Cis Trans Proline Isomerization Effects on Collagen Triple-Helix Stability are Limited

Cis Trans Proline Isomerization Effects on Collagen Triple-Helix Stability are Limited an Dai and Felicia A. Etzkorn* Department of Chemistry, Virginia Tech, Blacksburg, VA 24061-0212 Supporting Information. Synthesis: Fmoc Pro Ψ[(E)CH=C] Pro H 1 and Fmoc Gly Pro Pro H 17. Scheme S1. Synthesis of Alcohol 9. H + H DCC, DIEA DMAP, CH 2 Cl 2 75% 7 I s-buli, THF 49% 8 abh 4, CeCl 3 THF, MeH 86% H (R)-9: 49% H (S)-9: 37% We used our well-developed method for the stereoselective synthesis of the Ser Ψ[(E)CH=C] Pro and Gly Ψ[(E)CH=C] Pro isosteres 1,2 to synthesize dipeptide alkene isostere Fmoc Pro Ψ[(E)CH=C] Pro H 1. A similar synthesis for dipeptide isostere 1 has been reported. 3 Starting from commercially available Pro H, the Pro Weinreb amide 7 was synthesized by DCC coupling with,-dimethylhydroxylamine (Scheme S1). The Pro Weinreb amide 7 was reacted with cyclopentenyl lithium, which was synthesized by mixing iodocyclopentene and sec-butyl lithium at 40 C to give α,β-unsaturated ketone 8. α,β- Unsaturated ketone 8 was reduced by abh 4 CeCl 3 to give alcohol 9. This reaction was only slightly stereoselective, and the resulting two diastereomers were separated by flash chromatography. The absolute stereochemistry was determined by Mosher s method reported by Williams + S1

et al. 4 Cyclic carbamates (R)-10 and (S)-10 were obtained, and the coupling constant (J) between the C H proton of the α-carbon and C H proton adjacent to the hydroxyl group was used to determine the absolute stereochemistry (Scheme S2). 4-6 Compound (R)-10 had the synconfiguration with a J value of 7.8 Hz, while compound (S)-10 had the anti-configuration with a J value of 4.1 Hz. The synthetic route to give the Fmoc-Pro-trans-Pro isostere 1 from alcohol (R)-9 is shown in Scheme S3. Alcohol 9 was coupled with 2-(tert-butyldimethylsilyloxy)acetic acid with HBTU as the coupling reagent, and ester 11 was obtained. α-hydroxycarboxylic acid 12 was Scheme S2. Proof of stereochemistry at alcohol carbon of 9. (R)-9 H 1) TFA/CH 2 Cl 2 2) carbonyl diimidazole, THF J = 7.8 Hz (R)-10 (S)-9 H 1) TFA/CH 2 Cl 2 2) carbonyl diimidazole, THF J = 4.1 Hz (S)-10 Scheme S3. Synthesis of the Pro trans Pro alkene isostere 1. H (R)-9 + H TBS HBTU, DMAP DIEA, CH 2 Cl 2 59% 11 TBS 1) LDA, pyridine TMS-Cl, THF 2) TBAF, THF 63% 1) Pb(Ac) 4 1) TFA/CH 2 Cl 2 12 H H 2) Cr 3, H 2 S 4 acetone 56% 13 CH 2) Fmoc-Su 93% Fmoc 1 CH S2

synthesized from ester 11 by an Ireland Claisen rearrangement, followed by TBS-deprotection with TBAF. The stereochemistry was retained through the chair-form 6-membered ring transition state, and the alkene in the product adopted the (E)-configuration, which was confirmed by E spectroscopy. α-hydroxycarboxylic acid 12 was decarboxylated with Pb(Ac) 4 to give the aldehyde, which was oxidized to acid 13 by freshly prepared Jones reagent. The group was removed with TFA, and the Fmoc protecting group was added by stirring with Fmoc Su in saturated ahc 3. The dipeptide isostere Fmoc Pro Ψ[(E)CH=C] Pro H 1 was purified by flash chromatography. The tripeptide building block 17 was synthesized in 5 steps as shown in Scheme S4. Scheme S4. Synthesis of tripeptide building block Fmoc Gly Pro Pro H 17. H H BnH, TsH Bn CCl 4, 100% H TsH 14 -Pro-H HBTU, HBt DIEA, DMAP CH 2 Cl 2, 57% 15 Bn 1) TFA/CH 2 Cl 2 2) HBTU, HBt DIEA, DMAP Fmoc-Gly-H 94% FmocH 16 Bn H 2, 10% Pd-C AcH, MeH 75% FmocH 17 H Experimental Section. General Information. Amino acid derivatives, resins, and reagents were purchased. Unless otherwise indicated, all reactions were carried out under 2 sealed from moisture. Anhydrous THF was obtained by refluxing from a-benzophenone. Anhydrous CH 2 Cl 2 was dried by passage through a dry alumina column. ther anhydrous solvents were used directly from sealed S3

bottles, which were stored under Ar. Brine (acl), ahc 3, a 2 S 2 3, and H 4 Cl refer to saturated aqueous solutions unless otherwise noted. Flash chromatography was performed on 32-63 μm or 230-400 mesh silica gel with reagent grade solvents. Analytical HPLC were obtained on a MetaSil Si 2 5 µm 4.6 mm 10 cm column in isocratic 5% i-prh in hexanes, or Xbridge C18 2.5 µm 4.6 mm 15 cm column with a 5% to 95% CH 3 C/H 2 gradient over 10 min, flow rate 1.0 ml/min, λ = 210 nm, with exceptions noted. 1 H and 13 C MR spectra were obtained at 400 and 100 MHz, respectively, at ambient temperature in CDCl 3 unless otherwise noted. Minor rotamer chemical shifts are shown in parenthesis. Pro (Me)Me, 7. Pro H (6.41 g, 29.8 mmol),,-dimethyl hydroxylamine hydrochloride (5.82 g, 59.6 mmol), HBt (5.47 g, 35.7 mmol) and DCC (7.41 g, 35.9 mmol) were dissolved in CH 2 Cl 2 (300 ml) and cooled to 0 C. DIEA (20.8 ml, 119 mmol) and DMAP (182 mg, 1.48 mmol) were added, and the reaction was stirred for 24 h. The mixture was filtered to remove dicyclohexyl urea and concentrated. The resulting slurry was diluted with EtAc (300 ml), and washed with H 4 Cl (2 100 ml), ahc 3 (2 100 ml), and brine (100 ml). The organic layer was dried over MgS 4, concentrated in vacuo, and purified by flash chromatography (20% EtAc in hexanes). A pale yellow solid was obtained (5.75 g, 74.8 %). mp 46-47 C. HPLC: Si 2 retention time 8.95 min, 97.8%. 1 H MR: δ 4.64 (dd, J = 8.3, 2.9, 0.5H), 4.55 (dd, J = 8.3, 3.7, 0.5H), 3.72 (s, 1.5H), 3.66 (s, 1.5H), 3.51 (m, 1H), 3.35 (m, 1H), 3.14 (s, 3H), 2.14 (m, 1H), 1.80 (m, 3H), 1.40 (s, 4.5H), 1.36 (s, 4.5H). 13 C MR: δ 173.9 (173.3), 153.9 (154.5), 79.5 (79.4), 61.3 (61.4), 56.8 (56.5), 46.9 (46.6), 32.4 (32.3), 30.5 (29.6), 28.4 (28.5), 23.4 (24.1). Rotamers: 1:1. HRMS (m/z): [M + H] + calcd. for C 12 H 23 2 4, 259.1658; S4

found, 259.1655. α, β-unsaturated ketone, 8. 1-Iodocyclopentene (4.35 g, 22.4 mmol) was dissolved in THF (80 ml) at 40 C, and s-buli (1.4 M in cyclohexane, 32.0 ml, 44.8 mmol) was added. The reaction was stirred at 40 C for 3 h to generate cyclopentenyl lithium. In another flask, Pro (Me)Me 7 (3.84 g, 14.9 mmol) was dissolved in THF (30 ml). The solution was cooled to 78 C, and the cyclopentenyl lithium solution was added via cannula. The mixture was stirred at 78 C for 1 h, warmed slowly to room temperature, and stirred for another 10 h. The reaction was quenched with H 4 Cl (10 ml). The resulting solution was diluted with EtAc (100 ml), washed with H 4 Cl (2 20 ml), ahc 3 (20 ml), brine (20 ml), dried over MgS 4, and concentrated. Chromatography with 10% EtAc in hexanes gave a pale yellow solid (1.91 g. 48.7 %). mp 45-49 C. HPLC: Si 2 retention time 2.93 min, 94.8%. 1 H MR: δ 6.82 (m, 0.5H), 6.76 (m, 0.5H), 4.95 (dd, J = 9.0, 3.6, 0.5H), 4.77 (dd, J = 8.8, 4.4, 0.5H), 3.53 (m, 1.5H), 3.41 (m, 0.5H), 2.58 (m, 4H), 2.18 (m, 1H), 1.89 (m, 5H), 1.44 (s, 4H), 1.33 (s, 5H). 13 C MR: δ 197.6 (197.1), 154.0 (154.6), 144.12 (143.52), 143.34 (143.47), 79.7 (79.6), 62.0 (61.5), 46.7 (46.9), 34.32 (34.35), 31.3 (31.1), 30.3, 28.4 (28.6), 23.8 (24.3), 22.63 (22.55). Rotamers: 5:4. HRMS (m/z): [M + H] + calcd. for C 15 H 24 3, 266.1756; found, 266.1759. Alcohol, 9. Ketone 8 (1.20 g, 4.52 mmol) was dissolved in THF:MeH (2.5:1, 65 ml) and cooled to 0 C. CeCl 3 7H 2 (4.21 g, 11.3 mmol) was added, followed by abh 4 (860 mg, 22.7 mmol). After stirring at 0 C for 2.5 h, the reaction was quenched with H 4 Cl (50 ml). The resulting solution was diluted with EtAc (150 ml), washed with H 4 Cl (2 50 ml), and brine (50 ml), dried over MgS 4, concentrated in vacuo, and purified by chromatography (20% S5

EtAc in hexanes). Both (R)-9 (596.1 mg, 49.4 %) and (S)-9 (445.7 mg, 36.9 %) were obtained 25 as pale yellow oils. Diastereomer (R)-9: HPLC: Si 2 retention time 2.03 min, 98.3%. [α] D 46.3 (c 0.36, CHCl 3 ). 1 H MR: δ 5.62 (m, 1H), 4.49 (m, 1H), 4.10 (m, 1H), 3.46 (m, 1H), 3.20 (m, 1H), 2.31 (m, 4H), 1.86 (m, 6H), 1.48 (s, 9H). 13 C MR: δ 156.4, 144.4, 127.3, 80.1, 73.9, 62.0, 48.1, 32.2, 32.1, 28.6, 27.5, 24.3, 24.0. HRMS (m/z): [M + H] + calcd. for C 15 H 26 3, 25 268.1913; found, 268.1908. Diastereomer (S)-9: [α] D 62.6 (c 0.26, CHCl 3 ). HPLC: Si 2 retention time 1.90 min, 96.6%. 1 H MR: δ 5.61 (m, 1H), 5.21 (br s, 1H), 4.15 (m, 1H), 3.98 (m, 1H), 3.42 (m, 1H), 3.33 (m, 1H), 2.52 (m, 1H), 2.31 (m, 2H), 2.21 (m, 1H), 1.83 (m, 5H), 1.61 (m, 1H), 1.48 (s, 9H). 13 C MR: δ 158.2, 145.0, 128.6, 80.7, 76.1, 61.3, 47.5, 32.1, 30.1, 28.6, 28.5, 24.0, 23.4. HRMS (m/z): [M + H] + calcd. for C 15 H 26 3, 268.1913; found, 268.1927. Bicyclic carbamates, (R)-10 and (S)-10. Alcohol (S)-9 (27 mg, 0.099 mmol) was dissolved in CH 2 Cl 2 (3.0 ml), and TFA (1.0) ml was added. The solution was stirred at room temperature for 30 min, and the solvent was removed in vacuo. The resulting oil was dissolved in THF (3.0 ml), and carbonyl diimidazole (0.33 ml, 0.099 mmol) was added. The solution was stirred at room temperature for 24 h, concentrated in vacuo, diluted with EtAc (50 ml), washed with 1 M HCl (2 10 ml), ahc 3 (2 10 ml) and brine (10 ml), dried over a 2 S 4, and purified by chromatography (20% EtAc in hexanes). A colorless oil was obtained (17.8 mg, 92.6 %). Diastereomer (S)-10: [α] 25 D 82.4 (c 0.64, CHCl 3 ). HPLC: Si 2 retention time 5.47 min, 89.1%. 1 H MR: δ 5.80 (m, 1H), 4.88 (d, J = 4.1, 1H), 3.66 (m, 1H), 3.18 (dd, J = 9.2, 4.4, 1H), 3.15 (dd, J = 9.2, 4.6, 1H), 2.38 (m, 4H), 2.06 (m, 2H), 1.94 (m, 3H), 1.53 (m, 1H). 13 C MR: δ 161.3, 141.2, 129.5, 79.6, 63.7, 45.9, 32.5, 30.9, 30.6, 25.9, 23.4. HRMS (m/z): [M + H] + calcd. S6

for C 11 H 16 2, 194.1181; found, 194.1186. (R)-10 was prepared by a similar method. Diastereomer (R)-10: HPLC: Si 2 retention time 5.42 min, 95.3%. 1 H MR: δ 5.82 (dt, J = 4.0, 2.0, 1H), 5.22 (d, J = 7.8, 1H), 3.91 (ddd, J = 10.3, 7.7, 5.8, 1H), 3.67 (dt, J = 11.5, 7.9, 1H), 3.17 (ddd, J = 11.3, 9.5, 4.0, 1H), 2.37 (m, 2H), 2.27 (m, 2H), 1.94 (m, 3H), 1.70 (dddd, J = 12.6, 7.5, 5.5, 1.8, 1H), 1.55 (m, 1H), 1.42 (dddd, J = 12.4, 10.6, 10.6, 8.9, 1H). 13 C MR: δ 166.9, 138.1, 127.8, 75.7, 63.1, 46.1, 32.8, 32.3, 25.8, 25.2, 23.6. HRMS (m/z): [M + H] + calcd. for C 11 H 16 2, 194.1181; found, 194.1187. Ester, 11. Alcohol (R)-9 (263 mg, 0.984 mmol) was dissolved in CH 2 Cl 2 (20 ml) and cooled to 0 C. HBTU (559 mg, 1.47 mmol), DMAP (5.9 mg, 0.048 mmol) and 2-(tertbutyldimethylsilyloxy)acetic acid (385 mg, 2.02 mmol) were added. The mixture was stirred at 0 C for 10 min. DIEA (0.70 ml, 4.0 mmol) was added and the solution was stirred at room temperature for 10 h. The solvent was removed in vacuo and the resulting slurry was diluted with EtAc (50 ml). The organic layer was washed with 1 M HCl (2 25 ml), ahc 3 (25 ml), brine (25 ml), dried over MgS 4, and concentrated. Chromatography with 10% EtAc in hexanes gave a pale yellow solid (254 mg, 58.8 %). [α] 25 D 67.5 (c 0.55, CHCl 3 ). HPLC: Si 2 isocratic hexanes, retention time 1.53 min, 96.4%. 1 H MR: δ 5.93 (br s, 0.5H), 5.90 (br s, 0.5H), 5.51 (m, 1H), 4.25 (d, J = 16.6, 1H), 4.19 (d, J = 16.8, 1H), 4.10 (m, 0.5H), 3.94 (m, 0.5H), 3.46 (m, 0.5H), 3.32 (m, 0.5H), 3.24 (m, 1H), 2.29 (m, 4H), 1.87 (m, 6H), 1.48 (s, 5H), 1.43 (s, 4H), 0.90 (s, 9H), 0.09 (s, 6H). 13 C MR: δ 170.8 (170.7), 154.3 (154.6), 140.8 (140.9), 126.3 (126.8), 79.9 (79.4), 74.4 (73.9), 61.9 (62.0), 58.5 (58.3), 46.9 (47.0), 33.1 (32.9), 32.3, 28.6, 26.2 (25.6), 25.9, 23.9 (24.5), 23.3 (23.4), 18.5, 5.3 ( 5.4). Rotamers: 5:4. HRMS (m/z): S7

[M + H] + calcd. for C 23 H 42 5 Si, 440.2832; found, 440.2805. Pro Ψ[(E)CH=C] Pro H, 13. A solution of LDA (2.0 M in THF, 1.2 ml, 2.3 mmol) was diluted with THF (8.0 ml) and stirred at 100 C for 30 min. A mixture of Me 3 SiCl (0.80 ml, 6.4 mmol) and pyridine (0.56 ml, 6.9 mmol) in THF (4.0 ml), cooled to 100 C, was added dropwise to the LDA solution. After 5 min, a solution of ester 11 (254 mg, 0.581 mmol) in THF (4.0 ml), cooled to 100 C, was added dropwise and the reaction was stirred at 100 C for 25 min, then warmed slowly to room temperature over 2.5 h, and stirred at room temperature for 10 h. Then the mixture was heated to 45 C for 1.5 h. The reaction was quenched with 1 M HCl (10 ml), and the aqueous layer was extracted with Et 2 (2 100 ml). The organic layer was dried over MgS 4 and concentrated to give 271 mg of a yellow oil. Without further purification, the product was dissolved in THF (10 ml), n-bu 4 F (547 mg, 1.73 mmol) in THF (4.0 ml) was added, and the mixture was stirred at 0 C for 5 min, then at room temperature for 3 h. The reaction was quenched with 0.5 M HCl (10 ml). The solution was extracted with EtAc (50 ml), dried over MgS 4 and concentrated. The resulting oil was purified by chromatography with 5% methanol in CHCl 3 and α-hydroxy acid 12 was obtained as a yellow oil (118 mg, 62.9 %). 1 H MR (CD 3 D): δ 5.30 (d, J = 8.5, 1H), 4.33 (m, 1H), 4.07 (m, 1H), 3.34 (t, J = 6.9, 1H), 3.23 (m, 0.5H), 3.12 (m, 0.5H), 2.86 (m, 1H), 2.54 (br s, 1H), 2.17 (m, 1H), 2.04 (m, 1H), 1.84 (m, 4H), 1.69 (m, 3H), 1.56 (m, 1H), 1.41 (s, 9H). Pb(Ac) 4 (162 mg, 0.365 mmol) in CHCl 3 (2.0 ml) was added dropwise to a solution of α-hydroxy acid 12 (118 mg, 0.365 mmol) in EtAc (5.0 ml) at 0 C. The reaction was stirred for 15 min at 0 C, then quenched with ethylene glycol (1.5 ml). The solution was diluted with EtAc (40 ml), washed S8

with H 2 (2 10 ml), and brine (10 ml), dried over anhydrous a 2 S 4, and concentrated to give a yellow oil. The crude product was dissolved in acetone (10 ml), and cooled to 0 C. Freshly prepared Jones reagent (2.7 M Cr 3, 2.7 M H 2 S 4, 0.27 ml, 0.73 mmol) was added dropwise. The reaction was stirred at 0 C for 30 min, quenched with isopropanol (1 ml), and stirred for another 10 min. The precipitate was removed by filtration, and the solvent was evaporated. The resulting residue was extracted with EtAc (3 10 ml), and the organic layer was washed with H 2 (5 ml) and brine (5 ml), dried over a 2 S 4, and concentrated. Chromatography with 5% MeH in CHCl 3 gave a white solid (59.6 mg, 55.6 %). [α] 25 D 62.5 (c 0.20, CHCl 3 ). HPLC: C18 retention time 5.95 min, 88.3%. 1 H MR: δ 5.44 (d, J = 7.5, 1H), 4.28 (m, 1H), 3.34 (m, 3H), 2.52 (m, 1H), 2.24 (m, 1H), 1.90 (m, 6H), 1.61 (m, 2H), 1.40 (s, 9H). 13 C MR: δ 180.1, 154.8, 139.7, 126.3, 79.2, 56.7, 49.4, 46.5, 33.0, 30.1, 28.9, 28.7, 25.3, 23.7. HRMS (m/z): [M + H] + calcd. for C 16 H 26 4, 296.1862; found, 296.1857. Fmoc Pro Ψ[(E)CH=C] Pro H, 1. Pro Ψ[(E)CH=C] Pro H 13 (72.1 mg, 0.245 mmol) was dissolved in CH 2 Cl 2 (3.0 ml) and TFA (1.0 ml) was added. The solution was stirred at room temperature for 40 min. The solvent was evaporated and the excess TFA was removed by high vacuum. The resulting dark yellow oil was dissolved in ahc 3 (8.0 ml) and stirred at 0 C for 20 min. Fmoc Su (124 mg, 0.366 mmol) was dissolved in 1,4-dioxane (3.0 ml) and added dropwise. The solution was stirred at room temperature for 14 h. A solution of 1 M HCl was added to give ph 1. The aqueous solution was extracted with CH 2 Cl 2 (4 50 ml). The organic layers were combined, washed with water (50 ml), and brine (50 ml). After drying with MgS 4, the organic layer was concentrated in vacuo. Chromatography with 5% MeH in CHCl 3 S9

25 gave a pale yellow solid (94.8 mg, 93.0 %). [α] D 40.1 (c 0.83, CHCl 3 ). HPLC: C18 retention time 7.92 min, 97.1%. 1 H MR: δ 7.76 (d, J = 7.7, 2H), 7.61 (d, J = 7.3, 2H), 7.39 (t, J = 7.4, 2H), 7.30 (t, J = 7.5, 2H), 5.59 (m, 0.5H), 5.53 (m, 0.5H), 4.39 (m, 3H), 4.23 (m, 1H), 3.48 (m, 2H), 3.40 (m, 1H), 2.73 (m, 1H), 2.32 (m, 2H), 1.96 (m, 6H), 1.69 (m, 1H). 13 C MR: δ 180.4, (180.1), (155.5), 155.0, 144.3, (142.0), 141.4, (140.9), 127.7, (127.1), 126.2, 125.2, (124.7), 120.0, 67.2 (67.1), 57.2 (56.8), 49.4, 47.5, (47.0), 46.5, (33.2), 32.1, 30.0, (29.1), 25.3, 24.4 23.7, (20.9). Rotamers: 1:1. HRMS (m/z): [M + H] + calcd. for C 26 H 28 4, 418.2018; found, 418.1987. H Pro Bn TsH, 14. H Pro H (3.22 g, 28.0 mmol) was dissolved in a mixture of CCl 4 (25 ml) and BnH (20 ml). TsH (6.39 g, 33.6 mmol) was added. The solution was refluxed for 24 h and then cooled to room temperature. The solution was concentrated in vacuo, and the remaining BnH was removed by Et 2 wash. After drying under high vacuum, a colorless oil (9.4 g, 100 %) was obtained. 1 H MR: δ 7.74 (d, J = 8.0, 2H), 7.31 (m, 5H), 7.14 (d, J = 7.9, 2H), 5.16 (d, J = 12.2, 1H), 5.08 (d, J = 12.2, 1H), 4.56 (m, 1H), 3.48 (m, 2H), 2.34 (s, 3H), 1.97 (m, 4H). 13 C MR: δ 169.0, 141.3, 140.7, 134.7, 129.1, 128.7, 128.5, 126.1, 68.3, 59.6, 46.5, 28.8, 23.6, 21.5. HRMS (m/z): [M + H] + calcd. for C 12 H 16 2, 206.1181; found, 206.1192. Pro Pro Bn, 15. Pro H (6.62 g, 30.8 mmol) was dissolved in CH 2 Cl 2 (200 ml). HBTU (11.7 g, 30.7 mmol), HBt (4.29 g, 30.8 mmol), DMAP (170 mg, 1.4 mmol) and DIEA (19.5 ml, 0.112 mol) were added, and the solution was stirred at 0 C for 10 min. H Pro Bn 14 (9.4 g, 28 mmol) was added, and the solution was stirred at room temperature for 14 h. The resulting solution was concentrated in vacuo, diluted with EtAc (250 ml), washed with 1 M HCl (2 75 ml), H 2 (75 ml), ahc 3 (2 75 ml) and brine (75 ml), dried over a 2 S 4, S10

and purified by chromatography (33% EtAc/hexanes). A colorless oil was obtained (6.40 g, 57%). 1 H MR: δ 7.33 (m, 5H), 5.22 (d, J = 12.2, 0.5H), 5.21 (d, J = 12.4, 0.5H), 5.06 (d, J = 12.2, 0.5H), 5.04 (d, J = 12.3, 0.5H), 4.64 (m, 1H), 4.49 (dd, J = 8.4, 2.9, 0.5H), 4.37 (dd, J = 8.4, 4.2, 0.5H), 3.76 (ddd, J = 9.2, 7.3, 7.3, 0.5H), 3.58 (m, 2.5H), 3.42 (m, 1H), 2.01 (m, 7H), 1.80 (m, 1H), 1.45 (s, 5H), 1.38 (s, 4H). 13 C MR: δ 172.3 (172.0), 171.2 (171.7), 154.7 (153.8), 135.75 (135.67), 128.61 (128.59), 128.30 (128.36), 128.22 (128.26), 79.6 (79.5), 66.8 (67.0), 58.9, 57.8 (57.7), 46.9 (46.7), 46.5 (46.6), 29.1 (30.0), 28.8 (28.9), 28.6 (28.4), 25.0 (25.1), 24.1 (23.6). Rotamers: 5:4. Fmoc Gly Pro Pro Bn, 16. Pro Pro Bn 15 (6.40 g, 15.9 mmol) was dissolved in CH 2 Cl 2 (100 ml), and TFA (30 ml) was added. The solution was stirred at room temperature for 30 min, and the solvent was removed in vacuo. The resulting slurry was dissolved in CH 2 Cl 2 (200 ml), and HBTU (7.35 g, 19.4 mmol), HBt (2.92 g, 19.1 mmol), DMAP (97 mg, 0.79 mmol) and DIEA (11.1 ml, 63.6 mmol) were added, and stirred at 0 C for 20 min. Fmoc Gly H (5.50 g, 18.5 mmol) was added, and the solution was stirred at room temperature for 14 h. The resulting solution was concentrated in vacuo, diluted with EtAc (250 ml), washed with 1 M HCl (2 100 ml), H 2 (100 ml), ahc 3 (2 100 ml) and brine (100 ml), dried over a 2 S 4, and purified by chromatography (2% MeH/CHCl 3 ). A white solid (8.67 g, 93.8 %) was obtained. 1 H MR: δ 7.75 (d, J = 7.4, 2H), 7.59 (dd, J = 7.4, 3.0, 2H), 7.39 (t, J = 7.3, 2H), 7.33 (m, 7H), 5.75 (t, J = 4.0, 1H), 5.21 (d, J = 12.3, 1H), 5.06 (d, J = 12.2, 1H), 4.66 (ddd, J = 15.4, 8.0, 3.6, 2H), 4.35 (m, 2H), 4.21 (t, J = 7.3, 1H), 4.10 (dd, J = 17.3, 5.4, 1H), 3.97 (dd, J = 17.3, 3.6, 1H), 3.80 (m, 1H), 3.59 (m, 2H), 3.44 (m, 1H), 2.16 (m, 3H), 1.98 (m, 5H). 13 C MR: S11

δ 172.0, 170.2, 166.9, 156.3, 144.0, 141.4, 135.7, 128.6, 128.4, 128.2, 127.8, 127.2, 125.3, 120.0, 67.2, 67.0, 59.0, 58.2, 47.2, 46.8, 46.3, 43.4, 28.9, 28.2, 25.0, 24.6. Fmoc Gly Pro Pro H, 17. Fmoc Gly Pro Pro Bn 16 (8.56 g, 14.7 mmol) was dissolved in a mixture of MeH (75 ml) and AcH (75 ml), and 10% (w/w) Pd/C (520 mg) was added. H 2 was introduced with a balloon, and the suspension was stirred at room temperature for 2 h. The solution was filtered through Celite, and concentrated in vacuo. The resulting oil was purified by chromatography (5% MeH/CHCl 3 ), and a white solid was obtained (5.42 g, 75.0 %). 1 H MR: δ 10.02 (br s, 1H). 7.74 (d, J = 7.4, 2H), 7.60 (t, J = 6.6, 2H), 7.37 (t, J = 7.4, 2H), 7.28 (app. t, J =7.4, 2H), 5.95 (m, 1H), 4.62 (m, 1H), 4.56 (app. dd, J = 8.2, 3.2, 1H), 4.35 (d, J = 7.4, 2H), 4.20 (t, J = 7.1, 1H), 4.09 (dd, J = 17.1, 6.1, 1H), 3.90 (dd, J = 17.0, 3.0, 1H), 3.76 (m, 1H), 3.57 (m, 2H), 3.42 (m, 1H), 2.15 (m, 2H), 2.01 (m, 5H), 1.90 (m, 1H). 13 C MR: δ 174.0, 171.1, 167.4, 156.5, 143.9 (143.8), 141.17 (141.20), 127.6, 127.0, 125.2 (125.1), 120.0, 67.0, 59.1, 58.1, 47.0, 46.9, 46.3, 43.2, 38.6, 28.5, 28.1, 24.8 (24.6). HRMS (m/z): [M + H] + calcd. for C 27 H 30 3 6, 492.2135; found, 492.2130. References (1) Dai,.; Wang, X. J.; Etzkorn, F. A. J. Am. Chem. Soc. 2008, 130, 5396-5397. (2) Wang, X. J.; Xu, B.; Mullins, A. B.; eiler, F. K.; Etzkorn, F. A. J. Am. Chem. Soc. 2004, 126, 15533-15542. (3) Bandur,. G.; Harms, K.; Koert, U. Synlett 2005, 2005, 773-776. (4) Williams, T. M.; Crumbie, R.; Mosher, H. S. J. rg. Chem. 1985, 50, 91-97. (5) Hart, S. A.; Sabat, M.; Etzkorn, F. A. J. rg. Chem. 1998, 63, 7580-7581. (6) Wang, X. J.; Hart, S. A.; Xu, B.; Mason, M. D.; Goodell, J. R.; Etzkorn, F. A. J. rg. Chem. 2003, 68, 2343-2349. S12

Table S1. T m values of isostere peptide 2 and control peptide 3 in different concentrations of TMA. Data plots are shown in Figure 4 of article. Peptide 2 [TMA] (M) 0 calcd. 2.5 3.0 3.2 3.5 T m ( C) 22.0 ± 1.9 14.4 22.1 24.6 29.3 Peptide 3 [TMA] (M) 0 calcd. 0 0.5 1.0 3.0 T m ( C) 31.6 31.5 34.2 37.3 48.3 60 50 40 Det 168-210nm DAI-VI-P91-PURE ame Retention Time Area Percent 3.067 0.566 7.350 11.739 9.400 0.698 60 50 40 30 30 mau 20 10 2.000 1.245 2.817 0.826 20 10 mau 0 0-10 -20 8.283 10.101 11.800 13.607-10 -20 0 1 2 3 4 5 6 7 8 9 10 11 12 Minutes 2: H (Pro Pro Gly) 4 Pro Ψ[(E)CH=C]Pro Gly (Pro Pro Gly) 5 H 120 Det 168-210nm DAI-VI-P89-PURE ame Retention Time Area Percent 120 100 100 80 80 mau 60 60 mau 40 40 20 20 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 Minutes 3: H (Pro Pro Gly) 10 -H Figure S1. Analytical HPLC chromatograms of peptide 2 (top), 3 (bottom) after purification. S13

800 Det 168-210nm DAI-III-P9 ame Retention Time Area Percent 800 700 700 600 600 500 500 mau 400 7 400 mau 300 300 200 6.450 2.202 200 100 0 8.950 97.798 100 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Minutes 200 180 Det 168-210n m DAI-V-P29-I Retention Time Area Percent 200 180 160 160 140 8 140 120 120 100 100 mau mau 80 80 60 60 40 40 20 0 1.300 0.402 1.533 0.564 1.783 0.332 1.983 2.069 2.933 94.771 7.233 1.670 8.333 0.192 20 0-20 -20 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Minutes S14

1000 Det 168-210nm DAI-VI-P103-crude ame Retention Time Area Percent 1000 800 800 600 1.867 33.853 H H Mixture of (R)-9 + (S)-9 600 mau mau 400 400 200 0 2.017 65.263 2.300 0.885 200 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Minutes 1200 1000 800 Det 168-210nm DAI-VI-P103-II-R ame Retention Time Area Percent (R)-9 H 1200 1000 800 mau 600 600 mau 400 400 200 0 1.283 1.721 2.033 98.279 200 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Minutes S15

1200 Det 168-210nm DAI-VI-P103-I-S ame Retention Time Area Percent 1200 1000 800 (S)-9 H 1000 800 mau 600 600 mau 400 400 200 0 1.317 3.359 1.900 96.641 200 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Minutes 350 300 250 Det 168-210nm DAI-VI-P111-II ame Retention Time Area Percent (S)-10 350 300 250 200 200 mau mau 150 150 100 100 50 0 2.033 0.326 2.300 0.241 2.750 0.157 4.533 0.754 5.467 89.090 7.050 1.077 8.733 1.646 10.667 1.458 12.267 0.733 14.250 1.272 50 0 0 2 4 6 8 10 12 14 16 18 20 22 Minutes S16

40 35 30 25 Det 168-210nm DAI-VI-P117-II ame Retention Time Area Percent (R)-10 40 35 30 25 20 20 mau 15 15 mau 10 5 4.250 4.035 4.917 0.116 10 5 0-5 5.417 81.463 0-5 -10-10 0 2 4 6 8 10 12 14 16 18 20 22 Minutes 600 Det 168-210nm DAI-VI-P109-I ame Retention Time Area Percent 600 500 TBS 500 11 400 400 mau 300 300 mau 200 200 100 0 1.333 3.578 1.533 96.422 100 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Minutes S17

250 200 Det 168-210nm DAI-VI-P121 ame Retention Time Area Percent 250 200 CH 150 13 150 mau 100 100 mau 50 0 0.733 0.492 0.983 0.641 1.183 0.562 1.683 1.210 1.967 10.985 4.267 0.904 4.550 1.300 4.817 1.153 5.033 0.608 5.217 1.507 5.950 56.720 6.233 1.664 6.500 1.010 6.717 5.993 7.833 0.730 7.967 1.496 8.233 2.268 8.667 1.759 9.000 1.019 9.317 2.984 9.917 3.714 11.500 0.380 12.083 0.070 12.700 0.034 13.750 0.024 14.433 0.009 14.667 0.022 14.967 0.183 15.350 0.008 15.567 0.013 16.150 0.017 16.417 0.010 16.600 0.012 16.933 0.011 17.217 0.016 17.550 0.017 17.883 0.011 18.183 0.042 18.500 0.012 19.833 0.178 50 0-50 -50 0 2 4 6 8 10 12 14 16 18 20 Minutes 2000 1750 1500 Det 168-210nm DAI-VI-P123 ame Retention Time Area Percent Area Height Percent Fmoc CH 2000 1750 1500 1250 1 1250 1000 1000 mau mau 750 500 250 0 5.900 0.110 32867 0.461 6.600 0.168 49893 0.741 7.917 97.124 28917470 90.370 8.667 0.406 120795 0.975 9.483 1.770 526988 6.349 11.117 0.289 86076 0.746 12.750 0.016 4760 0.059 13.600 0.117 34767 0.300 750 500 250 0-250 -250 0 2 4 6 8 10 12 14 16 18 20 22 24 Minutes S18

7 S19

7 S20

8 S21

8 S22

(R)-9 H S23

H (R)-9 S24

H (S)-9 S25

H (S)-9 S26

(S)-10 S27

(S)-10 S28

(S)-10 CSY S29

(S)-10 HSQC: CH 2 CH S30

(R)-10 S31

(R)-10 S32

(R)-10 CSY S33

11 TBS S34

11 TBS S35

12 H H S36

13 CH S37

13 CH S38

13 CH S39

CH 13 HSQC red: CH blue: CH 2 S40

d c a 13 a b CH c b d S41

Fmoc 1 CH S42

CSY Fmoc 1 CH S43

Fmoc 1 CH S44

Fmoc 1 CH HSQC: CH 2 red CH blue S45

h i g f e d c b a CH e j k f j a b g,h TMS S46

H TsH 14 Bn S47

H TsH 14 Bn S48

15 Bn S49

15 Bn S50

FmocH 16 Bn S51

FmocH 16 Bn S52

FmocH 17 H S53

FmocH 17 H S54