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

Transcription

1 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 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

2 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

3 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

4 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 μm or 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 C 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 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 (173.3), (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 , ; S4

5 found, 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 %). mp 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 (197.1), (154.6), (143.52), (143.47), 79.7 (79.6), 62.0 (61.5), 46.7 (46.9), (34.35), 31.3 (31.1), 30.3, 28.4 (28.6), 23.8 (24.3), (22.55). Rotamers: 5:4. HRMS (m/z): [M + H] + calcd. for C 15 H 24 3, ; found, 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

6 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, HRMS (m/z): [M + H] + calcd. for C 15 H 26 3, ; found, 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, HRMS (m/z): [M + H] + calcd. for C 15 H 26 3, ; found, Bicyclic carbamates, (R)-10 and (S)-10. Alcohol (S)-9 (27 mg, 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, 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, HRMS (m/z): [M + H] + calcd. S6

7 for C 11 H 16 2, ; found, (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, HRMS (m/z): [M + H] + calcd. for C 11 H 16 2, ; found, Ester, 11. Alcohol (R)-9 (263 mg, 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, 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 (170.7), (154.6), (140.9), (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

8 [M + H] + calcd. for C 23 H 42 5 Si, ; found, 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, 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, mmol) in CHCl 3 (2.0 ml) was added dropwise to a solution of α-hydroxy acid 12 (118 mg, 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

9 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, HRMS (m/z): [M + H] + calcd. for C 16 H 26 4, ; found, Fmoc Pro Ψ[(E)CH=C] Pro H, 1. Pro Ψ[(E)CH=C] Pro H 13 (72.1 mg, 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, 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

10 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, , (20.9). Rotamers: 1:1. HRMS (m/z): [M + H] + calcd. for C 26 H 28 4, ; found, 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, HRMS (m/z): [M + H] + calcd. for C 12 H 16 2, ; found, 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, 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

11 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 (172.0), (171.7), (153.8), (135.67), (128.59), (128.36), (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

12 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, 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 (br s, 1H) (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, (143.8), (141.20), 127.6, 127.0, (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 , ; found, References (1) Dai,.; Wang, X. J.; Etzkorn, F. A. J. Am. Chem. Soc. 2008, 130, (2) Wang, X. J.; Xu, B.; Mullins, A. B.; eiler, F. K.; Etzkorn, F. A. J. Am. Chem. Soc. 2004, 126, (3) Bandur,. G.; Harms, K.; Koert, U. Synlett 2005, 2005, (4) Williams, T. M.; Crumbie, R.; Mosher, H. S. J. rg. Chem. 1985, 50, (5) Hart, S. A.; Sabat, M.; Etzkorn, F. A. J. rg. Chem. 1998, 63, (6) Wang, X. J.; Hart, S. A.; Xu, B.; Mason, M. D.; Goodell, J. R.; Etzkorn, F. A. J. rg. Chem. 2003, 68, S12

13 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 T m ( C) 22.0 ± Peptide 3 [TMA] (M) 0 calcd T m ( C) Det nm DAI-VI-P91-PURE ame Retention Time Area Percent mau mau Minutes 2: H (Pro Pro Gly) 4 Pro Ψ[(E)CH=C]Pro Gly (Pro Pro Gly) 5 H 120 Det nm DAI-VI-P89-PURE ame Retention Time Area Percent mau mau Minutes 3: H (Pro Pro Gly) 10 -H Figure S1. Analytical HPLC chromatograms of peptide 2 (top), 3 (bottom) after purification. S13

14 800 Det nm DAI-III-P9 ame Retention Time Area Percent mau mau Minutes Det n m DAI-V-P29-I Retention Time Area Percent mau mau Minutes S14

15 1000 Det nm DAI-VI-P103-crude ame Retention Time Area Percent H H Mixture of (R)-9 + (S) mau mau Minutes Det nm DAI-VI-P103-II-R ame Retention Time Area Percent (R)-9 H mau mau Minutes S15

16 1200 Det nm DAI-VI-P103-I-S ame Retention Time Area Percent (S)-9 H mau mau Minutes Det nm DAI-VI-P111-II ame Retention Time Area Percent (S) mau mau Minutes S16

17 Det nm DAI-VI-P117-II ame Retention Time Area Percent (R) mau mau Minutes 600 Det nm DAI-VI-P109-I ame Retention Time Area Percent TBS mau mau Minutes S17

18 Det nm DAI-VI-P121 ame Retention Time Area Percent CH mau mau Minutes Det nm DAI-VI-P123 ame Retention Time Area Percent Area Height Percent Fmoc CH mau mau Minutes S18

19 7 S19

20 7 S20

21 8 S21

22 8 S22

23 (R)-9 H S23

24 H (R)-9 S24

25 H (S)-9 S25

26 H (S)-9 S26

27 (S)-10 S27

28 (S)-10 S28

29 (S)-10 CSY S29

30 (S)-10 HSQC: CH 2 CH S30

31 (R)-10 S31

32 (R)-10 S32

33 (R)-10 CSY S33

34 11 TBS S34

35 11 TBS S35

36 12 H H S36

37 13 CH S37

38 13 CH S38

39 13 CH S39

40 CH 13 HSQC red: CH blue: CH 2 S40

41 d c a 13 a b CH c b d S41

42 Fmoc 1 CH S42

43 CSY Fmoc 1 CH S43

44 Fmoc 1 CH S44

45 Fmoc 1 CH HSQC: CH 2 red CH blue S45

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

47 H TsH 14 Bn S47

48 H TsH 14 Bn S48

49 15 Bn S49

50 15 Bn S50

51 FmocH 16 Bn S51

52 FmocH 16 Bn S52

53 FmocH 17 H S53

54 FmocH 17 H S54