Azidoproline containing Helices Stabilization of the Polyproline II Structure by a Functionalizable Group

Transcription

1 Azidoproline containing Helices Stabilization of the Polyproline II Structure by a Functionalizable Group Michael Kümin, Louis-Sebastian Sonntag, Helma Wennemers* Department of Chemistry, University of Basel St. Johanns Ring 19, CH-4056 Basel (Switzerland) Fax: Helma.Wennemers@unibas.ch Supporting information Table of contents: General aspects General protocols for solid phase syntheses and CD-spectroscopic analyses Syntheses and analytical data of of 2R, 2S and Ac-[Pro] 9 -H Syntheses and analytical data of 3R, 4R and 5R Syntheses and analytical data of 6R S2 S2 S4 S6 S11 S1

2 General aspects: Materials and reagents were of the highest commercially available grade and used without further purification. Reactions were monitored by thin layer chromatography using Merck silica gel 60 F 254 plates. Compounds were visualized by UV and ninhydrin. Flash chromatography was performed using Merck silica gel 60, particle size µm. 1 H and 13 C MR spectra were recorded on Bruker DPX 500 and DPX 400 spectrometers. Chemical shifts are reported in ppm using TMS as a reference. Finnigan MAT LCQ and TSQ 700 instruments were used for electrospray ionization (ESI) mass spectrometry measurements. Analytical HPLC was performed using a LiChrospher 100 RP-18e 5 µm (250 mm x 4 mm) column from Merck. Preparative HPLC was carried out on a LiChrospher RP-18e 5 µm (250 mm x 10 mm) column from Merck. DS62 spectropolarimeter (Aviv Associates, Lakewood, J) and Chirascan (Applied Biophysics Ltd, Leatherhead, UK) were used for CD measurements. For automated peptide synthesis, a Syro I Peptide Synthesizer (MultiSynTech GmbH, Witten, Germany) was employed. General protocols for solid phase syntheses and CD-spectroscopic analyses: General procedure for peptide couplings: Fmoc-Xxx-H (3 eq) and HCTU (3 eq) dissolved in DMF followed by i Pr 2 Et (9 eq) were added to the amino-functionalized resin in DMF ( 100 mm concentration). The mixture was agitated for 1.5 h and washed with DMF (3x) and CH 2 Cl 2 (5x). For couplings on the peptide synthesizer, Hünig`s base was dissolved in -methylpyrrolidone (3M) and the washing with CH 2 Cl 2 was omitted. General procedure for peptide couplings on 2-chlorotrityl chloride resin: Fmoc-Xxx- H (4 eq) and i Pr 2 Et (4 eq) were added to a suspension of the resin in CH 2 Cl 2, agitated for 1 h and washed with a mixture of CH 2 Cl 2 /MeH/ i Pr 2 Et (17:2:1, 3x), CH 2 Cl 2 (3x), DMF (3x) and again CH 2 Cl 2 (5x). The loading was determined with a quantitative Fmoc test. S2

3 General procedure for Fmoc-deprotections: 20% piperidine in DMF was added to the resin (preswollen in DMF) and the reaction mixture was agitated for 3 min, drained and the piperidine treatment repeated for 10 min. Finally the resin was washed with DMF (7x) and CH 2 Cl 2 (5x). Couplings and deprotections were monitored by the qualitative Chloranil test. 1 General procedure for acetylation: Et 3 (20 eq) and 20 eq Ac 2 (20 eq) were added to the resin in CH 2 Cl 2 ( 120 mm). The mixture was agitated for 1 h and washed with CH 2 Cl 2 (7x). General procedure for cleavage of peptides from the solid support: The solid supported peptides were cleaved off the resin by stirring in a mixture of acid in CH 2 Cl 2 (Wang resin (TFA:CH 2 Cl 2 1:2), Rink Amide resin (TFA:CH 2 Cl 2 2:1) and 2-chlorotrityl chloride resin (CH 2 Cl 2 :AcH:TFE 8:1:1)) for 1 h and a second time for 20 min. Pooling of filtrates and removal of all volatiles under reduced pressure followed by precipitation with Et 2 afforded the peptides. General procedure for CD-spectroscopic analysis: CD spectra were recorded using a spectral bandwidth of 1.5 nm (AVIV) and 1 nm (Chirascan) respectively, at 25 C with a time constant of 5 s and a step resolution of 1 nm. CD data are given as mean residual molar ellipticities (Θ MRW in deg cm 2 dmol -1 ). The spectra are the result of 2-4 accumulations. A Quartz cell with a path length of 2 mm was used with solutions containing approximately 70 µgml -1 (6x10-4 M per residue) peptide solutions. For the spectra in buffer and n-prh the blank spectrum of the solution was subtracted. All samples were equilibrated for at least 12 h before measurement. S3

4 Syntheses and analytical data of 2R, 2S and Ac-[Pro] 9 -H Ac[(4R)Azp] 9 H (2R) and Ac[(4S)Azp] 9 H (2S) were prepared on 2-chlorotrityl chloride resin on a 32 µmol scale on a Syro I peptide synthesizer from MultiSynTech using Fmoc-(4R)Azp-H and Fmoc-(4S)Azp-H, respectively. The syntheses were accomplished following the general procedures for solid phase syntheses using HATU instead of HCTU as coupling reagent after an initial coupling onto chlorotrityl resin. After the initial coupling and capping of unreacted sites, a quantitative Fmoc test revealed a loading of 0.40 mmolg -1. After cleavage from the solid support, the crude peptides were further purified by preparative HPLC on a LiChrospher RP-18e 5 µm (250 mm x 10 mm) column: gradient: 75% to 45% of solvent B (0.1% TFA in H 2 ) in solvent A (CH 3 C) over 30 min at a flow rate of 5 ml/min. Analytical HPLC: t R = 22.9 min (2R), 16.6 min (2S),; gradient: 80% to 50% solvent B over 25 min at a flow rate of 1 ml/min. 2R: 1 H-MR (400 MHz, D 2, 25 C): δ = 4.40 (m, 9H; Hγ), 3.89 (m, 8H; Hδ), 3.76 (m, 1H; Hδ), 3.71 (m, 8H; Hδ), 3.61 (m, 1H; Hδ), 2.36 (m, 9H; Hβ), (m, 9H; Hβ), 1.98 (s, 3H, Ac), the signals for Hα are together with the solvent signal at ~ MS (ESI, neg): m/z (%): (100) [M-H] -. 2S: 1 H-MR (400 MHz, D 2, 25 C): δ = 4.31 (m, 9H; Hγ), 4.01 (m, 7H; Hδ), 3.92 (m, 1H; Hδ), 3.81 (m, 1H; Hδ), 3.51 (m, 1H; Hδ), 3.41 (m, 8H; Hδ) 2.59 (m, 8H; Hβ), 2.44 (m, 1H; Hβ), 2.12 (m, 1H; Hβ), 1.97 (s, 3H, Ac), 1.91 (m, 8H; Hβ), the signals for Hα are together with the solvent signal at ~ MS (ESI, neg): m/z (%): (100) [M-H] -. Ac-[Pro] 9 -H: Ac[Pro] 9 H was prepared on 2-chlorotrityl chloride resin on a 120 µmol scale following the standard protocols for coupling and Fmoc-deprotection on a Syro I peptide synthesizer from MultiSynTech using only DMF for the washing steps. After acetylation the peptide was cleaved from the resin purified by preparative HPLC: gradient: 95% to 77% solvent B over 23 min at a flow rate of 5 ml/min. Analytical HPLC: t R = 14.2 min; gradient: 93% to 70% solvent B over 23 min at a flow rate of 1 ml/min. 1 H- S4

5 MR (400 MHz, D 2, 25 C): δ = 4.58 (m, overlap with solvent signal; Hα), 4.29 (dd, J = 8.7 Hz, 4.4 Hz, 1H; Hα), 3.70 (m, 8H; Hδ), 3.50 (m, 10H; Hδ), 2.22 (m, 9H; Hβ), 1.96 (s, 3H; Ac) (m, 27H; Hγ, Hβ). 13 C-MR (100 MHz, D 2, 22 C): δ = 172.0, 58.9, 48.0, 28.3, 24.9, (main signals) MS (ESI): m/z (%) = (50) [M+a] +, (100) [M+2a-H] +. CD spectra of Ac-[Pro] 9 -H CD-spectra in aqueous phosphate buffer (10 mm, ph 7.2) ( ), 25% vol/vol n-prh in buffer ( ), 50% n-prh ( ), 75% n-prh ( ), 85% n-prh ( ), 90% n-prh ( ), 95% n-prh ( ), and n-prh ( ). Spectra were recorded at concentrations of 6x10-4 M per residue at 25 C [Θ] (10 3 deg cm 2 dmol -1 ) Wavelength (nm) S5

6 Syntheses and analytical data of 3R, 4R and 5R 1.) Synthesis of the tripeptidic building block Fmoc-Pro-(4R)Azp-Pro-H 5R 3R was prepared by successive couplings of Fmoc-Pro-(4R)Azp-Pro-H 5R on Wang resin following the general procedures for solid phase synthesis. 5R was prepared by standard peptide synthesis in solution phase, following the route depicted in Scheme A. H Boc C 2CH 3 a) 95% 3 Boc C 2CH 3 7R b) 82% Boc 8R 3 C 2 CH 3 3 c) 42% Fmoc 5R C 2 H Scheme A: Synthesis of the tripeptide 5R. a) i) 1.2 eq CH 3 S 3 H, 2.0 eq DIAD, 1.8 eq PPh 3, 0.4 eq Et 3, toluene, 0-70 C; ii) 5.0 eq a 3, DMF, 80 C; b) i) 4M HCl in dioxane, RT; ii) 1.2 eq Boc-Pro-H, 1.5 eq EDC, 1.2 eq i Pr 2 Et, CH 2 Cl 2, RT; c) i) 2.1 eq ah, H 2, THF, CH 3 H, RT; ii) 1.2 eq H-Pro-CH 3, 1.5 eq EDC, 1.2 eq i Pr 2 Et, CH 2 Cl 2, RT; iii) repeat steps bi) and ci); iv) 1.2 eq Fmoc-Cl, H 2, dioxane, ahc 3, RT. Boc-(4R)Azp-CH 3, (7R): 2,3 Methanesulfonic acid (3.2 ml, 49.3 mmol) and Et 3 (2.3 ml, 16.5 mmol) were successively added to a solution of PPh 3 (19.3 g, 73.6 mmol) in dry toluene (40 ml). The solution was cooled with an ice bath and a suspension of Boc- (4R)Hyp-CH 3 8 (10.0 g, 40.8 mmol) in dry toluene (50 ml) was added followed by slow addition of DIAD (15.8 ml, 81.5 mmol). Cooling was removed and the reaction mixture was heated for 3h at 70 C. The mixture was extracted with CH 2 Cl 2 (200 ml) and sat. ahc 3 (150 ml). The aqueous layer was extracted with CH 2 Cl 2 (2x 150 ml), the combined organic layers were dried over a 2 S 4 and all volatiles were removed at reduced pressure. The residual yellow oil was dissolved in DMF (100 ml), a 3 (13.5 g, 208 mmol) was added and the reaction mixture was stirred over night at 80 C. The S6

7 solvent was removed by evaporation at reduced pressure, Et 2 (200 ml) was added and the mixture was extracted with sat. ahc 3 (200 ml). The aqueous layer was extracted with Et 2 (3x 150 ml) and the combined organic layers were dried over a 2 S 4. The volume of the solution was reduced in vacuo and after addition of pentane and seed crystals of triphenylphosphine oxide the bulk triphenylphosphine oxide was removed by filtration. All volatiles were removed at reduced pressure and the residue was filtered through silica gel (pentanes:etac 4:1 then 3:1). 7R (11.1 g, 41.0 mmol, 95%) was obtained as a colorless oil. ( 1 H MR- and 13 C MR- spectra show a double set of peaks ( 1.5 : 1) due to the s-cis and s-trans conformers around the tertiary carbamate.). major conformer: 1 H-MR (400 MHz, CDCl 3, 25 C): δ = 4.31 (dd, J = 8.8 Hz, 4.4 Hz, 1H; Hα), 4.15 (m, 1H; Hγ), 3.75 (s, 3H; CH 3 ), 3.70 (m, 1H; Hδ), 3.45 (m, 1H; Hδ), 2.49 (m, 1H; Hβ) 2.16 (m, 1H; Hβ), 1.41 (s, 9H; tbu). 13 C-MR (100 MHz, CDCl 3, 22 C): δ = 172.9, 153.3, 80.6, 58.7, 57.7, 52.1, 51.2, 36.2, minor conformer: 1 H-MR (400 MHz, CDCl 3, 25 C): δ = 4.42 (dd, J = 8.9 Hz, 3.8 Hz, 1H; Hα), 4.15 (m, 1H; Hγ), 3.75 (s, 3H; CH 3 ), 3.70 (m, 1H; Hδ), 3.45 (m, 1H; Hδ), 2.49 (m, 1H; Hβ) 2.16 (m, 1H; Hβ), 1.47 (s, 9H; tbu); 13 C-MR (100 MHz, CDCl 3, 22 C): δ = 172.7, 153.9, 80.6, 59.2, 57.3, 52.3, 51.3, 35.3, MS (ESI): m/z (%): (100) [M+a] +. Boc-Pro-(4R)Azp-CH 3, (8R): Boc-Pro-(4R)Azp-CH 3 8R was prepared starting from Boc-(4R)Azp-CH 3 7R by standard protocols for Boc-deprotection followed by coupling with Boc-Pro-H using EDC as coupling reagent. 8R and was obtained as a white solid in a yield of 82 % (20.2 g, 55 mmol) over two steps. (f the four possible conformers due to cis-trans isomerism around the tertiary amide and carbamate only two are visible in the 1 H MRand 13 C MR-spectra in a ratio of 1 : 1) conformer A: 1 H-MR (500 MHz, CDCl 3, 23 C): δ = 4.65 (dd, J = 8.9 Hz, 4.3 Hz, 1H; Azp-Hα), 4.45 (dd, J = 8.2 Hz, 3.1 Hz, 1H; Pro-Hα), 4.29 (m, 1H; Azp-Hγ), 3.85 (m, 1H; Azp-Hδ), 3.79 (m, 1H; Azp-Hδ), 3.72 (s, S7

8 3H; CH 3 ), 3.54 (m, 1H; Pro-Hδ), 3.39 (m, 1H; Pro-Hδ), 2.26 (m, 1H; Azp-Hβ), 2.20 (m, 1H; Azp-Hβ), 2.11 (m, 1H; Pro-Hβ), 2.06 (m, 1H, Pro-Hβ), 1.97 (m, 1H; Pro-Hγ), 1.86 (m, 1H; Pro-Hγ), 1.44 (s, 9H; tbu). conformer B: 1 H-MR (500 MHz, CDCl 3, 23 C): δ = 4.65 (dd, J = 8.9 Hz, 4.3 Hz, 1H; Azp-Hα), 4.35 (m, 2H; Pro-Hα, Azp-Hγ), 3.79 (m, 1H; Azp-Hδ), 3.74 (s, 3H; CH 3 ), 3.63 (dd, J = 10.0 Hz, 3.6 Hz, 1H; Azp-Hδ), 3.58 (m, 1H; Pro-Hδ), 3.46 (m, 1H; Pro-Hδ), 2.26 (m, 1H; Azp-Hβ), 2.20 (m, 1H; Azp- Hβ), 2.18 (m, 1H; Pro-Hβ), 2.16 (m, 1H, Pro-Hβ), 1.97 (m, 1H; Pro-Hγ), 1.86 (m, 1H; Pro-Hγ), 1.41 (s, 9H; tbu). 13 C-MR (126 MHz, CDCl 3, 23 C): both conformers δ = 172.3, 172.1, 171.5, 171.3, 154.5, 153.7, 79.8, 79.6, 59.8, 59.2, 57.9, 57.6, 57.5, 52.5, 52.4, 51.4, 51.0, 46.8, 46.5, 34.1, 34.0, 29.9, 29.7, 28.5, 28.3, 24.1, MS (ESI): m/z (%): (100) [M+a] +. Fmoc-Pro-(4R)Azp-Pro-H, (5R): Fmoc-Pro-(4R)Azp-Pro-H 5R was prepared starting from Boc-Pro-(4R)Azp-CH 3 8R by standard protocols for methyl ester hydrolysis, followed by coupling with H-Pro- CH 3 using EDC as coupling reagent to obtain Boc-Pro-(4R )Azp-Pro-CH 3. Methylester hydrolysis and Boc deprotection followed by protection of the resulting secondary amine with Fmoc-Cl yielded 5R as a white solid in a yield of 42 % over five steps on a 18 mmol scale. (f the eight possible conformers due to cis-trans isomerism around the tertiary amides and carbamate only two are visible in the 1 H MR- and 13 C MR-spectra in a ratio of 2.5 : 1) major conformer: 1 H-MR (500 MHz, CDCl 3, 23 C): δ = 7.76 (m, 2H; Fmoc), 7.57 (m, 2H; Fmoc), 7.39 (m, 2H; Fmoc), 7.30 (m, 2H; Fmoc), 4.82 (dd, J = 7.9 Hz, 5.8 Hz, 1H; Azp-Hα), (m, 4H; Pro C -Hα, Fmoc, Pro -Hα, Azp-Hγ), 4.38 (m, 1H; Fmoc), 4.25 (dd, J = 7.3 Hz, 7.3 Hz, 1H; Fmoc), 3.90 (dd, J = 10.3 Hz, 6.1 Hz, 1H; Azp-Hδ), 3.84 (m, 1H; Pro C -Hδ), 3.79 (m, 1H; Azp-Hδ), 3,69 (m, 1H; Pro -Hδ), (m, 2H; Pro -Hδ, Pro C -Hδ), (m, 10H; Azp- Hβ, Pro C -Hβ, Azp-Hβ, Pro C -Hβ, Pro -Hβ, Pro -Hγ, Pro C -Hγ, Pro -Hγ, Pro C -Hγ, Pro - S8

9 Hβ); 13 C-MR (126 MHz, CDCl 3, 23 C): δ = 173.6, 171.7, 171.3, 155.1, 144.3, 144.0, 141.4, 127.8, 127.2, 127.2, 127.2, 125.4, 125.3, 120.1, 67.7, 59.8, 59.6, 58.3, 56.9, 51.7, 47.7, 47.5, 47.4, 46.9, 33.7, 29.1, 28.2, 25.1, 24.6, minor conformer: 1 H-MR (500 MHz, CDCl 3, 23 C): δ = 7.76 (m, 2H; Fmoc), 7.57 (m, 2H; Fmoc), 7.39 (m, 2H; Fmoc), 7.30 (m, 2H; Fmoc), 4.56 (m, 1H; Azp-Hα), 4.53 (m, 1H; Pro C -Hα,), 4.39 (m, 1H; Azp-Hγ), (m, 2H; Fmoc), (m, 2H; Pro -Hα, Fmoc), 3.79 (m, 1H, Pro-Hδ), 3.69 (m, 1H; Azp-Hδ), (m, 3H; Pro -Hδ, Pro C -Hδ, Pro-Hδ), 3.19 (dd, J = 10.4 Hz, 5.3 Hz, 1H; Azp-Hδ), (m, 10H; Azp-Hβ, Pro C -Hβ, Azp-Hβ, Pro C -Hβ, Pro -Hβ, Pro -Hγ, Pro C -Hγ, Pro -Hγ, Pro C -Hγ, Pro -Hβ). 13 C-MR (126 MHz, CDCl 3, 23 C): δ = 173.6, 171.4, 171.1, 154.4, 144.6, 144.0, 141.3, 127.6, 127.0, 125.0, 125.0, 120.0, 66.7, 59.5, 57.8, 56.8, 51.2, 47.7, 47.5, 47.4, 46.9, 33.6, 29.9, 28.1, 25.1, 24.6, MS (ESI, neg): m/z (%): (100) [M-H] -. 2.) Solid-phase synthesis of 3R The synthesis was accomplished following the general protocol for peptide coupling, Fmoc-deprotection, acetylation and removal from Wang resin. For the initial coupling on Wang-H resin (2.00 g, 1.86 mmol), 1 eq of HCTU (0.78 g, 1.86 mmol), 1 eq of Fmoc-[Pro-(4R)Azp-Pro]-H 5R (1.06 g, 1.86 mmol), 3 eq of i Pr 2 Et (0.96 ml, 5.58 mmol) and a catalytic amount of DMAP was used. A quantitative Fmoc test after the couplings revealed loadings of 0.39 mmolg -1 that proved ideal for further couplings. The use of resins with loadings higher than 0.4 mmolg -1 led to lower coupling yields. The crude peptide was further purified by preparative HPLC on a LiChrospher RP-18e 5 µm (250 mm x 10 mm) column (gradient of 79% to 70% of solvent B (0.1% TFA in H 2 ) in solvent A (CH 3 C) over 30 min at a flow rate of 3 ml/min. Analytical HPLC: t R = 12.8 min; gradient: 80% to 60% solvent B over 21 min at a flow rate of 1 ml/min. 1 H-MR (400 MHz, D 2, 25 C): δ = 4.70 (m, overlap with solvent signal, ~ 17H), 4.50 (m, 6H), 4.42 (m, 1H), 3.96 (m, 6H), 3.82 (m, 18H), 3.61 (m, 12H), 2.49 (m, 6H), 2.32 S9

10 (m, 12H), (m, 45H). 13 C-MR (126 MHz, CDCl 3, 23 C): δ = 175.9, 172.5, 172.4, 171.8, 171.4, 171.1, 170.6, 60.1, 60.1, 59.5, 58.6, 58.4, 58.1, 57,0, 52.2, 52.1, 48.5, 47.6, 47.4, 33.1, 33.0, 28.7, 28.4, 27.8, 27.7, 24.4, 24.2, MS (ESI, neg): m/z (%) = (100) [M-H] -, calculated for C 92 H CD spectra of 3R in phosphate buffer (10 mm, ph 7.2) (filled dots) and n-prh (open circles) [Θ] (10 3 deg cm 2 dmol -1 ) Wavelength (nm) The model of the PPII helix shown in Figure 2 was generated using the programs Maestro 4 and Spartan 5 based on the dihedral angles taken from the crystal structure of polyproline. 6 The energetics were not optimized. 3. Synthesis of 4R 16 mg (7.8 µmol) of peptide 3R and 12 µl (144 µmol) methyl propiolate were suspended in tbuh (30 µl) and H 2 (240 µl). Sodium ascorbate (17 µl of a 1M solution in water, 19 µmol) was added followed by CuS 4 5H 2 (23 µl of a 0.4 M solution in water, 9 µmol). The heterogeneous reaction mixture was stirred overnight and then extracted with CHCl 3 (4x 10 ml). The organic layers were dried over a 2 S 4, filtered and all volatiles were removed at reduced pressure. The residue was taken up in CHCl 3 ( 0.3 ml) and the product precipitated upon addition of Et 2 ( 5 ml). The product was isolated as a white powder (8 mg, 40 %). S10

11 Analytical HPLC: t R = 14.6 min; gradient: 80% to 50% solvent B (0.1% TFA in H 2 ) in solvent A (CH 3 C) over 30 min at a flow rate of 1 ml/min. MS (ESI, neg): m/z (%) = (100) [M-H] -, calculated for C 116 H CD spectra of 4R in phosphate buffer (10 mm, ph 7.2) (filled dots) and in n-prh (open circles). 10 [Θ] (10 3 deg cm 2 dmol -1 ) Wavelength (nm) Syntheses and analytical data of 6R 6R was prepared by successive couplings of Fmoc-Pro-(4R)Azp-Pro-H 5R and cycloaddition steps on Rink Amid resin on a 30 µmol scale (Scheme B). H-Pro 1) Fmoc-Pro-(4R)Azp-Pro-H, HCTU, i Pr 2 Et, DMF Fmoc-Pro-Azp-Pro-Pro 2) C 2 Me CuI, i Pr 2 Et, DMF/THF 1:1 C 2 Me Fmoc-Pro-Pro-Pro-Pro 3) DMF/Piperidin (4:1) 4) repeat 1) 5) repeat 2) with Ph Ph C 2 Me 6R Fmoc-Pro-Pro-Pro-Pro-Pro-Pro CH 3 Ph C 2 CH 3 Ph C 2 CH 3 H H 2 Scheme B: Synthesis of peptide 6R. 6R S11

12 The first coupling was performed following the general procedure for peptide couplings with a 1:1 mixture of Fmoc-Pro-H and AcH (1.5 eq each) to reduce the loading of the Rink Amide resin from 0.71 mmolg -1 to 0.3 mmolg -1. Following the general procedures the Fmoc-protecting group was removed and the tripeptidic building block Fmoc-Pro-(4R)Azp-Pro-H 5R was coupled onto 200 mg of resin. The resin was then agitated with methylpropiolate (26 µl, 310 µmol), CuI (1.0 mg, 5.2 µmol), i Pr 2 Et (39 µl, 236 µmol) in DMF (500 µl) and THF (500 µl) at RT. The progression of the reaction was monitored by IR spectroscopy. After disappearance of the signal around 2103 cm -1, the suspension was filtered and rinsed with DMF, MeH and CH 2 Cl 2. The Fmocdeprotection and the coupling of 5R were repeated as described. The 2 nd click reaction was performed with phenylacetylene (33 µl, 310 µmol), the 3 rd with 4-ethynylanisole (39 µl, 308 µmol) (repeated twice), the 4 th with methylpropiolate (26 µl, 310 µmol) and the 5 th with phenylacetylene (33 µl, 310 µmol) under otherwise identical conditions. Fmoc-deprotection and cleavage from Rink Amid resin following the general procedure yielded 74 mg (52 %) of crude 6R. (After each coupling and click chemistry step a small portion of the resin ( 5 mg) was removed from the synthesis for IR- and MS- ESI- analysis, thus, the effective yield would be higher.) The crude peptide was further purified by preparative HPLC on a LiChrospher RP-18e 5 µm (250 mm x 10 mm) column (gradient of 75% to 55% of solvent B (0.1% TFA in H 2 ) in solvent A (CH 3 C) over 30 min at a flow rate of 4 ml/min. Analytical HPLC: t R = 17.0 min; gradient: 80% to 40% solvent B over 25 min at a flow rate of 1 mlmin -1. MS (ESI): m/z (%) = (10) [M+H] +, (100) [M+2H] 2+. S12

13 CD spectra of 6R in phosphate buffer (10 mm, ph 7.2) (filled dots) and in n-prh (open circles). 10 [Θ] (10 3 deg cm 2 dmol -1 ) Wavelength (nm) References (1) Vojkovsky, T. Pept. Res. 1995, 8, (2) Gangamani, B. P.; Kumar, V. A.; Ganesh, K.. Tetrahedron 1996, 52, (3) Anderson,. G.; Lust, D. A.; Colapret, K. A.; Simpson J. H.; Malley, M. F.; Gougoutas, J. Z. J. rg. Chem. 1996, 61, (4) Maestro 7.5, Schrödinger, LLC, (5) Spartan`02, Wavefunction, Inc., Irvine, CA. (6) Kakinoki, S.; Hirano, Y.; ka, M. Polymer Bull. 2005, 53, S13