9 mmol, 1.0 eq) in dry CH 2 Cl 2 (50 ml). The residue was purified using a Biotage Horizon automated flash column chromatography system (silica gel, 20-100% EtAc in hexanes gradient, R f = 0.51 in EtAc) to yield 23 as a white solid (596.2 mg, 83% yield): 1 H MR (600 MHz, CDCl 3 ): δ 7.21-7.32 (m, 4 H), 6.18 (br. s., 1 H), 5.34 (br. s., 1 H), 4.30 (m, 2 H), 3.58 (m, 4 H), 3.50 (t, J = 5.4 Hz, 2 H), 3.41 (q, J = 5.4 Hz, 2 H), 3.29 (t, J = 4.8 Hz, 2 H), 1.42 (s, 9 H); 13 C MR (150 MHz, CDCl 3 ): δ 171.0, 156.2, 137.7, 133.5, 131.1, 129.8, 128.3, 128.1, 79.6, 77.0, 69.8, 50.8, 42.5, 40.9, 39.5, 28.6. m/z = 278.2 [M+H, -Boc] +. S-18

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry Boc H 3 H 24. Prepared according to General Procedure C using 23 (596.2 mg, 1.58 mmol, 1.0 eq) and 4.0 M HCl in 1,4-dioxane (3.0 ml, 12.0 mmol, 7.6 eq). The peptide coupling was carried out using Boc-deprotected 23 (150.0 mg, 0.48 mmol, 1.1 eq), PyBP (230 mg, 0.44 mmol, 1.0 eq), diisopropylethylamine (380 µl, 2.2 mmol, 5.0 eq) and 22 (117.0 mg, 0.44 mmol, 1.0 eq) in dry CH 2 Cl 2 (30 ml). The residue was purified (without filtering off PyBP impurities) using a Biotage Horizon automated flash column chromatography system (silica gel, 20-100% EtAc in hexanes gradient, R f = 0.49 in EtAc) to yield 24 as a colorless oil (232.6 mg, >99% yield): 1 H MR (600 MHz, CD 3 D): δ 7.17-7.42 (m, 4 H), 4.35-4.56 (m, 2 H), 4.15-4.34 (m, 4 H), 3.54-3.77 (m, 8 H), 3.30-3.43 (m, 3 H), 2.86 (m, 1 H), 2.39 (m, 1 H), 2.02 (m, 1 H), 1.27-1.48 (m, 9 H); 13 C MR (asterisks denote minor rotamer peaks, 150 MHz, CD 3 D): δ 175.0, 174.8*, 173.9*, 173.8, 156.4*, 156.0, 138.3, 137.9*, 135.6, 135.3*, 131.61, 131.57*, 130.7, 130.1*, 139.0, 128.9*, 128.6, 128.49*, 129.46, 81.7, 81.5*, 80.6*, 80.5, 78.0*, 77.2, 76.05, 76.02*, 70.9, 70.4*, 60.9, 60.5*, 57.03*, 56.96, 55.8, 53.5*, 53.0*, 51.8, 43.8, 42.3*, 42.1, 40.8, 40.7*, 40.60, 40.56*, 38.0, 37.2*, 37.0*, 28.7*, 28.5. m/z = 529.3 [M+H] +. H S-19

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 25. Prepared according General Procedure A using (1R,2S)-(-)-ephedrine (660.0 mg, 4.0 mmol, 1.0 eq), PyBP (2.1 g, 4.4 mmol, 1.0 eq), diisopropylethylamine (2.1 ml, 12.0 mmol, 3.0 eq) and (R)-(+)-3-methylsuccinic acid 1-monomethyl ester (640 mg, 4.4 mmol, 1.1 eq). The residue was purified (without filtering off PyBP impurities) using a Biotage Horizon automated flash column chromatography system (silica gel, 10-100% EtAc in hexanes gradient, R f = 0.29 in 1:1 hexanes/etac) to yield 25 as a colorless oil (722 mg, 62% yield): 1 H MR (4.0:1 rotamer ratio, asterisks denote minor rotamer peaks, 600 MHz, CDCl 3 ): δ 7.12-7.30 (m, 5 H), 4.60 (m, 1 H), 4.06 (m, 1 H), 3.55* (s, 3 H), 3.54 (s, 3 H), 2.91 (m, 1 H), 2.28 (m, 4 H), 1.94 (m, 1 H), 1.24* (d, J = 7.2 Hz, 3 H), 1.15 (d, J = 7.2 Hz, 3 H), 0.93* (d, J = 7.2 Hz, 3 H), 0.74 (d, J = 7.2 Hz, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 150 MHz, CDCl 3 ): δ 176.4, 175.6*, 173.5*, 173.1, 141.91*, 141.90, 128.4*, 128.1, 127.7*, 127.6, 126.5, 126.2*, 77.2, 76.5*, 57.8, 55.9*, 51.7*, 51.2, 37.7, 37.5*, 36.7, 32.2*, 31.6*, 29.9, 17.5*, 16.7, 14.5*, 12.9. m/z = 294.2 [M+H] +. H 26. 25 (722 mg, 2.46 mmol, 1.0 eq) was dissolved in dry THF (50 ml) at room temperature. ah (dry, 65 mg, 2.7 mmol, 1.1 eq) and tetrabutylammonium iodide (45 mg, 0.12 mmol, 0.05 eq) were added to the reaction mixture and allowed to stir until gas evolution ceased. Propargyl bromide (80% w/w in toluene, 330 µl, 3.1 mmol, 1.2 eq) was added drop wise and allowed to stir overnight. S-20

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry The reaction was not complete by TLC after having been stirred overnight, so additional doses of ah and propargyl bromide were added to the reaction mixture at room temperature and stirred overnight. The reaction was deemed complete by TLC and was quenched by the addition of H (15 ml). LiH monohydrate (450 mg, 10.7 mmol, 4.3 eq) dissolved in 10 ml DI H 2 was added to the reaction and stirred at room temperature until deemed complete by TLC. The reaction was concentrated and the resultant residue was acidified using 5 M HCl. The residue was partitioned between DI H 2 and CH 2 Cl 2, and the aqueous phase extracted with CH 2 Cl 2 (2x 25 ml). The organic phase was dried over a 2 S 4, concentrated and dried in vacuo to yield 26 as a colorless oil (600 mg, 77% yield): 1 H MR (3.0:1 rotamer ratio, asterisks denote minor rotamer peaks, 500 MHz, CDCl 3 ): δ 7.16-7.35 (m, 5 H), 4.76 (br. s., 1 H), 4.54 (m, 1 H), 4.09 (m, 1 H), 4.09* (s, 1 H), 3.77 (m, 1 H), 2.82-2.89 (m, 4 H), 2.67 (m, 1 H), 2.40 (m, 1 H), 2.34 (t, J = 2.5 Hz, 1 H), 1.38* (d, J = 7.5 Hz, 3 H), 1.18 (d, J = 7.0 Hz, 3 H), 1.03* (d, J = 7.5 Hz, 3 H), 0.75 (d, J = 7.5 Hz, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 125 MHz, CDCl 3 ): δ 177.1*, 176.4, 175.7, 174.9*, 138.1, 137.8*, 129.04, 129.98*, 128.5, 128.4*, 127.6, 127.0*, 82.9, 82.1*, 79.9, 79.2*, 75.1*, 74.6, 64.6*, 57.9, 56.4*, 56.0, 38.3, 38.2*, 32.7, 32.2*, 31.2*, 29.1, 19.3, 16.8*, 13.9*, 12.8. m/z = 318.2 [M+H] +. H BocH 27. -Boc-tyramine (1.0 g, 4.2 mmol, 1.0 eq), K 2 C 3 (1.71 g, 12.4 mmol, 3.0 eq) and methyl bromoacetate (500 µl, 5.3 mmol, 1.3 eq) were stirred in DMF (30 ml) overnight at room temperature. The reaction was concentrated in vacuo and the residue dissolved in DI H 2 (50 S-21

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry ml) and EtAc (50 ml). The aqueous layer was extracted with EtAc (3x 30 ml). The combined organic extracts were dried over a 2 S 4, filtered, concentrated and dried. The residue was dissolved in THF/H (25 ml, 3:2 v/v). LiH monohydrate (530 mg, 12.6 mg, 3.0 eq) was dissolved in DI H 2 (5 ml) and added drop wise to the solution and allowed to stir at RT until deemed complete by TLC. The reaction was concentrated and the ph of the resultant residue was adjusted to ph 5-6 using 1 M HCl. The residue was partitioned between DI H 2 and CH 2 Cl 2, and the aqueous phase extracted with CH 2 Cl 2 (3x 25 ml). The organic phase was dried over a 2 S 4, concentrated and dried in vacuo to yield 27 as a white solid (1.24 mg, >99% yield): 1 H MR (600 MHz, CD 3 D): δ 7.13 (d, J = 8.4 Hz, 2 H), 6.86 (d, J = 9.0 Hz, 2 H), 4.62 (s, 2 H), 3.21 (t, J = 7.2 Hz, 2 H), 2.71 (t, J = 7.2 Hz, 2 H), 1.42 (s, 9 H); 13 C MR (asterisks denote minor rotamer peaks, 150 MHz, CD 3 D): δ 173.0, 158.6, 158.2, 133.8, 131.0, 115.8, 80.1, 66.1, 43.3, 36.4, 28.9. m/z = 294.1 [M+H] +. BocH H 3 28. Prepared according General Procedure A using 9 (350.0 mg, 2.1 mmol, 1.1 eq), PyBP (990 mg, 1.9 mmol, 1.0 eq), diisopropylethylamine (1.66 ml, 10.5 mmol, 5.0 eq) and 27 (560.0 mg, 1.9 mmol, 1.0 eq) in dry CH 2 Cl 2 (40 ml). The residue was purified using a Biotage Horizon automated flash column chromatography system (silica gel, 10-100% EtAc in hexanes gradient, R f = 0.57 in EtAc) to yield 28 as a viscous oil (685.7 mg, 89% yield): 1 H MR (600 MHz, CD 3 D): δ 7.15 (d, J = 7.8 Hz, 2 H), 6.91 (d, J = 8.4 Hz, 2 H), 4.48 (s, 2 H), 3.63 (t, J = 4.2 Hz, 2 H), 3.59 (t, J = 6.6 Hz, 2 H), 3.48 (t, J = 4.8 Hz, 2 H), 3.34 (t, J = 4.2 Hz, 2 H), 3.21 (t, J = 7.2 Hz, 2 H), 2.70 (t, J = 7.2 Hz, 2 H), 1.42 (s, 9 H); 13 C MR (150 MHz, CD 3 D): δ 171.5, S-22

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 158.6, 157.8, 134.1, 131.1, 116.0, 80.1, 71.2, 70.4, 69.4, 51.9, 43.3, 40.0, 36.5, 28.9. m/z = 308.2 [M+H, -Boc] +. 3 H H 29. Prepared according to General Procedure C using 28 (685.7 mg, 1.68 mmol, 1.0 eq) and 4.0 M HCl in 1,4-dioxane (3.0 ml, 12.0 mmol, 7.1 eq). The peptide coupling was carried out using Boc-deprotected 28 (238.0 mg, 0.69 mmol, 1.1 eq), PyBP (327 mg, 0.63 mmol, 1.0 eq), diisopropylethylamine (550 µl, 3.14 mmol, 5.0 eq) and 26 (200.0 mg, 0.63 mmol, 1.0 eq) in dry CH 2 Cl 2 (40 ml). The residue was purified (without filtering off PyBP impurities) using a Biotage Horizon automated flash column chromatography system (silica gel, 25-100% EtAc in hexanes gradient, R f = 0.29 in EtAc) to yield 29 as a colorless oil (219.0 mg, 59% yield): 1 H MR (4.1:1 rotamer ratio, asterisks denote minor rotamer peaks, 600 MHz, CD 3 D): δ 8.31 (m, 1 H), 7.79 (m, 5 H), 7.61 (m, 2 H), 7.36 (m, 2 H), 5.34 (br. s., 1 H), 5.04 (m, 1 H), 4.95 (m, 2 H), 4.64 (m, 1 H), 4.28 (m, 1 H), 4.11 (t, J = 6.0 Hz, 2 H), 4.07 (t, J = 5.4 Hz, 2 H), 3.97 (t, J = 5.4 Hz, 2 H), 3.82 (t, J = 5.4 Hz, 2 H), 3.80 (m, 3 H), 3.46 (m, 1 H), 3.41 (s, 3 H), 3.17 (t, J = 7.8 Hz, 2 H), 2.59-2.87 (m, 1 H), 2.56 (m, 1 H), 1.87* (d, J = 6.0 Hz, 3 H), 1.74 (d, J = 6.6 Hz, 3 H), 1.44* (d, J = 6.0 Hz, 3 H), 1.03 (d, J = 6.0 Hz, 3 H); 13 C MR (150 MHz, CD 3 D): δ 177.7, 174.0, 171.2, 157.7, 139.5, 133.8, 130.9, 129.3, 128.8, 128.4, 115.8, 83.9, 80.5, 76.0, 70.9, 70.3, 68.3, 56.5, 51.5, 42.1, 40.5, 39.9, 35.64, 35.62, 33.87, 33.86, 16.9, 13.8. m/z = 607.3 [M+H] +. S-23

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry H 30. Prepared according General Procedure A using (1R,2S)-(-)-ephedrine (485 mg, 2.93 mmol, 1.0 eq), PyBP (1.53 g, 2.93 mmol, 1.0 eq), diisopropylethylamine (1.53 ml, 8.8 mmol, 3.0 eq) and -(4-methoxybenzyl)-iminodiacetic acid mono t-butyl ester (1.00 g, 3.23 mmol, 1.1 eq) in dry CH 2 Cl 2 (40 ml). The crude reaction mixture was purified (without filtering off PyBP impurities) using a Biotage Horizon automated flash column chromatography system (silica gel, 10-100% EtAc in hexanes gradient, R f = 0.35 in 1:1 hexanes/etac) to yield 30 as a colorless oil (1.23 g, 91% yield): 1 H MR (2.4:1 rotamer ratio, asterisks denote minor rotamer peaks, 600 MHz, CDCl 3 ): δ 7.32 (m, 1 H), 7.25 (m, 2 H), 7.16-7.20 (m, 3 H), 6.83* (d, J = 9.0 Hz, 2 H), 6.79 (d, J = 7.2 Hz, 2 Hz), 4.77 (d, J = 4.2 Hz, 1 H), 4.48* (d, J = 7.8 Hz, 1 H), 4.39 (br. s., 1 H), 4.13* (br. s., 1 H), 3.76 (m, 3 H), 3.64 (m, 2 H), 3.34 (m, 2 H), 3.20 (m, 2 H), 2.72 (s, 3 H), 2.60* (s, 3 H), 1.41 (m, 9 H), 1.26 (d, J = 6.6 Hz, 3 H), 1.14* (d, J = 7.2 Hz, 3 H); 13 C MR (150 MHz, CDCl 3 ): δ 171.9, 170.8, 170.5*, 170.4*, 159.2*, 159.0, 141.9, 141.8*, 130.9*, 130.5, 130.4, 129.8*, 128.4, 128.3*, 128.0*, 127.6, 126.5, 126.4*, 113.9*, 113.8, 81.2*, 81.1, 76.9, 75.9*, 58.0*, 57.7, 56.5, 55.8*, 55.4*, 55.34, 55.33*, 55.27, 32.7*, 28.7*, 28.29, 28.28*, 15.2*, 12.1. m/z = 457.3 [M+H] +. S-24

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 31. 30 (1.23 g, 2.7 mmol, 1.0 eq), propargyl bromide (80% w/w in toluene, 356 µl, 3.3 mmol, 1.2 eq) and tetrabutylammonium iodide (10 mg, 0.03 mmol, 0.01 eq) were dissolved in dry THF (50 ml) at 0 C. ah (dry, 71 mg, 3.0 mmol, 1.1 eq) was added to the reaction mixture and allowed to stir overnight at room temperature. The reaction had proceeded to only about 50-60% conversion, but was quenched carefully with DI H 2 and concentrated. The resulting residue dissolved in DI H 2 (50 ml) and EtAc (50 ml). The organic phase was dried over a 2 S 4, concentrated and purified using a Biotage Horizon automated flash column chromatography system (silica gel, 10-100% EtAc in hexanes gradient, R f = 0.50 in 1:1 hexanes/etac) to yield 31 as a colorless oil (581.2 mg, 43% yield). Residual starting material was also isolated: 1 H MR (1.8:1 rotamer ratio, asterisks denote minor rotamer peaks, 600 MHz, CDCl 3 ): δ 7.15-7.33 (m, 6 H), 6.97 (m, 1 H), 6.83* (d, J = 8.4 Hz, 2 Hz), 6.80 (d, J = 8.4 Hz, 2 Hz), 4.71 (br. s., 1 H), 4.63 (d, J = 5.4 Hz, 1 H), 4.36* (d, J = 7.8 Hz, 1 H), 4.09 (m, 1 H), 3.76 (m, 4 H), 3.60 (m, 2 H), 2.87-3.32 (m, 7 H), 2.39* (t, J = 2.4 Hz, 1 H), 2.30 (t, J = 2.4 Hz, 1 H), 1.42 (m, 9 H), 1.30* (d, J = 6.6 Hz, 3 H), 1.15 (d, J = 7.2 Hz, 3 H); 13 C MR (150 MHz, CDCl 3 ): δ 171.0, 170.6*, 170.5*, 170.3, 159.1*, 158.9, 138.4, 138.3*, 130.8, 130.6*, 130.5, 130.4*, 128.7, 128.50*, 128.47, 128.2*, 127.4, 127.2*, 113.9*, 113.7, 82.8, 82.0*, 81.0*, 80.9, 79.9, 79.6*, 74.7*, 74.4, 57.9*, 57.3, 56.9*, 56.1, 56.0*, 55.8, 55.5*, 55.48, 55.45*, 55.39, 55.1, 28.4*, 28.33, 28.32, 15.2, 12.4*. m/z = 495.3 [M+H] +. S-25

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry H 32. 31 (573 mg, 1.16 mmol, 1.0 eq) was dissolved in dry CH 2 Cl 2 (5 ml). Trifluoroacetic acid (3 ml, excess) was added at room temperature and allowed to stir for 4 hours. The reaction appeared to stall and an addition dose of trifluoroacetic acid (3 ml) was added and the reaction was allowed to stir overnight. Volatiles were removed under a stream of nitrogen and the residue was allowed to dry under vacuum. 32, isolated as a light brown oil, was determined to be pure by LC/MS and was used without further purification or characterization. 3 H H 33. Prepared according to General Procedure C using 23 (685.7 mg, 1.68 mmol, 1.0 eq) and 4.0 M HCl in 1,4-dioxane (3.0 ml, 12.0 mmol, 7.1 eq). The peptide coupling was carried out using Boc-deprotected 23 (200.0 mg, 0.64 mmol, 1.1 eq), PyBP (307 mg, 0.59 mmol, 1.0 eq), diisopropylethylamine (511 µl, 2.93 mmol, 5.0 eq) and 32 (255.8 mg, 0.58 mmol, 1.0 eq) in dry CH 2 Cl 2 (30 ml). The residue was purified (without filtering off PyBP impurities) using a Biotage Horizon automated flash column chromatography system (silica gel, 20-100% EtAc in S-26

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry hexanes gradient then 10% H in EtAc, R f = 0.41 in EtAc) to yield 33 as a colorless oil (318.2 mg, 79% yield): 1 H MR (600 MHz, CD 3 D): δ 7.04-7.42 (m, 12 H), 6.94 (br. s., 1 H), 6.70-6.83 (m, 2 H), 4.76 (br. s., 1 H), 4.58 (m, 1 H), 4.42 (m, 2 H), 4.09 (m, 2 H), 3.68-3.86 (m, 4 H), 3.58-3.66 (m, 5 H), 3.54 (m, 3 H), 3.32-3.45 (m, 6 H), 3.14 (m, 2 H), 3.06 (br. s., 1 H), 2.79 (m, 2 H), 1.18 (m, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 150 MHz, CD 3 D): δ 173.8*, 173.69, 173.65, 173.59*, 172.73, 172.5* 160.7*, 160.6, 139.53, 139.52*, 138.25, 135.20*, 135.18, 131.8*, 131.7, 130.1, 130.0*, 129.73*, 129.69, 129.53,* 129.46, 128.8, 128.7*, 128.55, 128.54*, 128.52, 128.49, 128.4, 127.8, 114.9*, 114.8, 85.5, 82.3*, 80.5, 80.3*, 70.88*, 70.87, 70.35*, 70.32, 58.3, 58.2*, 56.9*, 56.8, 56.1*, 55.8*, 55.73, 55.67, 51.7, 47.38, 47.35*, 45.82*, 43.78, 41.70*, 41.6*, 40.8, 40.57*, 40.56, 37.2, 28.9*, 27.4*, 27.3, 15.6*, 13.2. m/z = 698.4 [M+H] +. 3 H H 34. Prepared according to General Procedure C using 28 (685.7 mg, 1.68 mmol, 1.0 eq) and 4.0 M HCl in 1,4-dioxane (3.0 ml, 12.0 mmol, 7.1 eq). The peptide coupling was carried out using Boc-deprotected 28 (180.0 mg, 0.53 mmol, 1.1 eq), PyBP (250 mg, 0.48 mmol, 1.0 eq), diisopropylethylamine (420 µl, 2.41 mmol, 5.0 eq) and 32 (210.0 mg, 0.63 mmol, 1.0 eq) in dry CH 2 Cl 2 (40 ml). The residue was purified (without filtering off PyBP impurities) using a Biotage Horizon automated flash column chromatography system (silica gel, 25-100% EtAc in S-27

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry hexanes gradient then 10% H in EtAc, R f = 0.29 in EtAc) to yield 34 as a colorless oil (232.0 mg, 65% yield): 1 H MR (600 MHz, CD 3 D): δ 7.21-7.34 (m, 5 H), 7.11-7.17 (m, 3 H), 6.95-7.04 (m, 2 H), 6.78-6.88 (m, 4 H), 4.70 (br. s., 1 H), 4.61 (m, 1 H), 4.39 (s, 2 H), 4.09 (m, 1 H), 3.75-3.80 (m, 4 H), 3.56-3.63 (m, 4 H), 3.38-3.50 (m, 5 H), 3.33-3.36 (m, 4 H), 2.85-3.81 (m, 4 H), 2.67-2.80 (m, 6 H), 1.23-1.35 (m, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 150 MHz, CD 3 D): δ 173.7, 173.6*, 172.4*, 172.1, 160.6*, 160.5, 157.64*, 157.63, 139.6, 139.5*, 133.63, 133.58*, 131.62*, 131.55, 131.01, 130.98*, 129.70*, 129.65, 129.62*, 129.4, 128.6, 138.3*, 115.9*, 115.8, 114.9*, 114.8, 83.5, 82.8*, 80.6, 80.3*, 76.2*, 75.9, 71.0, 70.3*, 68.1, 59.6, 59.3*, 68.1, 59.6*, 59.3*, 58.73*, 58.69, 58.1, 56.9*, 56.7, 56.1*, 55.8*, 55.76, 55.70, 51.7, 41.38, 41.36*, 39.9, 35.3, 28.0, 15.6, 13.5*. m/z = 729.3 [M+H] +. Synthesis of Macrocycles. H 1a: Preparative Scale Flow Macrocyclization, General Procedure E. Azido-alkyne 11 (0.10 M in EtH, 100 µl, 0.010 mmol, 1.0 eq), TTTA (0.01 M in EtH, 100 µl, 0.001 mmol, 0.10 eq), DIPEA (0.1 M in EtH, 200 µl, 0.020 mmol, 2.0 eq) and EtH (200 µl) were aspirated from their respective source vials, mixed through a PFA mixing tube (0.2 mm inner diameter), and loaded into an injection loop. The reaction segment was injected into the flow reactor set at 150 C, passed through the reactor at 300 µl min -1 (5 minute residence time). A total of 40 S-28

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry reaction segments prepared in this manner were collected in a round bottom flask. Upon completion, the reaction mixture was concentrated and dried in vacuo. The crude reaction mixture was purified using a Biotage Horizon automated flash column chromatography system (silica gel, EtAc, R f = 0.17) to yield 1a as a white solid (103.8 mg, 87% yield): 1 H MR (600 MHz, CD 3 D): δ 8.06 (br. s., 1 H), 7.64 (br. s., 1 H), 7.26-7.39 (m, 4 H), 7.19-7.25 (m, 1 H), 4.88 (d, J = 13.2 Hz, 1 H), 4.73 (br. s., 1 H), 4.39 (br. s., 1 H), 4.21 (d, J = 13.2 Hz, 2 H), 3.53 (d, J = 6.6 Hz, 1 H), 2.20-2.58 (m, 3 H), 1.97 (br. s., 1 H), 0.79 (d, J = 6.6 Hz, 3 H); 13 C MR (150 MHz, CD 3 D): δ 173.3, 147.3, 141.0, 129.5, 128.4, 128.3, 127.4, 77.5, 63.3, 52.8, 52.2, 34.2, 26.1, 11.6; HRMS (ESI-TF): C 16 H 20 4 2 : [M+H] + : calculated 301.1659, found 301.1664. 2a. Prepared according to General Procedure E using 40 reaction segments containing azidoalkyne 13 (0.10 M in EtH, 100 µl, 0.010 mmol, 1.0 eq), TTTA (0.01 M in EtH, 100 µl, 0.001 mmol, 0.10 eq), DIPEA (0.1 M in EtH, 200 µl, 0.020 mmol, 2.0 eq) and EtH (200 µl). The crude reaction mixture was purified using a Biotage Horizon automated flash column chromatography system (silica gel, EtAc, R f = 0.23) to yield 2a as a white solid (109.4 mg, 80% yield): 1 H MR (600 MHz, CDCl 3 ): δ 7.46 (m, 3 H), 7.37 (t, J = 7.5 Hz, 2 H), 7.25-7.31 (m, 1 H), 4.90 (d, J = 13.6 Hz, 1 H), 4.52-4.61 (m, 2 H), 4.52 (s, 1 H), 4.22 (d, J = 13.6 Hz, 2 H), 2.85 (s, 3 H), 2.22-2.32 (m, 1 H), 1.98-2.10 (m, 2 H), 1.87-1.96 (m, 1 H), 1.76-1.87 (m, 1 H), 1.52-1.62 (m, 1 H), 1.23 (d, J = 6.6 Hz, 1 H), 0.96 (m, 4 H); 13 C MR (150 MHz, S-29

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry CDCl 3 ): δ 172.1, 143.9, 139.1, 128.3, 127.3, 126.5, 123.7, 80.2, 60.9, 54.3, 50.1, 31.6, 30.9, 28.5, 23.5, 23.4, 9.4; HRMS (ESI-TF): C 19 H 26 4 2 : [M+H] + : calculated 343.2128, found 343.2127. H 3a. Prepared according to General Procedure E using 40 reaction segments containing azidoalkyne 17 (0.10 M in EtH, 100 µl, 0.010 mmol, 1.0 eq), TTTA (0.01 M in EtH, 100 µl, 0.001 mmol, 0.10 eq), DIPEA (0.1 M in EtH, 200 µl, 0.020 mmol, 2.0 eq) and EtH (200 µl). The crude reaction mixture was purified using a Biotage Horizon automated flash column chromatography system (KP-H cartridge, 0-10% H in EtAc, R f = 0.44 in 10% H in EtAc) to yield 3a as an off-white solid (96.7 mg, 57% yield): 1 H MR (5.0:1 rotamer ratio, asterisks denote minor rotamer peaks, 600 MHz, CD 3 D): δ 8.06 (s, 1 H), 8.02* (s, 1 H), 7.20-7.35 (m, 5 H), 4.58 (m, 3 H), 4.21 (m, 1 H), 3.76-3.90 (m, 3 H), 3.63 (m, 2 H), 3.37-3.57 (m, 6 H), 3.07 (m, 1 H), 2.96 (m, 1 H), 2.51 (m, 1 H), 2.15 (m, 1 H), 1.15 (d, J = 6.6 Hz, 3 H), 1.11* (d, J = 6.0 Hz, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 150 MHz, CD 3 D): δ 176.8, 176.2*, 173.9*, 173.8, 146.6, 144.4*, 141.4, 139.5*, 129.9, 129.8*, 128.8, 128.5*, 128.4, 128.2*, 127.5*, 126.0, 86.5, 80.0*, 71.0, 70.7*, 70.2*, 70.1, 64.2, 63.6*, 52.5, 52.0*, 51.9, 51.5*, 50.9*, 50.8*, 50.6, 49.6, 41.3*, 41.2, 40.5*, 40.4, 36.1, 35.7*, 17.2; HRMS (ESI-TF): C 22 H 29 5 4 : [M+H] + : calculated 428.2292, found 428.2283. S-30

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry H 4a. Prepared according to General Procedure E using 30 reaction segments containing azidoalkyne 21 (0.10 M in EtH, 100 µl, 0.010 mmol, 1.0 eq), TTTA (0.01 M in EtH, 100 µl, 0.001 mmol, 0.10 eq), DIPEA (0.1 M in EtH, 200 µl, 0.020 mmol, 2.0 eq) and EtH (200 µl). The crude reaction mixture was purified using a Biotage Horizon automated flash column chromatography system (silica gel, 2% H in EtAc, R f = 0.14) to yield 4a as an off-white solid (105.2 mg, 71% yield): 1 H MR (600 MHz, CDCl 3 ): δ 7.70 (d, J=7.9 Hz, 2 H), 7.37 (t, J=8.1 Hz, 4 H), 7.34 (s, 1 H), 7.30 (m, 2 H), 7.21 (m, 1 H), 7.04-7.10 (m, 1 H), 6.20-6.29 (m, 1 H), 4.58-4.68 (m, 2 H), 4.49 (t, J = 4.6 Hz, 2 H), 4.33 (d, J = 11.8 Hz, 1 H), 4.10 (d, J = 11.4 Hz, 1 H), 3.81-3.91 (m, 2 H), 3.61-3.67 (m, 1 H), 3.56-3.61 (m, 2 H), 3.50-3.55 (m, 1 H), 3.27-3.38 (m, 1 H), 3.02-3.10 (m, 1 H), 2.96 (m, 1 H), 2.68 (s, 1 H), 2.59 (s, 3 H), 2.53-2.60 (m, 1 H), 0.91 (d, J=7.5 Hz, 3 H); 13 C MR (asterisks denote minor rotamer peaks, 150 MHz, CDCl 3 ): δ 172.9, 172.7*, 167.4*, 166.9, 145.6, 144.6, 139.2, 132.6*, 132.1, 129.4*, 129.1, 128.4*, 128.3, 127.3*, 127.27, 127.22, 126.4*, 126.2, 123.3, 85.2*, 84.9, 70.0*, 69.4, 69.3*, 68.5, 63.6*, 63.4, 54.6, 50.4*, 50.1, 39.7*, 39.4, 34.5, 32.7, 32.2*, 31.9, 29.7*, 9.7; HRMS (ESI- TF): C 27 H 33 5 4 : [M+H] + : calculated 492.2605, found 492.2609. S-31

Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry Boc H H 5a. Prepared according to General Procedure E using 30 reaction segments containing azidoalkyne 24 (0.10 M in EtH, 100 µl, 0.010 mmol, 1.0 eq), TTTA (0.01 M in EtH, 100 µl, 0.001 mmol, 0.10 eq), DIPEA (0.1 M in EtH, 200 µl, 0.020 mmol, 2.0 eq) and EtH (200 µl). The crude reaction mixture was purified using a Biotage Horizon automated flash column chromatography system (KP-H cartridge, 0-10% H in EtAc, R f = 0.41 in 10% H in EtAc) to yield 5a as an off-white solid (99.7 mg, 63% yield): 1 H MR (1.6:1 rotamer ratio, asterisks denote minor rotamer peaks, 600 MHz, CD 3 D): δ 7.99 (s, 1 H), 7.90* (s, 1 H), 7.17-7.30 (m, 4 H), 4.44-4.67 (m, 5 H), 4.12-4.30 (m, 4 H), 3.85 (m, 2 H), 3.70 (m, 1 H), 3.39-3.62 (m, 5 H), 3.21 (m, 2 H), 2.01 (m, 2 H), 1.40* (s, 9 H), 1.29 (s, 9 H); 13 C MR (asterisks denote minor rotamer peaks, 150 MHz, CD 3 D): δ 174.3, 174.1*, 173.92*, 173.90, 156.1*, 155.6, 145.5*, 145.4, 137.9, 137.8*, 136.3, 136.1*, 131.8, 131.7, 131.62*, 131.58*, 129.4, 129.2*, 128.61, 128.59*, 126.1, 125.9, 81.6, 81.4*, 75.9*, 75.6, 70.4, 70.2*, 70.1, 63.2, 62.9*, 60.8, 60.4*, 52.7*, 52.5, 51.5, 51.4*, 42.94*, 42.88, 40.8, 40.6, 40.58*, 40.55*, 37.5, 36.6*, 28.6, 26.5 ; HRMS (ESI-TF): C 26 H 36 6 6 : [M+H] + : calculated 529.2769, found 529.2774. S-32