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1 Supporting Information for Kinetically controlled chemoselective cyclization simplifies the access to cyclic and branched peptides Emmanuelle Boll, a Hervé Drobecq, a Elizabeth Lissy, a François-Xavier Cantrelle, b Oleg Melnyk a *. a Univ. Lille, CNRS, Institut Pasteur de Lille, UMR M3T Mechanisms of Tumorigenesis and Targeted Therapies, F-59 Lille, France. b Univ. Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, F 59 Lille, France Corresponding author : Dr. Oleg Melnyk, oleg.melnyk@ibl.cnrs.fr Centre National de la Recherche Scientifique (CNRS) Institut de Biologie de Lille 1 rue du Pr Calmette, CS 5447, 5921 Lille cedex, France Tel: S1

2 Contenu 1. General Methods...4 Reagents and solvents...4 Analyses...4 HPLC purification Peptide synthesis Synthesis of linear SEA off peptide segments Characterization of the linear SEA off peptides Characterization of peptide C(StBu)ILKED(SEA off )VRGA-SEA off 12a...6 Characterization of peptide C(StBu)ILKED(SEA off )VRGS-SEA off 12b Characterization of peptide C(StBu)ILKED(SEA off )VRGL-SEA off 12c Characterization of peptide C(StBu)ILKEE(SEA off )VRGA-SEA off 12d Characterization of peptide C(StBu)ILKEE(SEA off )VRGS-SEA off 12e Characterization of peptide C(StBu)ILKEE(SEA off )VRGL-SEA off 12f Synthesis of peptide Characterization of peptide CGGTLPSPLALLTVH-NH Kinetically controlled cyclization/ligation sequence... 4 Typical experimental procedure (illustrated with peptide 12a and 15)... 4 Characterization of peptide 16a Characterization of peptide 16b Characterization of peptide 16c Characterization of peptide 16d Characterization of peptide 16e Characterization of peptide 16g... 5 NMR analysis of peptide 16g Proteomic analysis General procedure illustrated with peptide 16a Peptide 16a Peptide 17a (analytical sample)... 6 Peptide 16b Peptide 16c Peptide 16d Peptide 16e References S2

3 S3

4 1. General Methods Reagents and solvents 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium fluorophosphate (HBTU) and N-Fmoc protected amino acids were obtained from Iris Biotech GmbH. Side-chain protecting groups used for the amino acids were Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(StBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc- Gly-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH. Non-standard Fmoc-protected L-amino acids Fmoc-Asp(SEA off )-OH and Fmoc-Glu(SEA off )-OH were prepared as described elsewhere. 1 Synthesis of bis(2-sulfanylethyl)aminotrityl polystyrene (SEA PS) resin was carried out as described elsewhere. 2 For a detailed protocol see ref 3. 4-mercaptophenylacetic acid (MPAA) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich. All other reagents were purchased from Acros Organics or Merck and were of the purest grade available. Peptide synthesis grade N,N-dimethylformamide (DMF), dichloromethane (CH 2 Cl 2 ), diethylether (Et 2 O), acetonitrile (CH 3 CN), heptane, LC MS-grade acetonitrile (CH 3 CN,.1% TFA), LC MSgrade water (H 2 O,.1% TFA), N,N-diisopropylethylamine (DIEA), acetic anhydride (Ac 2 O) were purchased from Biosolve and Fisher-Chemical. Trifluoroacetic acid (TFA) was obtained from Biosolve. Water was purified with a Milli-Q Ultra-Pure Water Purification System. Analyses The reactions were monitored by analytical LC MS (Waters 2695 LC/ZQ 2 quadripole) on an reverse phase column XBridge BEH3 C18 (3.5 µm, 3 Å, mm) unless otherwise stated. The elutions were carried out at 3 C using a linear gradient of eluent B in eluent A over 3 min at a flow rate of 1 ml/min (-1%, eluent A =.1% TFA in H 2 O; eluent B =.1% TFA in CH 3 CN/H 2 O: 4/1 by vol). The column eluate was monitored by UV at 215 nm and by evaporative light scattering (ELS, Waters 2424 detector). The peptide masses were measured by on-line LC MS: Ionization mode: ES+, range 35 24, capillary voltage 3 kv, cone voltage 3 V, extractor voltage 3 V, RF lens.2 V, source temperature 12 C, dessolvation temperature 35 C. Samples were prepared using 1 µl aliquots of the reaction mixtures. The aliquots were quenched by adding 9 L of 1% aqueous TFA, extracted with Et 2 O to remove MPAA or MPA before analysis. MALDI-TOF mass spectra were recorded with a BrukerAutoflex Speed using alpha-cyano-4- hydroxycinnaminic acid as matrix. The observed corresponded to the monoisotopic ions, unless otherwise stated. 1 H and 13 C NMR spectra were recorded on a Bruker Advance-3 spectrometer operating at 3 MHz and 75 MHz respectively. The spectra are reported as parts per million (ppm) down field shift using tetramethylsilane or dimethylselenide as internal references. The data are reported as chemical shift (δ), multiplicity, relative integral, coupling constant (J Hz) and assignment where possible. HPLC purification Preparative reverse phase HPLCs of crude peptides were performed with an Autopurification prep HPLC MS Waters system using a reverse phase column XBridge ODB prep C-18 (5 m, 3 Å, 19 1 mm) and appropriate gradient of increasing concentration of eluent B in eluent A (flow rate of 25 ml/min). The fractions containing the purified target peptide were identified on-line using MS (ZQ 2 quadripole). Selected fractions were combined, frozen at -2 C and lyophilized. S4

5 2. Peptide synthesis 2.1 Synthesis of linear SEA off peptide segments 12 Typical procedures for the synthesis of SEA off peptide segments were described in previous papers. 1,2 For a detailed protocol see the protocol article. 3 General protocol Scheme S1. SEA off peptides 12 synthesized in this study. Peptide elongation was performed on SEA PS resin (.1 mmol,.16 mmol/g) using standard Fmoc/tert-butyl chemistry. The Fmoc-L-Asp(SEA off )-OH (.11 mmol) or Fmoc-L-Glu(SEA off )-OH (.11 mmol) were coupled manually using HATU (.15 mmol)/diea (.1 mmol) activation in DMF for 2 h. The other amino acids were incorporated using an automated peptide synthesizer (.1 mmol scale). Couplings were performed using 5-fold molar excess of each Fmoc-L- amino acid, 4.5-fold molar excess of HBTU, and 1-fold molar excess of DIEA. A capping step was performed after each coupling with Ac 2 O/DIEA in DMF. At the end of the synthesis, the peptidyl resin was washed with CH 2 Cl 2 (2 2 min) and Et 2 O (2 2 min) and dried in vacuo. The peptide was cleaved from the resin using a mixture of trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/dimethylsulfide (DMS)/thioanisol/water: 92.5/2.5/2.5/2.5/2.5 by vol (1 ml) for 2 h. The crude peptide was precipitated in ice-cold diethyl ether/heptane: 1/1 by vol (1 ml), solubilized in deionized water and lyophilized. The crude peptide was oxidized and purified as follows. The crude peptide was dissolved in AcOH/water : 1/4 by vol. Iodine (73 mm solution in DMSO, 2 equiv) was added to the peptide solution. The solution becomes yellow due to the excess of iodine. After 3 seconds of mixing, dithiothreitol (DTT, 81 µm solution in AcOH/water: 1/4 by vol, 2 equiv) was added to decompose the excess of iodine. The peptide solution was then purified immediately by RP-HPLC. HPLC column XBridge Prep column C18 OBD 13 Å, 19 1 mm, 5 µm. Eluent A : water containing.1% TFA by vol, eluent B: water/acetonitrile : ¼ by vol containing.1% TFA by vol. For 12a,b: linear gradient 2% - 45% B in 25 min, flow rate 25 ml/min. For 12c : linear gradient 2% - 6% B in 25 min, flow rate 25 ml/min. S5

6 For 12d-f : linear gradient 25% - 5% B in 25 min, flow rate 2 ml/min. Table S1. Yields for purified SEA off peptides 12 Peptide Sequence Isolated yield (%) 12a C(StBu)ILKED(SEA off )VRGA-SEA off 31% 12b C(StBu)ILKED(SEA off )VRGS-SEA off 23% 12c C(StBu)ILKED(SEA off )VRGL-SEA off 24% 12d C(StBu)ILKEE(SEA off )VRGA-SEA off 28% 12e C(StBu)ILKEE(SEA off )VRGS-SEA off 27% 12f C(StBu)ILKEE(SEA off )VRGL-SEA off 24% 2.2 Characterization of the linear SEA off peptides 12 Characterization of peptide C(StBu)ILKED(SEA off )VRGA-SEA off 12a 55. Intensity, light scattering (AU) Intensity (AU) [M+2H] Time (min) Figure S1. LC-MS analysis of peptide C(StBu)ILKED(SEA off )VRGA-SEA off 12a. LC trace, eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient -1 % B in 3 min (1 ml/min, detection 215 nm). MS trace: [M+H] + calculated (mean) , found S6

7 x StBu Figure S2. MALDI-TOF analysis of peptide C(StBu)ILKED(SEA off )VRGA-SEA off 12a. Matrix : α- cyano-4-hydroxycinnamic acid, [M+H] + calcd (monoisotopic) , found The peak at is due to the partial deprotection of the cysteine residue during MS analysis. NMR analysis of peptide C(StBu)ILKED(SEA off )VRGA-SEA off 12a 1 H NMR (3 MHz, H 2 O+D 2 O) δ (m, 1H), (m, 1H), (m, 1H), (m, 4H), (m, 1H), (m, 1H), (m, 1H), 7.25 (t, 1H), 6.72 (s, 1H), (m, 2H), (m, 4H), (m, 1H), (m, 1H), (m, 2H), (m, 1H), 2.47 (t, J = 7.5 Hz, 2H), (m, 5H), (m, 1H), (m, 3H), (m, 12H), (m, 1H), (m, 18H). 13 C NMR (75 MHz, H 2 O+D 2 O) δ ; (m) ; (m) ; (s) ; (m) ; (s) ; (m) ; (m) ; (s) ; ; ; ; ; ; (s) ; (m) ; (s) ; (s) ; (s) ; (s) ; (s) ; (m) ; 55.9 (s) ; (s) ; (m) ; (s) ; ; (s) ; (m) ; (s) ; (s) ; (m) ; (s) ; 4.96 ; 4.72 (s) ; (s) ; (s) ; (m) ; (m) ; (m) ; (s) ; 33.4 (s) ; (s) ; (m) ; (m) ; 29.2 (s), (s) ; (s) ; (s) ; (s) ; 24.5 (s) ; (s) ; 2.31 (s) ; (s) ; (s) ; (s). S7

8 5E+7 4E+7 C(StBu)ILKED(SEA off )VRGA-SEA off 4E+7 12a 4E+7 3E+7 2E+7 2E+7 2E+7 1E+7 5E+6-5E f1 (ppm) Figure S3. 1 H NMR spectrum of peptide C(StBu)ILKED(SEA off )VRGA-SEA off 12a C(StBu)ILKED(SEA off )VRGA-SEA off 12a Figure S4. 13 C NMR spectrum of peptide C(StBu)ILKED(SEA off )VRGA-SEA off 12a. S8

9 C(StBu)ILKED(SEA off )VRGA-SEA off 12a f1 (ppm) Figure S5. 1 H- 1 H COSY spectrum of peptide C(StBu)ILKED(SEA off )VRGA-SEA off 12a. f1 (ppm) C(StBu)ILKED(SEA off )VRGA-SEA off 12a Figure S6. 1 H- 1 H DIPSI spectrum of peptide C(StBu)ILKED(SEA off )VRGA-SEA off 12a. S9

10 C(StBu)ILKED(SEA off )VRGA-SEA off 12a f1 (ppm) Figure S7. 1 H- 1 H ROESY spectrum of peptide C(StBu)ILKED(SEA off )VRGA-SEA off 12a. C(StBu)ILKED(SEA off )VRGA-SEA off 12a f1 (ppm) Figure S8. 1 H- 13 C HSQC spectrum of peptide C(StBu)ILKED(SEA off )VRGA-SEA off 12a. S1

11 Characterization of peptide C(StBu)ILKED(SEA off )VRGS-SEA off 12b Intensity, light scattering (AU) Time (min) 1 Intensity (AU) [M+2H] [M+H] Figure S9.LC-MS analysis of peptide C(StBu)ILKED(SEA off )VRGS-SEA off 12b. LC trace, eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient -1 % B in 3 min (1 ml/min, detection 215 nm). MS trace: [M+H] + calculated (mean) , found S11

12 StBu Figure S1. MALDI-TOF analysis of peptide C(StBu)ILKED(SEA off )VRGS-SEA off 12b. Matrix : α- cyano-4-hydroxycinnamic acid, [M+H] + calcd (monoisotopic) , found The peak at is due to the deprotection of the cysteine residue during MS analysis. NMR analysis of peptide C(StBu)ILKED(SEA off )VRGS-SEA off 12b 1 H NMR (3 MHz, H 2 O+D 2 O) δ 8.69 (d, J = 7.9 Hz, 1H), 8.58 (d, J = 7.4 Hz, 1H), 8.5 (d, J = 6.4 Hz, 1H), (m, 4H), 8.25 (d, J = 7.6 Hz, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.25 (s, 1H), (m, 6H), (m, 1H), (m, 6H), (m, 4H), (m, 11H), 2.45 (d, J = 7.2 Hz, 2H), (m, 16H), (m, 3H), 1.39 (s, 9H), (m, 1H), (m, 18H). 13 C NMR: 13 C NMR (75 MHz, H 2 O+D 2 O) δ , , , , , 175.5, 175.2, 175.5, , , , , , 64.8, 62.4, 61.12, , , , 55.47, , , 54.46, 53.54, , 53.32, 51.52, , 43.47, , , 42.23, 41.7, 4.72, 39.26, , 37.27, , , 33.15, , 31.73, , , 29.2, 27.27, 27.12, 24.9, 24.84, 24.5, 21.17, 2.31, 17.44, , S12

13 C(StBu)ILKED(SEA off )VRGS-SEA off 12b Figure S11. 1 H NMR spectrum of peptide C(StBu)ILKED(SEA off )VRGS-SEA off 12b C(StBu)ILKED(SEA off )VRGS-SEA off 12b Figure S C NMR spectrum of peptide C(StBu)ILKED(SEA off )VRGS-SEA off 12b. S13

14 C(StBu)ILKED(SEA off )VRGS-SEA off 12b Figure S13. 1 H- 1 H COSY spectrum of peptide C(StBu)ILKED(SEA off )VRGS-SEA off 12b. f1 (ppm) f1 (ppm) C(tBu)ILKED(SEA off )VRGS-SEA off 12b Figure S14. 1 H- 1 H DIPSI spectrum of peptide C(StBu)ILKED(SEA off )VRGS-SEA off 12b. S14

15 C(StBu)ILKED(SEA off )VRGS-SEA off 12b f1 (ppm) Figure S15. 1 H- 1 H ROESY spectrum of peptide C(StBu)ILKED(SEA off )VRGS-SEA off 12b. C(StBu)ILKED(SEA off )VRGS-SEA off 12b f1 (ppm) Figure S16. 1 H- 13 C HSQC spectrum of peptide C(StBu)ILKED(SEA off )VRGS-SEA off 12b. S15

16 Characterization of peptide C(StBu)ILKED(SEA off )VRGL-SEA off 12c Intensity, light scattering (AU) Time (min) 1 [M+2H] [M+H] Figure S17. LC-MS analysis of peptide C(StBu)ILKED(SEA off )VRGL-SEA off 12c. LC trace, eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient -1 % B in 3 min (1 ml/min, detection 215 nm). MS trace: [M+H] + calculated (mean) 1469., found S16

17 x StBu Figure S18. MALDI-TOF analysis of peptide C(StBu)ILKED(SEA off )VRGL-SEA off 12c. Matrix : α- cyano-4-hydroxycinnamic acid, [M+H] + calcd (monoisotopic) , found The peak at is due to the partial deprotection of the cysteine residue during MS analysis. S17

18 NMR analysis of peptide C(StBu)ILKED(SEA off )VRGL-SEA off 12c 1 H NMR (3 MHz, H 2 O+D 2 O) δ (m, 1H), (m, 1H), (m, 1H), (m, 4H), (m, 1H), (m, 1H), (m, 1H), (m, 5H), (m, 1H), (m, 4H), 3.92 (s, 6H), (m, 2H), (m, 2H), (m, 1H), (m, 2H), (m, 18H), 1.34 (s, 3H), (m, 1H), (m, 24H). 13 C NMR (75 MHz, H 2 O+D 2 O) δ (s), (s), (s), (s), (s), (s), (s), (s), (s), 175. (s), (s), (s), (s), (s), 61.9 (s), (s, J = 15.4 Hz), (s), (s), (s, J = 6.8 Hz), (s), (s), 55.4 (s, J = 13. Hz), (s, J = 16.3 Hz), (s), (s, J = 17.3 Hz), 51.2 (s), 45.9 (s), (s, J = 16.4 Hz), (s), (s), (s), (s), 4.85 (s, J = 18.5 Hz), 4.61 (s), (s), (s, J = 8.3 Hz), (s), 33.4 (s), 33.1 (s), (s), 3.68 (s), 29.5 (s), (s), (s, J = 11.2 Hz), 27.4 (s), 25.2 (s), (s), (s), (s), 21.2 (s), 2.22 (s), (s), (s) C(StBu)ILKED(SEA off )VRGL-SEA off 12c Figure S19. 1 H NMR spectrum of peptide C(StBu)ILKED(SEA off )VRGL-SEA off 12c. S18

19 C(StBu)ILKED(SEA off )VRGL-SEA off 12c Figure S2. 13 C NMR spectrum of peptide C(StBu)ILKED(SEA off )VRGL-SEA off 12c. C(StBu)ILKED(SEA off )VRGL-SEA off 12c f1 (ppm) Figure S21. 1 H- 1 H COSY spectrum of peptide C(StBu)ILKED(SEA off )VRGL-SEA off 12c. S19

20 C(StBu)ILKED(SEA off )VRGL-SEA off 12c f1 (ppm) Figure S22. 1 H- 1 H DIPSI spectrum of peptide C(StBu)ILKED(SEA off )VRGL-SEA off 12c. C(StBu)ILKED(SEA off )VRGL-SEA off 12c f1 (ppm) Figure S23. 1 H- 13 C HSQC spectrum of peptide C(StBu)ILKED(SEA off )VRGL-SEA off 12c. S2

21 C(StBu)ILKED(SEA off )VRGL-SEA off 12c f1 (ppm) Figure S24. 1 H- 1 H ROESY spectrum of peptide C(StBu)ILKED(SEA off )VRGL-SEA off 12c. S21

22 Characterization of peptide C(StBu)ILKEE(SEA off )VRGA-SEA off 12d 1. Intensity, light scattering (AU) Time (min) 1 Intensity (AU) [M+2H] [M+H] Figure S25. LC-MS analysis of peptide C(StBu)ILKEE(SEA off )VRGA-SEA off 12d. LC trace, eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient -1 % B in 3 min (1 ml/min, detection 215 nm). MS trace: [M+H] + calcd (mean) 144.9, found S22

23 x Figure S26. MALDI-TOF analysis of peptide C(StBu)ILKEE(SEA off )VRGA-SEA off 12d. Matrix : α- cyano-4-hydroxycinnamic acid, [M+H] + calcd (monoisotopic) , found NMR analysis of peptide C(StBu)ILKEE(SEA off )VRGA-SEA off 12d 1 H NMR (3 MHz, H 2 O+D 2 O) δ 8.62 (d, J = 8.1 Hz, 1H), (m, J = 1.4, 6.8 Hz, 2H), 8.4 (t, J = 5.8 Hz, 1H), (m, 3H), 8.2 (d, J = 6.9 Hz, 1H), 8.12 (d, J = 6.4 Hz, 1H), 7.19 (s, 1H), (m, J = 15.5 Hz, 6H), (m, 5H), (m, 2H), (m, 5H), (m, J = 11.1, 5.4 Hz, 6H), 2.51 (t, J = 7. Hz, 2H), 2.39 (t, J = 7.4 Hz, 2H), (m, 15H), (m, 3H), (m, 12H), (m, 1H), (m, J = 12.9, 6.9 Hz, 15H). 13 C NMR (75 MHz, H 2 O+D 2 O) δ 18.41, , , , , , , , , , 173.3, 17.72, , 62.38, 61.18, 56.44, 55.92, 55.74, 55.47, 55.38, , , , 51.52, 48.84, , , , , 42.23, , 4.96, , 39.29, 37.53, , 32.96, 31.73, , , , 29.2, 27.25, 27.12, , 24.86, 24.5, 21.21, 2.63, 19.65, 17.46, S23

24 1E+8 1E+8 C(StBu)ILKEE(SEA off )VRGA-SEA off 12d 9E+7 8E+7 7E+7 6E+7 5E+7 4E+7 3E+7 2E+7 1E+7-1E+7-2E+7-3E+7-4E+7-5E f1 (ppm) Figure S27. 1 H NMR spectrum of peptide C(StBu)ILKEE(SEA off )VRGA-SEA off 12d. 5E+8 4E+8 C(StBu)ILKEE(SEA off )VRGA-SEA off 12d 4E+8 4E+8 3E+8 2E+8 2E+8 2E+8 1E+8 5E+7-5E+7-1E+8-2E+8-2E+8-2E f1 (ppm) Figure S C NMR spectrum of peptide C(StBu)ILKEE(SEA off )VRGA-SEA off 12d. S24

25 C(StBu)ILKEE(SEA off )VRGA-SEA off 12d f1 (ppm) Figure S29. 1 H- 1 H COSY spectrum of peptide C(StBu)ILKEE(SEA off )VRGA-SEA off 12d. C(StBu)ILKEE(SEA off )VRGA- SEA off 12d f1 (ppm) Figure S3. 1 H- 13 C HSQC spectrum of peptide C(StBu)ILKEE(SEA off )VRGA-SEA off 12d. S25

26 C(StBu)ILKEE(SEA off )VRGA-SEA off 12d Figure S31. 1 H- 1 H DIPSI spectrum of peptide C(StBu)ILKEE(SEA off )VRGA-SEA off 12d f1 (ppm) 6 7 C(StBu)ILKEE(SEA off )VRGA- SEA off 12d f2 (ppm) Figure S32. 1 H- 1 H ROESY spectrum of peptide C(StBu)ILKEE(SEA off )VRGA-SEA off 12d. S26

27 Characterization of peptide C(StBu)ILKEE(SEA off )VRGS-SEA off 12e 9. Intensity, light scattering (AU) Time (mim 1 Intensity (AU) [M+2H] [M+H] Figure S33. LC-MS analysis of peptide C(StBu)ILKEE(SEA off )VRGS-SEA off 12e. LC trace, eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient -1 % B in 3 min (1 ml/min, detection 215 nm). MS trace: [M+H] + calcd (mean) , found S27

28 x Figure S34. MALDI-TOF analysis of peptide C(StBu)ILKEE(SEA off )VRGS-SEA off 12e. Matrix : α- cyano-4-hydroxycinnamic acid, [M+H] + calcd (monoisotopic) , found The peak at is due to the partial deprotection of the cysteine residue during MS analysis. S28

29 NMR analysis of peptide C(StBu)ILKEE(SEA off )VRGS-SEA off 12e 1 H NMR (3 MHz, H 2 O+D 2 O) δ 8.53 (d, J = 7.9 Hz, 1H), (m, 3H), (m, 3H), 8.13 (d, J = 7.6 Hz, 2H), 7.8 (s, 1H), (m, 6H), (m, 5H), (m, 4H), (m, 5H), (m, 2H), (m, 1H), 2.37 (d, J = 7.2 Hz, 2H), 2.25 (t, J = 7.3 Hz, 2H), (m, 5H), (m, 7H), (m, 9H), (m, 3H), 1.19 (s, 9H), (m, 1H), (m, 18H). 13 C NMR (75 MHz, H 2 O+D 2 O) δ (m), (s), (m), (m), 176. (s), (m), (m), (s), (m), (m), 64.5 (d, J = 15.5 Hz), (s), (s), (s), (s), (s), (m), (s), (m), (m), (m), (m), (m), (m), (m), 41.7 (s), 4.77 (s), (s), (m), (s), (m), (m), (s), (s), (m), (m), 29.2 (s), (s), (s), (m), (s), 24.5 (s), (s), 2.65 (s), (s), (s) C(StBu)ILKEE(SEA off )VRGS-SEA off 12e Figure S35. 1 H NMR spectrum of peptide C(StBu)ILKEE(SEA off )VRGS-SEA off 12e. S29

30 C(StBu)ILKEE(SEA off )VRGS-SEA off 12e Figure S C NMR spectrum of peptide C(StBu)ILKEE(SEA off )VRGS-SEA off 12e. C(StBu)ILKEE(SEA off )VRGS-SEA off 12e f1 (ppm) Figure S37. 1 H- 1 H COSY spectrum of peptide C(StBu)ILKEE(SEA off )VRGS-SEA off 12e. S3

31 C(StBu)ILKEE(SEA off )VRGS-SEA off 12e f1 (ppm) Figure S38. 1 H- 13 C HSQC spectrum of peptide C(StBu)ILKEE(SEA off )VRGS-SEA off 12e. C(StBu)ILKEE(SEA off )VRGS-SEA off 12e f1 (ppm) Figure S39. 1 H- 1 H DIPSI spectrum of peptide C(StBu)ILKEE(SEA off )VRGS-SEA off 12e. S31

32 f1 (ppm) C(StBu)ILKEE(SEA off )VRGS-SEA off 12e Figure S4. 1 H- 1 H ROESY spectrum of peptide C(StBu)ILKEE(SEA off )VRGS-SEA off 12e. S32

33 Characterization of peptide C(StBu)ILKEE(SEA off )VRGL-SEA off 12f Intensity, light scattering (AU) Time (min) 1 Intensity (AU) [M+2H] [M+H] + Figure S41. LC-MS analysis of peptide C(StBu)ILKEE(SEA off )VRGL-SEA off 12f. LC trace, eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient -1 % B in 3 min (1 ml/min, detection 215 nm). MS trace: [M+H] + calcd (mean) 1483., found S33

34 x Figure S42. MALDI-TOF analysis of peptide C(StBu)ILKEE(SEA off )VRGL-SEA off 12f. Matrix : α- cyano-4-hydroxycinnamic acid, [M+H] + calcd (monoisotopic) , found The peak at is due to the partial deprotection of the cysteine residue during MS analysis. NMR analysis of peptide C(StBu)ILKEE(SEA off )VRGL-SEA off 12f 1 H NMR (3 MHz, H 2 O+D 2 O) δ 8.59 (d, J = 8.1 Hz, 1H), 8.42 (d, J = 6.7 Hz, 2H), (m, 5H), 8.11 (d, J = 7.9 Hz, 1H), 7.16 (s, 1H), (m, 6H), (m, 7H), (m, 4H), (m, 8H), (m, 6H), 2.48 (t, J = 6.9 Hz, 2H), 2.33 (t, J = 7.4 Hz, 2H), (m, 5H), (m, 5H), (m, 1H), (m, 3H), 1.3 (s, 9H), (m, 1H), (m, 24H). 13 C NMR (75 MHz, H 2 O+D 2 O) δ (m), (m), (m), (m), (m), (s), (m), (m), (m), (m), (s), (s), (s), (s, J = 33. Hz), (s), (s), (m), (m), (d, J = 11.9 Hz), (m), (m), (m), (m), (m), (m), (m), (m), (m), (m), (m), (m), (m), 32.9 (s), (m), (m), (m), (m), 29.2 (s), (s), (s), (s), (m), 24.6 (s), (s, J = 53. Hz), (s), 2.66 (s), (s), 13.1 (s). S34

35 C(StBu)ILKEE(SEA off )VRGL-SEA off 12f Figure S43. 1 H NMR spectrum of peptide C(StBu)ILKEE(SEA off )VRGL-SEA off 12f C(StBu)ILKEE(SEA off )VRGL-SEA off 12f Figure S C NMR spectrum of peptide C(StBu)ILKEE(SEA off )VRGL-SEA off 12f. S35

36 C(StBu)ILKEE(SEA off )VRGL-SEA off 12f f1 (ppm) Figure S45. 1 H- 1 H COSY spectrum of peptide C(StBu)ILKEE(SEA off )VRGL-SEA off 12f. C(StBu)ILKEE(SEA off )VRGL-SEA off 12f f1 (ppm) Figure S46. 1 H- 13 C HSQC spectrum of peptide C(StBu)ILKEE(SEA off )VRGL-SEA off 12f. S36

37 C(StBu)ILKEE(SEA off )VRGL-SEA off 12f f1 (ppm) Figure S47. 1 H- 1 H DIPSI spectrum of peptide C(StBu)ILKEE(SEA off )VRGL-SEA off 12f. 2.3 Synthesis of peptide 15 Peptide elongation was performed using standard Fmoc/tert-butyl chemistry on an automated peptide synthesizer (.5 mmol scale, NovaSyn TGR resin,.25 mmol/g). Couplings were performed using 5- fold molar excess of each Fmoc-L- amino acid, 4.5-fold molar excess of HBTU, and 1-fold molar excess of DIEA. A capping step was performed after each coupling with Ac 2 O/DIEA in DMF. At the end of the synthesis, the resin was washed with CH2Cl2, diethylether (2 2 min) and dried in vacuo. The peptide was cleaved from the resin using a mixture of trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/ 1,2-ethanedithiol (EDT) /water: 94/1/2.5/2.5 by vol (1 ml) for 2 h. The crude peptide was precipitated in ice-cold diethyl ether/heptane: 1/1 by vol (1 ml), solubilized in deionized water and lyophilized. The crude peptide was dissolved in AcOH/water : 1/4 by vol. The peptide solution was then purified immediately by RP-HPLC. HPLC purification: HPLC column XBridge Prep column C18 OBD 13 Å, 19 1 mm, 5 µm. Eluent A : water containing.1% TFA by vol, eluent B: water/acetonitrile : ¼ by vol containing.1% TFA by vol. Linear gradient 2% - 55% B in 25 min, flow rate 25 ml/min. peptide sequence isolated yield (%) 15 CGGTLPSPLALLTVH-NH 2 64% S37

38 Characterization of peptide CGGTLPSPLALLTVH-NH 2 15,,,,, 4.e-1 Intensity, light scattering (AU) 3.6e-1 3.2e-1 2.8e-1 2.4e-1 2.e-1 1.6e-1 1.2e-1 8.e-2 4.e Time (min) 1 Intensity (AU) [M+2H] [M+H] Figure S48. LC-MS analysis of peptide CGGTLPSPLALLTVH-NH LC trace, eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient -1 % B in 3 min (1 ml/min, detection 215 nm). MS trace: [M+H] + calcd (mean) , found S38

39 Figure S49. MALDI-TOF analysis of peptide CGGTLPSPLALLTVH-NH Matrix : α-cyano-4- hydroxycinnamic acid, [M+H] + calcd (monoisotopic) , found S39

40 3. Kinetically controlled cyclization/ligation sequence Typical experimental procedure (illustrated with peptide 12a and 15) The experiment was carried out under nitrogen atmosphere. The reaction was monitored by HPLC or LC-MS : eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient -1 % B in 3 min (1 ml/min, detection 215 nm). Guanidinium hydrochloride (Gdn.HCl) (4.1 g, 42, mmol), MPAA (235.5 mg, 1.4 mmol) and TCEP (41.3 mg, 1.4 mmol) were dissolved in sodium phosphate buffer (.1 M, ph 7.2, 7 ml). The ph was adjusted to 7.48 by adding aqueous NaOH (6 M, 1.5 ml). Peptide 12a (37.57 mg, µmol) was dissolved in the above solution (6.76 ml). The mixture was stirred at 37 C for 48 h. Then, Cys peptide 15 (54.41 mg, 31.9 µmol) was added to the mixture and the ph was adjusted to 5.5 by adding aqueous HCl (1 N, 1.6 ml). The reaction mixture was further stirred for 144 h. Finally, the reaction mixture was diluted with water containing.1% TFA (6 ml). The mixture was acidified to ph 3 by adding aqueous TFA (1% by vol, 2 µl) and then extracted with Et 2 O (3 1 ml) and heptane (1 1 ml). The solution was filtered and purified by RP-HPLC (Vydac C18 column, eluent A : water containing.1% TFA by vol, eluent B: water/acetonitrile : ¼ by vol containing.1% TFA by vol, detection at 215 nm, flow rate 25 ml min, 25-45% eluent B in 45 min). The purified fractions were collected, frozen and lyophilized to give mg (32%) of cyclic and branched peptide 16a. S4

41 Characterization of peptide 16a Intensity, light scattering (AU) a Time (min) 1 Intensity (AU) [M+3H] a [M+2H] Figure S5. LC-MS analysis of peptide 16a. LC trace, eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient -1 % B in 3 min (1 ml/min, detection 215 nm). MS trace: M calcd (mean) 2545., found after deconvolution S41

42 Figure S51. MALDI-TOF analysis of peptide 16a. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H] + calcd (monoisotopic) , found S42

43 Characterization of peptide 16b 18. Intensity, light scattering (AU) b Time (min) 1 Intensity (AU) [M+3H] b [M+2H] Figure S52. LC-MS analysis of peptide 16b. LC trace, eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient -1 % B in 3 min (1 ml/min, detection 215 nm). MS trace: M calcd (mean) , found after deconvolution S43

44 4 x Figure S53. MALDI-TOF analysis of peptide 16b. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H] + calcd (monoisotopic) , found S44

45 Characterization of peptide 16c Intensity, light scattering (AU) c Time (min) Intensity (AU) [M+3H] c [M+2H] Figure S54. LC-MS analysis of peptide 16c. LC trace, eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient - 1 % B in 3 min (1 ml/min, detection 215 nm). MS trace: M calcd (mean) , found after deconvolution S45

46 x c Figure S55. MALDI-TOF analysis of peptide 16c. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H] + calcd (monoisotopic) , found S46

47 Characterization of peptide 16d,,,,, 18. Intensity, light scattering (AU) d Time (min) 1 Intensity (AU) [M+3H] d [M+2H] Figure S56. LC-MS analysis of peptide 16d. LC trace, eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient -1 % B in 3 min (1 ml/min, detection 215 nm). MS trace: M calcd (mean) , found after deconvolution S47

48 d Figure S57. MALDI-TOF analysis of peptide 16d. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H] + calcd (monoisotopic) , found S48

49 Characterization of peptide 16e 7. Intensity, light scattering (AU) e Time (min) 1 Intensity (AU) [M+3H] e [M+2H] Figure S58. LC-MS analysis of peptide 16e. LC trace, eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient - 1 % B in 3 min (1 ml/min, detection 215 nm). MS trace: M calcd (mean) , found after deconvolution S49

50 x e Figure S59. MALDI-TOF analysis of peptide 16e. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H] + calcd (monoisotopic) , found Characterization of peptide 16g Intensity, light scattering (AU) g Time (min) S5

51 1 Intensity (AU) [M+2H] g [M+H] Figure S6. LC-MS analysis of peptide 16g. LC trace, eluent A.1 % TFA in water, eluent B.1 % TFA in CH 3 CN/water: 4/1 by vol. C18 Xbridge BEH 3 Å 5 μm ( mm) column, gradient -1 % B in 3 min (1 ml/min, detection 215 nm). MS trace: M calcd (mean) , found after deconvolution S51

52 g g Figure S61. MALDI-TOF analysis of peptide 16g. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H] + calcd (monoisotopic) , found S52

53 NMR analysis of peptide 16g E+8 3.E+8 16g 2.5E+8 2.E+8 1.5E+8 1.E+8 5.E+7.E+ -5.E f1 (ppm) Figure S62. 1 H NMR spectrum of peptide 16g. 16g f1 (ppm) f2 (ppm) Figure S63. 1 H- 1 H NOESY spectrum of peptide 16g S53

54 f1 (ppm) f2 (ppm) Figure S64. 1 H- 1 H TOCSY spectrum of peptide 16g Figure S65. 1 H- 1 H TOCSY-NOESY spectrum of peptide 16g S54

55 Proteomic analysis General procedure illustrated with peptide 16a Peptide 16a (14 µg) was dissolved in ammonium bicarbonate buffer (25 mm, 14 µl) containing DTT (,1 mg/ml final concentration). An aliquot of this solution corresponding to 5 µg of peptide 16a was alkylated with iodoacetamide (1 mg/ml in 25 mm ammonium bicarbonate buffer, 5 µl) for 3 min at rt. Then trypsin was added (1 µg) and the peptides obtained by digestion were analyzed by MALDI-TOF mass spectrometry. Peptide 16a A) Before alkylation 4 x calcd. for [M+H] + (monoisotopic) , found S55

56 B) After alkylation x calcd. for [M+H] + (monoisotopic) , found C) After trypsin digestion 5 x calcd. for [M+H] + (monoisotopic) 234., found calcd. for [M+H] + (monoisotopic) 661.3, found S56

57 D) In source fragmentation of ion at E) In source fragmentation of ion at 234. S57

58 F) LC-MS analysis of the trypsin digest, LC trace 16. Intensity (Ligth scattering, AU) Time (min) G) LC-MS analysis of the trypsin digest, MS trace 1 Intensity (AU) % [M+H] + calcd.661.3, obs S58

59 H) LC-MS analysis of the trypsin digest, MS trace 1 Intensity (AU) [M+3H] [M+H] + calcd.234.7, obs [M+2H] [M+4H] Figure S66. Proteomic analysis of peptide 16a. A) Before alkylation, MALDI-TOF analysis, matrix : α-cyano-4-hydroxycinnamic acid. B) After alkylation. C) After trypsin digestion. D) In source fragmentation of ion at E) In source fragmentation of ion at F, G, H) LC-MS analysis of the trypsin digest. S59

60 Peptide 17a (analytical sample) We present here only the data for peptide 17a. The proteomic analysis for peptides 17b,e are available on request. A) Before alkylation x Peptide 17a, calcd. for [M+H] + (monoisotopic) , found B) After alkylation S6

61 x calcd.for [M+H] + (monoisotopic) , found C) After trypsin digestion x Contamination by 16a 3 calcd.for [M+H] + (monoisotopic) , found calcd.for [M+H] + (monoisotopic) found S61

62 D) In source fragmentation of ion at E) In source fragmentation of ion at F) LC-MS analysis of the trypsin digest S62

63 8. Intensity, light scattering (AU) a (contamination) Time (min) S63

64 G) LC-MS analysis of the trypsin digest, MS trace 1 Intensity (AU) [M+2H] [M+H] [M+H] + calcd , obs H) LC-MS analysis of the trypsin digest, MS trace 1 Intensity (AU) [M+2H] [M+H] + calcd , obs [M+3H] Figure S67. Proteomic analysis of peptide 17a (contaminated by some 16a),. A) Before alkylation, MALDI-TOF analysis. Matrix : α-cyano-4-hydroxycinnamic acid. B) After alkylation. C) After trypsin digestion. D) In source fragmentation of ion at E) In source fragmentation of ion at F, G, H) LC-MS analysis of the trypsin digest. S64

65 Peptide 16b A) Before alkylation x calcd. for [M+H] + (monoisotopic) , found B) After alkylation 4 x calcd. for [M+H] + (monoisotopic) , found S65

66 C) After trypsin digestion 4 x calcd. for [M+H] + (monoisotopic) 234.7, found D) In source fragmentation of ion at S66

67 E) LC-MS analysis of the trypsin digest, LC trace 1. Intensity, light scattering (AU) Time (min) F) LC-MS analysis of the trypsin digest, MS trace 1 Intensity (AU) [M+3H] [M+2H] calcd.for [M+H] + (monoisotopic) 234.7, found [M+4H] S67

68 1 G) LC-MS analysis of the trypsin digest, MS trace Intensity (AU) [M+H] calcd.for [M+H] + (monoisotopic) 677.4, found Figure S68. Proteomic analysis of peptide 16b,. A) Before alkylation, MALDI-TOF analysis, matrix : α-cyano-4-hydroxycinnamic acid. B) After alkylation. C) After trypsin digestion. D) In source fragmentation of ion at E, F, G) LC-MS analysis of the trypsin digest. S68

69 Peptide 16c A) Before alkylation x calcd. for [M+H] + (monoisotopic) , found B) After alkylation calcd. for [M+H] + (monoisotopic) 27.46, found S69

70 4 x1 C) After trypsin digestion calcd. for [M+H] + (monoisotopic) 234.7, found calcd. for [M+H] + (monoisotopic) 73.41, found D) In source fragmentation of ion at Abs. Int. * 1 b C I L y K L I C L G b y 1 b 2 y 2 b 3 y 3 y 4 b 5 y 5 y S7

E) In source fragmentation of ion at 234.

trace,,, 18. Intensity, light scattering (AU) 16.

71 E) In source fragmentation of ion at F) LC-MS analysis of the trypsin digest, LC trace,,, 18. Intensity, light scattering (AU) Time (min) S71

72 G) LC-MS analysis of the trypsin digest, MS trace 1 Intensity (AU) [M+H] calcd. for [M+H] + (monoisotopic) 73.4, found H) LC-MS analysis of the trypsin digest, MS trace 1 [M+3H] Intensity (AU) calcd. for [M+H] + (monoisotopic) 234.7, found [M+2H] Figure S69 Proteomic analysis of peptide 16c,. A) Before alkylation, MALDI-TOF analysis, matrix : α-cyano-4-hydroxycinnamic acid. B) After alkylation. C) After trypsin digestion. D) In source fragmentation of ion at E) In source fragmentation of ion at F, G, H) LC-MS analysis of the trypsin digest. S72

73 Peptide 16d A) Before alkylation calcd. for [M+H] + (monoisotopic) , found x1 B) After alkylation calcd. for [M+H] + (monoisotopic) , found S73

74 C) After trypsin digestion calcd. for [M+H] + (monoisotopic) 248.9, found calcd. for [M+H] + (monoisotopic) , found D) In source fragmentation of ion at Abs. Int. * 1 b C I L y K L I C A G 1 b b 4 y b 2 y 1 y 2 y 3 a 5 b 5 y 4 a 6 y S74

75 E) In source fragmentation of ion at Abs. Int. * 1 b C* G G T L A L L T y H V T L L A L P S P G G C* y 1 y 2 y 4 y 5 a 5 b 2 y 7 y 1 y 14 a 2 y 6 y 9 y 13 y 8 b 7 b 1 a 12 b 1 b 3 a 14 b 5 y 3 b 4 b 9 b 11 a 1 a 7 a 3 b 13 a 9 a 11 b 12 y 12 y F) LC-MS analysis of the trypsin digest, LC trace Intensity, light scattering (AU) NH 2 6. O H G A C I L K OH Time (min) S75

76 G) LC-MS analysis of the trypsin digest, MS trace [M+H] Intensity (AU) calcd. for [M+H] + (monoisotopic) 661.3, found H) LC-MS analysis of the trypsin digest, MS trace [M+3H] Intensity (AU) calcd. for [M+H] + (monoisotopic) 248.9, found [M+2H] [M+4H] Figure S69. Proteomic analysis of peptide 16d,. A) Before alkylation, MALDI-TOF analysis, matrix : α-cyano-4-hydroxycinnamic acid. B) After alkylation. C) After trypsin digestion. D) In source fragmentation of ion at E) In source fragmentation of ion at F, G, H) LC-MS analysis of the trypsin digest. S76

77 Peptide 16e A) Before alkylation 4 x calcd. for [M+H] + (monoisotopic) , found B) After alkylation calcd. for [M+H] + (monoisotopic) , found S77

78 x1 4 C) After trypsin digestion calcd. for [M+H] + (monoisotopic) 677.3, found calcd. for [M+H] + (monoisotopic) 248.9, found D) In source fragmentation of ion at Abs. Int. * 1 b I L K y K L I C 5 y b 3 b 4 1 y 1 y 2 y 3 y 4 b 5 b E) In source fragmentation of ion at Abs. Int. * 1 b C* G G T L A L y H V T L L A L P S P L T G G C* 5 4 y 15 3 y y 3 y 4 y 5 y 2 y b 6 1 b 2 b 3 y 7 y 8 b 4 y 1 b 5 y 11 y 9 y 12y 13 y 14 b 9 b 1 b S78

79 F) LC-MS analysis of the trypsin digest, LC trace 18. Intensity, light scattering (AU) Time (min) G) LC-MS analysis of the trypsin digest, MS trace [M+H] Intensity (AU) calcd. for [M+H] + (monoisotopic) 677.3, found S79

80 H) LC-MS analysis of the trypsin digest, MS trace [M+2H] Intensity (AU) [M+3H] calcd. for [M+H] + (monoisotopic) 248.9, found Figure S7. Proteomic analysis of peptide 16e,. A) Before alkylation, MALDI-TOF analysis, matrix : α-cyano-4-hydroxycinnamic acid. B) After alkylation. C) After trypsin digestion. D) In source fragmentation of ion at E) In source fragmentation of ion at F, G, H) LC-MS analysis of the trypsin digest. S8

81 References (1) Boll, E.; Dheur, J.; Drobecq, H.; Melnyk, O. Org. Lett. 212, 14, (2) Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.; Melnyk, O. Org. Lett. 21, 12, (3) Ollivier, N.; Raibaut, L.; Blanpain, A.; Desmet, R.; Dheur, J.; Mhidia, R.; Boll, E.; Drobecq, H.; Pira, S. L.; Melnyk, O. J. Pept. Sci. 214, 2, S81

Supporting Information for

Supporting Information for Native Chemical Ligation at Serine Revisited Benoît Snella, Vincent Diemer, Hervé Drobecq, Vangelis Agouridas,* Oleg Melnyk* University of Lille, Pasteur Institute of Lille,