Supporting Information for - Amazon S3 · 2018-07-04 · S 1 Supporting Information for Kinetically...

S1

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 Melnyka*.

aUniv. Lille, CNRS, Institut Pasteur de Lille, UMR 8161 - M3T – Mechanisms of

Tumorigenesis and Targeted Therapies, F-59000 Lille, France. b Univ. Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, F 59

000 Lille, France

Corresponding author : Dr. Oleg Melnyk, [email protected] Centre National de la Recherche Scientifique (CNRS)

Institut de Biologie de Lille

1 rue du Pr Calmette, CS 50447, 59021 Lille cedex, France

http://csb.ibl.fr Tel: +33 3 20 87 12 14

S2

Contenu 1. General Methods .............................................................................................................................4

Reagents and solvents .....................................................................................................................4

Analyses .........................................................................................................................................4

HPLC purification ...........................................................................................................................4

2. Peptide synthesis .............................................................................................................................5

2.1 Synthesis of linear SEAoff peptide segments 12 ..........................................................................5

2.2 Characterization of the linear SEAoff peptides 12 .......................................................................6

Characterization of peptide C(StBu)ILKED(SEAoff)VRGA-SEAoff 12a .......................................6

Characterization of peptide C(StBu)ILKED(SEAoff)VRGS-SEAoff 12b ...................................... 11

Characterization of peptide C(StBu)ILKED(SEAoff)VRGL-SEAoff 12c ...................................... 16

Characterization of peptide C(StBu)ILKEE(SEAoff)VRGA-SEAoff 12d ..................................... 22

Characterization of peptide C(StBu)ILKEE(SEAoff)VRGS-SEAoff 12e ...................................... 27

Characterization of peptide C(StBu)ILKEE(SEAoff)VRGL-SEAoff 12f ....................................... 33

2.3 Synthesis of peptide 15 ............................................................................................................ 37

Characterization of peptide CGGTLPSPLALLTVH-NH2 15 ..................................................... 38

3. Kinetically controlled cyclization/ligation sequence ...................................................................... 40

Typical experimental procedure (illustrated with peptide 12a and 15) ............................................ 40

Characterization of peptide 16a ................................................................................................. 41

Characterization of peptide 16b ................................................................................................. 43

Characterization of peptide 16c ................................................................................................. 45

Characterization of peptide 16d ................................................................................................. 47

Characterization of peptide 16e ................................................................................................. 49

Characterization of peptide 16g ................................................................................................. 50

NMR analysis of peptide 16g ........................................................................................................ 53

Proteomic analysis ............................................................................................................................ 55

General procedure illustrated with peptide 16a .............................................................................. 55

Peptide 16a ................................................................................................................................... 55

Peptide 17a (analytical sample) ..................................................................................................... 60

Peptide 16b ................................................................................................................................... 65

Peptide 16c ................................................................................................................................... 69

Peptide 16d ................................................................................................................................... 73

Peptide 16e ................................................................................................................................... 77

References ..................................................................................................................................... 81

S4

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(SEAoff)-OH and Fmoc-Glu(SEAoff)-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 (CH2Cl2), diethylether (Et2O), acetonitrile (CH3CN), heptane, LC–MS-grade acetonitrile (CH3CN, 0.1% TFA), LC–MS-grade water (H2O, 0.1% TFA), N,N-diisopropylethylamine (DIEA), acetic anhydride (Ac2O) 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 2000 quadripole) on an reverse phase column XBridge BEH300 C18 (3.5 µm, 300 Å, 4.6 × 150 mm) unless otherwise stated. The elutions were carried out at 30 °C using a linear gradient of eluent B in eluent A over 30 min at a flow rate of 1 mL/min (0-100%, eluent A = 0.1% TFA in H2O; eluent B = 0.1% TFA in CH3CN/H2O: 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+, m/z range 350–2040, capillary voltage 3 kV, cone voltage 30 V, extractor voltage 3 V, RF lens 0.2 V, source temperature 120 °C, dessolvation temperature 350 °C. Samples were prepared using 10 µL aliquots of the reaction mixtures. The aliquots were quenched by adding 90 L of 1% aqueous TFA, extracted with Et2O 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 m/z corresponded to the monoisotopic ions, unless otherwise stated. 1H and 13C NMR spectra were recorded on a Bruker Advance-300 spectrometer operating at 300 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, 300 Å, 19 × 100 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 2000 quadripole). Selected fractions were combined, frozen at -20 °C and lyophilized.

S5

2. Peptide synthesis

2.1 Synthesis of linear SEAoff peptide segments 12 Typical procedures for the synthesis of SEAoff peptide segments were described in previous papers.1,2 For a detailed protocol see the protocol article.3

Scheme S1. SEAoff peptides 12 synthesized in this study.

General protocol

Peptide elongation was performed on SEA PS resin (0.1 mmol, 0.16 mmol/g) using standard Fmoc/tert-butyl chemistry. The Fmoc-L-Asp(SEAoff)-OH (0.11 mmol) or Fmoc-L-Glu(SEAoff)-OH (0.11 mmol) were coupled manually using HATU (0.15 mmol)/DIEA (0.1 mmol) activation in DMF for 2 h. The other amino acids were incorporated using an automated peptide synthesizer (0.1 mmol scale). Couplings were performed using 5-fold molar excess of each Fmoc-L- amino acid, 4.5-fold molar excess of HBTU, and 10-fold molar excess of DIEA. A capping step was performed after each coupling with Ac2O/DIEA in DMF. At the end of the synthesis, the peptidyl resin was washed with CH2Cl2 (2 × 2 min) and Et2O (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 (10 mL) for 2 h. The crude peptide was precipitated in ice-cold diethyl ether/heptane: 1/1 by vol (100 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 30 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 130 Å, 19 × 100 mm, 5 µm. Eluent A : water containing 0.1% TFA by vol, eluent B: water/acetonitrile : ¼ by vol containing 0.1% TFA by vol. For 12a,b: linear gradient 20% - 45% B in 25 min, flow rate 25 mL/min. For 12c : linear gradient 20% - 60% B in 25 min, flow rate 25 mL/min.

S6

For 12d-f : linear gradient 25% - 50% B in 25 min, flow rate 20 mL/min.

Table S1. Yields for purified SEAoff peptides 12

Peptide Sequence Isolated yield (%)

12a C(StBu)ILKED(SEAoff)VRGA-SEAoff 31%

12b C(StBu)ILKED(SEAoff)VRGS-SEAoff 23%

12c C(StBu)ILKED(SEAoff)VRGL-SEAoff 24%

12d C(StBu)ILKEE(SEAoff)VRGA-SEAoff 28%

12e C(StBu)ILKEE(SEAoff)VRGS-SEAoff 27%

12f C(StBu)ILKEE(SEAoff)VRGL-SEAoff 24%

2.2 Characterization of the linear SEAoff peptides 12

Characterization of peptide C(StBu)ILKED(SEAoff)VRGA-SEAoff 12a

Figure S1. LC-MS analysis of peptide C(StBu)ILKED(SEAoff)VRGA-SEAoff 12a. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: [M+H]+ calculated (mean) 1426.9, found 1427.1.

Time (min)6.00 10.00 14.00 18.00 22.00 26.00

0.0

50.0

100.0

150.0

200.0

250.0

300.0

350.0

400.0

450.0

500.0

550.0Intensity, light scattering (AU)

m/z400 600 800 1000 1200 1400 1600 1800 20000

100713.9

1427.1

Intensity (AU)

[M+2H]2+

S7

Figure S2. MALDI-TOF analysis of peptide C(StBu)ILKED(SEAoff)VRGA-SEAoff 12a. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H]+ m/z calcd (monoisotopic) 1425.63, found 1425.68. The peak at m/z 1337.66 is due to the partial deprotection of the cysteine residue during MS analysis.

NMR analysis of peptide C(StBu)ILKED(SEAoff)VRGA-SEAoff 12a

1H NMR (300 MHz, H2O+D2O) δ 8.73 – 8.65 (m, 1H), 8.63 – 8.55 (m, 1H), 8.54 – 8.47 (m, 1H), 8.47 – 8.26 (m, 4H), 8.21 – 8.14 (m, 1H), 8.12 – 8.04 (m, 1H), 7.70 – 7.49 (m, 1H), 7.25 (t, 1H), 6.72 (s, 1H), 5.00 – 4.86 (m, 2H), 4.46 – 4.27 (m, 4H), 4.27 – 4.19 (m, 1H), 4.16 – 3.77 (m, 10H), 3.37 – 3.20 (m, 2H), 3.14 – 2.94 (m, 10H), 2.47 (t, J = 7.5 Hz, 2H), 2.25 – 2.07 (m, 5H), 2.06 – 1.58 (m, 10H), 1.57 – 1.44 (m, 3H), 1.45 – 1.34 (m, 12H), 1.31 – 1.10 (m, 1H), 1.03 – 0.83 (m, 18H).

13C NMR (75 MHz, H2O+D2O) δ 179.95 ;177.81 – 177.58 (m) ; 177.75 – 177.62 (m) ; 177.45 (s) ; 177.06 – 176.90 (m) ; 176.77 (s) ; 176.28 – 176.09 (m) ; 176.09 – 175.92 (m) ; 175.46 (s) ; 175.17 ; 174.98 ; 174.27 ; 172.91 ; 170.71 ; 159.70 (s) ; 62.26 – 62.00 (m) ; 61.18 (s) ; 56.57 (s) ; 56.33 (s) ; 55.81 (s) ; 55.47 (s) ; 55.45 – 55.32 (m) ; 55.09 (s) ; 53.47 (s) ; 53.40 – 53.33 (m) ; 53.32 (s) ; 51.46 ; 48.82 (s) ; 45.27 – 44.93 (m) ; 43.45 (s) ; 43.24 (s) ; 42.75 – 42.51 (m) ; 42.23 (s) ; 40.96 ; 40.72 (s) ; 39.26 (s) ; 37.53 (s) ; 37.47 – 37.35 (m) ; 36.75 – 36.66 (m) ; 36.30 – 36.22 (m) ; 33.17 (s) ; 33.04 (s) ; 31.73 (s) ; 30.80 – 30.61 (m) ; 29.56 – 29.30 (m) ; 29.20 (s), 27.25 (s) ; 27.12 (s) ; 24.84 (s) ; 24.84 (s) ; 24.05 (s) ; 21.24 (s) ; 20.31 (s) ; 19.65 (s) ; 17.46 (s) ; 12.97 (s).

1425.68

1337.60

0.0

0.5

1.0

1.5

x10 4

1320 1340 1360 1380 1400 1420 1440 1460 1480m/z

-StBu

S8

Figure S3. 1H NMR spectrum of peptide C(StBu)ILKED(SEAoff)VRGA-SEAoff 12a.

Figure S4. 13C NMR spectrum of peptide C(StBu)ILKED(SEAoff)VRGA-SEAoff 12a.

-1.5-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.5f1 (ppm)

-5E+06

0

5E+06

1E+07

2E+07

2E+07

2E+07

3E+07

4E+07

4E+07

4E+07

5E+07

12.9

717

.46

19.6

520

.31

21.2

424

.05

24.8

424

.90

27.1

227

.25

29.2

029

.41

30.7

131

.73

33.0

433

.17

36.2

636

.70

37.4

237

.53

39.2

640

.72

40.9

642

.23

42.6

343

.24

43.4

545

.12

48.8

251

.52

53.3

253

.35

53.4

755

.09

55.3

755

.47

55.8

156

.33

56.5

861

.18

62.1

4

159.

7017

0.72

172.

4517

2.97

173.

1217

4.34

174.

3917

5.01

175.

1417

5.20

175.

2917

5.46

176.

0017

6.20

176.

3617

6.77

177.

4517

7.67

180.

07

C(StBu)ILKED(SEAoff)VRGA-SEAoff

12a


12a

S9

Figure S5. 1H-1H COSY spectrum of peptide C(StBu)ILKED(SEAoff)VRGA-SEAoff 12a.

Figure S6. 1H-1H DIPSI spectrum of peptide C(StBu)ILKED(SEAoff)VRGA-SEAoff 12a.

f1 (p

pm)

f1 (p

pm)


12a

C(StBu)ILKED(SEAoff)VRGA-SEAoff 12a

S10

Figure S7. 1H-1H ROESY spectrum of peptide C(StBu)ILKED(SEAoff)VRGA-SEAoff 12a.

Figure S8. 1H-13C HSQC spectrum of peptide C(StBu)ILKED(SEAoff)VRGA-SEAoff 12a.

f1 (p

pm)

f1 (p

pm)



S11

Characterization of peptide C(StBu)ILKED(SEAoff)VRGS-SEAoff 12b

Figure S9.LC-MS analysis of peptide C(StBu)ILKED(SEAoff)VRGS-SEAoff 12b. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: [M+H]+ calculated (mean) 1442.9, found 1442.5.

Time (min)0.00 5.00 10.00 15.00 20.00 25.00 30.00

0.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

Intensity, light scattering (AU)

m/z500 700 900 1100 1300 1500 1700 1900 2100

0

100 721.9

1442.5

[M+2H]2+

Intensity (AU)

[M+H]+

S12

Figure S10. MALDI-TOF analysis of peptide C(StBu)ILKED(SEAoff)VRGS-SEAoff 12b. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H]+ m/z calcd (monoisotopic) 1441.6, found 1441.5. The peak at m/z 1353.5 is due to the deprotection of the cysteine residue during MS analysis.

NMR analysis of peptide C(StBu)ILKED(SEAoff)VRGS-SEAoff 12b

1H NMR (300 MHz, H2O+D2O) δ 8.69 (d, J = 7.9 Hz, 1H), 8.58 (d, J = 7.4 Hz, 1H), 8.50 (d, J = 6.4 Hz, 1H), 8.47 – 8.29 (m, 4H), 8.25 (d, J = 7.6 Hz, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.25 (s, 1H), 4.45 – 4.28 (m, 6H), 4.27 – 4.09 (m, 1H), 4.05 – 3.71 (m, 6H), 3.37 – 3.18 (m, 4H), 3.18 – 2.90 (m, 11H), 2.45 (d, J = 7.2 Hz, 2H), 2.24 – 1.58 (m, 16H), 1.58 – 1.43 (m, 3H), 1.39 (s, 9H), 1.31 – 1.12 (m, 1H), 1.07 – 0.84 (m, 18H).

13C NMR: 13C NMR (75 MHz, H2O+D2O) δ 180.45 – 180.05, 177.00 – 176.92, 176.79 – 176.69, 176.21, 176.08 – 175.98, 175.50, 175.20, 175.05, 174.47, 174.34, 173.45 – 173.37, 170.88 – 170.50, 159.62, 64.08, 62.04, 61.12, 56.61 – 56.49, 56.44 – 56.05, 55.94 – 55.79, 55.47, 55.42 – 55.33, 55.25 – 55.07, 54.46, 53.54, 53.42 – 53.34, 53.32, 51.52, 45.26 – 45.13, 43.47, 43.33 – 43.18, 42.71 – 42.54, 42.23, 41.07, 40.72, 39.26, 37.48 – 37.38, 37.27, 36.76 – 36.65, 33.23 – 33.17, 33.15, 33.12 – 33.02, 31.73, 30.81 – 30.70, 29.50 – 29.41, 29.20, 27.27, 27.12, 24.90, 24.84, 24.05, 21.17, 20.31, 17.44, 17.27 – 17.17, 12.97.

1441.5

1353.5

0

500

1000

1500

2000

2500

3000

1340 1360 1380 1400 1420 1440 1460 1480m/z

-StBu

S13

Figure S11. 1H NMR spectrum of peptide C(StBu)ILKED(SEAoff)VRGS-SEAoff 12b.

Figure S12. 13C NMR spectrum of peptide C(StBu)ILKED(SEAoff)VRGS-SEAoff 12b.

23.0

6

1.54

11.6

83.

53

18.7

7

2.31

13.0

6

4.48

6.80

1.51

1.96

1.00

1.01

1.21

4.37

1.08

1.06

0.81

0.89

0.92

0.93

0.96

0.97

1.17

1.20

1.22

1.24

1.27

1.39

1.46

1.48

1.51

1.64

1.71

1.73

1.76

1.83

1.87

1.90

1.99

2.01

2.04

2.07

2.13

2.15

2.17

2.19

2.44

2.46

2.95

3.06

3.11

3.16

3.21

3.23

3.26

3.28

3.30

3.33

3.76

3.78

3.81

3.84

3.88

3.90

3.94

3.95

4.00

4.01

4.13

4.18

4.20

4.22

4.25

4.31

4.37

4.84

4.92

6.71

7.25

8.10

8.12

8.24

8.27

8.31

8.33

8.35

8.37

8.40

8.43

8.44

8.49

8.51

8.57

8.59

8.68

8.70

-12.

9412

.97

17.2

217

.44

20.3

120

.61

21.2

424

.05

24.8

424

.90

27.1

227

.27

27.4

229

.20

29.4

730

.76

31.7

333

.08

33.1

533

.19

36.7

037

.27

37.4

239

.26

40.7

241

.07

42.2

342

.63

43.2

443

.47

45.1

951

.52

53.3

253

.36

53.5

454

.46

55.1

255

.19

55.3

655

.47

55.5

355

.85

56.3

156

.55

61.1

861

.55

62.1

764

.17

159.

6615

9.70

170.

7417

1.87

173.

4217

3.57

174.

3417

4.38

174.

4717

4.49

175.

0517

5.24

175.

5017

5.84

176.

0317

6.21

176.

7417

6.97

180.

24

C(StBu)ILKED(SEAoff)VRGS-SEAoff 12b


S14

Figure S13. 1H-1H COSY spectrum of peptide C(StBu)ILKED(SEAoff)VRGS-SEAoff 12b.

Figure S14. 1H-1H DIPSI spectrum of peptide C(StBu)ILKED(SEAoff)VRGS-SEAoff 12b.

f1 (p

pm)

f1 (p

pm)


C(tBu)ILKED(SEAoff)VRGS-SEAoff 12b

S15

Figure S15. 1H-1H ROESY spectrum of peptide C(StBu)ILKED(SEAoff)VRGS-SEAoff 12b.

Figure S16. 1H-13C HSQC spectrum of peptide C(StBu)ILKED(SEAoff)VRGS-SEAoff 12b.

f1 (p

pm)

f1 (p

pm)



S16

Characterization of peptide C(StBu)ILKED(SEAoff)VRGL-SEAoff 12c

Figure S17. LC-MS analysis of peptide C(StBu)ILKED(SEAoff)VRGL-SEAoff 12c. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: [M+H]+ calculated (mean) 1469.0, found 1468.4.

Time (min)0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00

0.0

100.0

200.0

300.0

400.0

500.0

600.0

700.0


m/z300 500 700 900 1100 1300 1500 1700 1900

0

100735.1

1468.4

[M+2H]2+

[M+H]+

S17

Figure S18. MALDI-TOF analysis of peptide C(StBu)ILKED(SEA off)VRGL-SEAoff 12c. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H]+ m/z calcd (monoisotopic) 1467.6, found 1467.8. The peak at m/z 1379.7 is due to the partial deprotection of the cysteine residue during MS analysis.

1467.8

1379.7

0

2

4

6

x104

600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600m/z

-StBu

S18

NMR analysis of peptide C(StBu)ILKED(SEAoff)VRGL-SEAoff 12c

1H NMR (300 MHz, H2O+D2O) δ 8.69 – 8.62 (m, 1H), 8.59 – 8.51 (m, 1H), 8.48 – 8.41 (m, 1H), 8.40 – 8.24 (m, 4H), 8.19 – 8.12 (m, 1H), 8.11 – 8.03 (m, 1H), 7.27 – 7.15 (m, 1H), 4.39 – 4.22 (m, 5H), 4.21 – 4.13 (m, 1H), 4.13 – 4.00 (m, 4H), 3.92 (s, 6H), 3.87 – 3.67 (m, 2H), 3.31 – 3.09 (m, 2H), 3.09 – 2.86 (m, 10H), 2.50 – 2.27 (m, 2H), 2.19 – 1.40 (m, 18H), 1.34 (s, 3H), 1.24 – 1.06 (m, 1H), 1.02 – 0.79 (m, 24H).

13C NMR (75 MHz, H2O+D2O) δ 180.51 (s), 177.30 (s), 176.91 (s), 176.57 (s), 176.14 (s), 175.92 (s), 175.84 (s), 175.47 (s), 175.15 (s), 175.00 (s), 174.27 (s), 173.18 (s), 170.66 (s), 62.14 (s), 61.09 (s), 56.44 (s, J = 15.4 Hz), 56.23 (s), 55.84 (s), 55.38 (s, J = 6.8 Hz), 55.29 (s), 55.22 (s), 55.04 (s, J = 13.0 Hz), 53.44 (s, J = 16.3 Hz), 53.22 (s), 51.43 (s, J = 17.3 Hz), 51.20 (s), 45.09 (s), 43.37 (s, J = 16.4 Hz), 43.15 (s), 42.78 (s), 42.52 (s), 42.13 (s), 40.85 (s, J = 18.5 Hz), 40.61 (s), 39.17 (s), 37.35 (s, J = 8.3 Hz), 37.24 (s), 33.40 (s), 33.01 (s), 31.64 (s), 30.68 (s), 29.50 (s), 29.11 (s), 27.19 (s, J = 11.2 Hz), 27.04 (s), 25.20 (s), 24.77 (s), 23.97 (s), 23.28 (s), 21.20 (s), 20.22 (s), 17.38 (s), 12.88 (s).

Figure S19. 1H NMR spectrum of peptide C(StBu)ILKED(SEAoff)VRGL-SEAoff 12c.

27.1

91.

5310

.40

22.6

5

2.00

10.4

04.

69

1.37

2.75

0.95

0.48

1.78

0.77

0.86

0.90

3.75

0.88

0.82

0.59

0.85

0.87

0.89

0.91

0.94

1.17

1.34

1.41

1.44

1.46

1.52

1.61

1.64

1.67

1.69

1.71

1.78

1.83

1.85

1.96

2.06

2.09

2.11

2.13

2.37

2.39

2.99

3.01

3.16

3.21

3.23

3.72

3.74

3.77

3.79

3.84

3.92

4.03

4.08

4.17

4.19

4.27

4.30

4.32

6.69

7.22

8.07

8.09

8.14

8.16

8.28

8.31

8.33

8.35

8.37

8.43

8.45

8.54

8.57

8.64

8.67

C(StBu)ILKED(SEAoff)VRGL-SEAoff 12c

S19

Figure S20. 13C NMR spectrum of peptide C(StBu)ILKED(SEAoff)VRGL-SEAoff 12c.

Figure S21. 1H-1H COSY spectrum of peptide C(StBu)ILKED(SEAoff)VRGL-SEAoff 12c.

12.8

817

.38

20.2

221

.20

23.2

823

.97

24.7

725

.20

27.0

427

.19

29.1

131

.64

33.0

133

.40

37.2

442

.78

51.2

051

.43

53.2

253

.44

55.0

455

.22

55.2

955

.38

55.8

456

.23

56.4

461

.09

62.1

4

170.

6617

3.18

174.

2717

5.00

175.

0017

5.15

175.

4717

5.84

175.

9217

6.14

176.

5717

6.91

177.

3018

0.51

f1 (p

pm)



S20

Figure S22. 1H-1H DIPSI spectrum of peptide C(StBu)ILKED(SEAoff)VRGL-SEAoff 12c.

Figure S23. 1H-13C HSQC spectrum of peptide C(StBu)ILKED(SEAoff)VRGL-SEAoff 12c.

f1 (p

pm)

f1 (p

pm)



S21

Figure S24. 1H-1H ROESY spectrum of peptide C(StBu)ILKED(SEAoff)VRGL-SEAoff 12c.

f1 (p

pm)

C(StBu)ILKED(SEAoff)VRGL-SEAoff

12c

S22

Characterization of peptide C(StBu)ILKEE(SEAoff)VRGA-SEAoff 12d

Figure S25. LC-MS analysis of peptide C(StBu)ILKEE(SEAoff)VRGA-SEAoff 12d. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: [M+H]+ calcd (mean) 1440.9, found 1441.3.

Time (min)2.00 6.00 10.00 14.00 18.00 22.00 26.00 30.00

0.0

100.0

200.0

300.0

400.0

500.0

600.0

700.0

800.0

900.0


m/z300 500 700 900 1100 1300 1500 1700 1900

0

100 721.1

1441.3

[M+2H]2+

[M+H]+

Intensity (AU)

S23

Figure S26. MALDI-TOF analysis of peptide C(StBu)ILKEE(SEAoff)VRGA-SEAoff 12d. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H]+ m/z calcd (monoisotopic) 1439.64, found 1439.84.

NMR analysis of peptide C(StBu)ILKEE(SEAoff)VRGA-SEAoff 12d

1H NMR (300 MHz, H2O+D2O) δ 8.62 (d, J = 8.1 Hz, 1H), 8.52 – 8.43 (m, J = 10.4, 6.8 Hz, 2H), 8.40 (t, J = 5.8 Hz, 1H), 8.35 – 8.23 (m, 3H), 8.20 (d, J = 6.9 Hz, 1H), 8.12 (d, J = 6.4 Hz, 1H), 7.19 (s, 1H), 4.41 – 4.20 (m, J = 15.5 Hz, 6H), 4.16 – 3.88 (m, 5H), 3.87 – 3.71 (m, 2H), 3.33 – 3.06 (m, 5H), 3.05 – 2.89 (m, J = 11.1, 5.4 Hz, 6H), 2.51 (t, J = 7.0 Hz, 2H), 2.39 (t, J = 7.4 Hz, 2H), 2.20 – 1.53 (m, 15H), 1.51 – 1.39 (m, 3H), 1.37 – 1.31 (m, 12H), 1.25 – 1.08 (m, 1H), 0.99 – 0.76 (m, J = 12.9, 6.9 Hz, 15H).

13C NMR (75 MHz, H2O+D2O) δ 180.41, 177.67, 177.24, 176.92, 176.72, 176.17 – 176.13, 176.10 – 176.05, 175.99, 175.88 – 175.80, 175.26 – 175.18, 173.03, 170.72, 159.78 – 159.54, 62.38, 61.18, 56.44, 55.92, 55.74, 55.47, 55.38, 55.24 – 55.09, 53.56 – 53.42, 53.42 – 53.34, 51.52, 48.84, 45.16 – 45.05, 43.52 – 43.38, 43.36 – 43.22, 42.68 – 42.57, 42.23, 42.17 – 42.07, 40.96, 40.84 – 40.71, 39.29, 37.53, 33.46 – 33.33, 32.96, 31.73, 31.31 – 31.16, 30.89 – 30.74, 29.71 – 29.53, 29.20, 27.25, 27.12, 24.97 – 24.88, 24.86, 24.05, 21.21, 20.63, 19.65, 17.46, 12.99.

1439.84

1461.83 1477.82

0.0

0.2

0.4

0.6

0.8

1.0

1.2

x104

1420 1430 1440 1450 1460 1470 1480m/z

S24

Figure S27. 1H NMR spectrum of peptide C(StBu)ILKEE(SEAoff)VRGA-SEAoff 12d.

Figure S28. 13C NMR spectrum of peptide C(StBu)ILKEE(SEAoff)VRGA-SEAoff 12d.

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)

-5E+07

-4E+07

-3E+07

-2E+07

-1E+07

0

1E+07

2E+07

3E+07

4E+07

5E+07

6E+07

7E+07

8E+07

9E+07

1E+08

1E+08

-50-40-30-20-100102030405060708090110130150170190210230250f1 (ppm)

-2E+08

-2E+08

-2E+08

-1E+08

-5E+07

0

5E+07

1E+08

2E+08

2E+08

2E+08

3E+08

4E+08

4E+08

4E+08

5E+08

C(StBu)ILKEE(SEAoff)VRGA-SEAoff 12d

C(StBu)ILKEE(SEAoff)VRGA-SEAoff12d

S25

Figure S29. 1H-1H COSY spectrum of peptide C(StBu)ILKEE(SEAoff)VRGA-SEAoff 12d.

Figure S30. 1H-13C HSQC spectrum of peptide C(StBu)ILKEE(SEAoff)VRGA-SEAoff 12d.

f1 (p

pm)

f1 (p

pm)

C(StBu)ILKEE(SEAoff)VRGA-SEAoff

12d


S26

Figure S31. 1H-1H DIPSI spectrum of peptide C(StBu)ILKEE(SEAoff)VRGA-SEAoff 12d.

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f2 (ppm)

0

1

2

3

4

5

6

7

8

9

f1 (

ppm

)

Figure S32. 1H-1H ROESY spectrum of peptide C(StBu)ILKEE(SEAoff)VRGA-SEAoff 12d.



S27

Characterization of peptide C(StBu)ILKEE(SEAoff)VRGS-SEAoff 12e

Figure S33. LC-MS analysis of peptide C(StBu)ILKEE(SEAoff)VRGS-SEAoff 12e. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: [M+H]+ calcd (mean) 1456.9, found 1456.3

Time (mim0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00

0.0

100.0

200.0

300.0

400.0

500.0

600.0

700.0

800.0

900.0


m/z400 800 1200 1600 2000

0

100728.8

1456.3

[M+2H]2+

Intensity (AU)

[M+H]+

S28

Figure S34. MALDI-TOF analysis of peptide C(StBu)ILKEE(SEAoff)VRGS-SEAoff 12e. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H]+ m/z calcd (monoisotopic) 1455.64, found 1455.79. The peak at m/z 1367.69 is due to the partial deprotection of the cysteine residue during MS analysis.

1455.79

1367.69

0.0

0.5

1.0

1.5

x104

600 1000 1400 1800 2200 2600m/z

S29

NMR analysis of peptide C(StBu)ILKEE(SEAoff)VRGS-SEAoff 12e

1H NMR (300 MHz, H2O+D2O) δ 8.53 (d, J = 7.9 Hz, 1H), 8.47 – 8.30 (m, 3H), 8.30 – 8.18 (m, 3H), 8.13 (d, J = 7.6 Hz, 2H), 7.08 (s, 1H), 4.26 – 4.08 (m, 6H), 4.05 – 3.91 (m, 5H), 3.87 – 3.78 (m, 4H), 3.77 – 3.45 (m, 5H), 3.16 – 2.99 (m, 2H), 2.98 – 2.75 (m, 10H), 2.37 (d, J = 7.2 Hz, 2H), 2.25 (t, J = 7.3 Hz, 2H), 2.04 – 1.77 (m, 5H), 1.75 – 1.60 (m, 7H), 1.60 – 1.39 (m, 9H), 1.39 – 1.25 (m, 3H), 1.19 (s, 9H), 1.10 – 0.93 (m, 1H), 0.88 – 0.64 (m, 18H).

13C NMR (75 MHz, H2O+D2O) δ 181.02 – 180.30 (m), 177.30 (s), 177.04 – 176.84 (m), 176.84 – 176.53 (m), 176.00 (s), 175.42 – 174.87 (m), 174.69 – 174.10 (m), 172.93 (s), 170.95 – 170.22 (m), 159.82 – 159.51 (m), 64.05 (d, J = 15.5 Hz), 62.41 (s), 61.18 (s), 56.39 (s), 55.78 (s), 55.47 (s), 55.26 – 55.08 (m), 54.46 (s), 53.63 – 53.47 (m), 53.46 – 53.27 (m), 45.27 – 45.10 (m), 43.56 – 43.41 (m), 43.37 – 43.21 (m), 42.72 – 42.55 (m), 42.28 – 42.14 (m), 41.07 (s), 40.77 (s), 39.29 (s), 37.56 – 37.42 (m), 37.29 (s), 33.68 – 33.54 (m), 33.23 – 33.04 (m), 32.95 (s), 31.73 (s), 31.32 – 31.12 (m), 31.00 – 30.78 (m), 29.20 (s), 27.25 (s), 27.12 (s), 24.97 – 24.86 (m), 24.84 (s), 24.05 (s), 21.21 (s), 20.65 (s), 17.46 (s), 12.99 (s).

Figure S35. 1H NMR spectrum of peptide C(StBu)ILKEE(SEAoff)VRGS-SEAoff 12e.

21.4

41.

6511

.12

3.32

8.73

5.14

5.85

2.16

1.92

11.0

34.

33

4.76

2.14

1.72

3.09

0.95

2.00

3.06

3.17

0.85

0.70

0.72

0.74

0.76

0.77

0.79

0.81

0.98

1.00

1.02

1.05

1.19

1.26

1.29

1.32

1.46

1.51

1.54

1.56

1.67

1.78

1.81

1.83

1.85

1.88

1.91

1.92

1.94

2.23

2.25

2.28

2.36

2.38

2.70

2.85

2.86

2.88

2.91

2.96

3.01

3.03

3.06

3.08

3.10

3.56

3.58

3.62

3.64

3.67

3.69

3.71

3.73

3.74

3.80

3.82

3.88

3.93

3.95

3.98

4.12

4.15

4.17

4.19

7.08

8.12

8.15

8.21

8.23

8.25

8.28

8.32

8.34

8.36

8.37

8.39

8.42

8.44

8.52

8.55

C(StBu)ILKEE(SEAoff)VRGS-SEAoff 12e

S30

Figure S36. 13C NMR spectrum of peptide C(StBu)ILKEE(SEAoff)VRGS-SEAoff 12e.

Figure S37. 1H-1H COSY spectrum of peptide C(StBu)ILKEE(SEAoff)VRGS-SEAoff 12e.

12.9

917

.46

20.6

521

.21

24.0

524

.90

24.9

027

.12

27.2

529

.20

29.2

030

.90

31.2

331

.73

32.9

533

.12

33.5

837

.29

37.4

839

.29

40.7

741

.07

42.2

242

.64

43.3

043

.46

53.3

953

.54

54.4

655

.19

55.4

755

.78

56.3

961

.18

62.4

164

.16

159.

7017

0.72

173.

4817

4.48

175.

2217

5.87

176.

0017

6.10

176.

6717

6.99

177.

3017

7.30

180.

73

f1 (p

pm)

C(StBu)ILKEE(SEAoff)VRGS-SEAoff

12e


S31

Figure S38. 1H-13C HSQC spectrum of peptide C(StBu)ILKEE(SEAoff)VRGS-SEAoff 12e.

Figure S39. 1H-1H DIPSI spectrum of peptide C(StBu)ILKEE(SEAoff)VRGS-SEAoff 12e.

f1 (p

pm)

f1 (p

pm)



S32

Figure S40. 1H-1H ROESY spectrum of peptide C(StBu)ILKEE(SEAoff)VRGS-SEAoff 12e.

f1 (p

pm)


S33

Characterization of peptide C(StBu)ILKEE(SEAoff)VRGL-SEAoff 12f

Figure S41. LC-MS analysis of peptide C(StBu)ILKEE(SEAoff)VRGL-SEAoff 12f. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: [M+H]+ calcd (mean) 1483.0, found 1483.4

Time (min)

0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00

0.0

100.0

200.0

300.0

400.0

500.0

600.0

700.0


m/z300 500 700 900 1100 1300 1500 1700 1900

0

100742.1

1483.4

[M+2H]2+

Intensity (AU)

[M+H]+

S34

Figure S42. MALDI-TOF analysis of peptide C(StBu)ILKEE(SEAoff)VRGL-SEAoff 12f. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H]+ m/z calcd (monoisotopic) 1481.6, found 1481.9. The peak at m/z 1393.8 is due to the partial deprotection of the cysteine residue during MS analysis.

NMR analysis of peptide C(StBu)ILKEE(SEAoff)VRGL-SEAoff 12f

1H NMR (300 MHz, H2O+D2O) δ 8.59 (d, J = 8.1 Hz, 1H), 8.42 (d, J = 6.7 Hz, 2H), 8.35 – 8.14 (m, 5H), 8.11 (d, J = 7.9 Hz, 1H), 7.16 (s, 1H), 4.36 – 4.16 (m, 6H), 4.12 – 3.86 (m, 7H), 3.86 – 3.54 (m, 4H), 3.29 – 3.05 (m, 8H), 3.05 – 2.86 (m, 6H), 2.48 (t, J = 6.9 Hz, 2H), 2.33 (t, J = 7.4 Hz, 2H), 2.16 – 1.88 (m, 5H), 1.87 – 1.70 (m, 5H), 1.70 – 1.50 (m, 10H), 1.50 – 1.33 (m, 3H), 1.30 (s, 9H), 1.22 – 1.02 (m, 1H), 0.98 – 0.74 (m, 24H).

13C NMR (75 MHz, H2O+D2O) δ 181.28 – 180.96 (m), 177.37 – 177.20 (m), 177.05 – 176.97 (m), 176.60 – 176.50 (m), 176.26 – 176.13 (m), 176.04 (s), 175.32 – 175.13 (m), 173.38 – 173.18 (m), 170.83 – 170.65 (m), 159.75 – 159.53 (m), 62.49 (s), 61.18 (s), 56.34 (s), 55.92 (s, J = 33.0 Hz), 55.48 (s), 55.28 (s), 53.62 – 53.31 (m), 51.58 – 51.45 (m), 51.37 (d, J = 11.9 Hz), 45.28 – 45.06 (m), 43.52 – 43.41 (m), 43.35 – 43.22 (m), 43.01 – 42.79 (m), 42.73 – 42.53 (m), 42.33 – 42.11 (m), 41.04 – 40.89 (m), 40.88 – 40.70 (m), 39.40 – 39.15 (m), 37.57 – 37.42 (m), 37.42 – 37.27 (m), 34.08 – 33.87 (m), 33.24 – 33.01 (m), 32.90 (s), 31.82 – 31.58 (m), 31.36 – 31.16 (m), 31.01 – 30.85 (m), 29.94 – 29.66 (m), 29.20 (s), 27.27 (s), 27.14 (s), 25.27 (s), 25.03 – 24.86 (m), 24.06 (s), 23.36 (s, J = 53.0 Hz), 21.24 (s), 20.66 (s), 17.46 (s), 13.01 (s).

1481.9

1393.8

0

2

4

6

8

x10

500 750 1000 1250 1500 1750 2000 2250 2500 2750m/z

S35

Figure S43. 1H NMR spectrum of peptide C(StBu)ILKEE(SEAoff)VRGL-SEAoff 12f.

Figure S44. 13C NMR spectrum of peptide C(StBu)ILKEE(SEAoff)VRGL-SEAoff 12f.

14.5

71.

045.

402.

225.

762.

692.

861.

120.

99

4.77

2.58

1.41

2.14

1.03

0.43

0.57

2.61

1.00

0.25

0.80

0.83

0.85

0.87

0.87

0.89

0.92

1.10

1.13

1.15

1.30

1.34

1.37

1.40

1.42

1.46

1.48

1.51

1.57

1.59

1.62

1.64

1.67

1.72

1.74

1.76

1.78

1.80

1.92

1.94

1.97

1.99

2.02

2.04

2.06

2.09

2.31

2.33

2.36

2.46

2.48

2.50

2.95

2.97

2.99

3.01

3.08

3.11

3.14

3.16

3.17

3.18

3.19

3.21

3.65

3.72

3.79

3.89

3.90

3.92

3.97

3.99

4.04

4.06

4.26

4.28

4.30

7.16

8.10

8.12

8.17

8.19

8.21

8.24

8.27

8.29

8.31

8.41

8.43

13.0

117

.46

20.6

621

.24

23.3

624

.06

24.9

125

.27

27.1

427

.27

29.2

029

.80

30.9

431

.26

31.7

332

.90

33.1

133

.98

37.3

537

.47

39.2

939

.29

40.7

840

.95

42.2

342

.61

42.9

043

.30

43.4

645

.17

51.2

951

.45

53.3

855

.28

55.4

855

.92

56.3

461

.18

62.4

9

159.

6917

0.74

173.

3017

5.24

176.

0417

6.19

176.

5617

7.01

177.

2818

1.16

C(StBu)ILKEE(SEAoff)VRGL-SEAoff

12f

C(StBu)ILKEE(SEAoff)VRGL-SEAoff 12f

S36

Figure S45. 1H-1H COSY spectrum of peptide C(StBu)ILKEE(SEAoff)VRGL-SEAoff 12f.

Figure S46. 1H-13C HSQC spectrum of peptide C(StBu)ILKEE(SEAoff)VRGL-SEAoff 12f.

f1 (p

pm)

f1 (p

pm)



S37

Figure S47. 1H-1H DIPSI spectrum of peptide C(StBu)ILKEE(SEAoff)VRGL-SEAoff 12f.

2.3 Synthesis of peptide 15 Peptide elongation was performed using standard Fmoc/tert-butyl chemistry on an automated peptide synthesizer (0.5 mmol scale, NovaSyn TGR resin, 0.25 mmol/g). Couplings were performed using 5-fold molar excess of each Fmoc-L- amino acid, 4.5-fold molar excess of HBTU, and 10-fold molar excess of DIEA. A capping step was performed after each coupling with Ac2O/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 (10 mL) for 2 h. The crude peptide was precipitated in ice-cold diethyl ether/heptane: 1/1 by vol (100 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 130 Å, 19 × 100 mm, 5 µm. Eluent A : water containing 0.1% TFA by vol, eluent B: water/acetonitrile : ¼ by vol containing 0.1% TFA by vol. Linear gradient 20% - 55% B in 25 min, flow rate 25 mL/min.

peptide sequence isolated yield (%)

15 CGGTLPSPLALLTVH-NH2 64%

f1 (p

pm)


S38

Characterization of peptide CGGTLPSPLALLTVH-NH2 15

Figure S48. LC-MS analysis of peptide CGGTLPSPLALLTVH-NH2 15. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: [M+H]+ calcd (mean) 1478.8, found 1479.1

,,,,,

Time (min)2.00 6.00 10.00 14.00 18.00 22.00 26.00

0.0

4.0e-2

8.0e-2

1.2e-1

1.6e-1

2.0e-1

2.4e-1

2.8e-1

3.2e-1

3.6e-1

4.0e-1 Intensity, light scattering (AU)

m/z500 700 900 1100 1300 1500 1700 1900 2100

0

100740.0

1479.1

Intensity (AU)

[M+2H]2+

[M+H]+

S39

Figure S49. MALDI-TOF analysis of peptide CGGTLPSPLALLTVH-NH2 15. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H]+ m/z calcd (monoisotopic) 1477.8, found 1477.8.

1477.8

0

500

1000

1500

2000

1000 1500 2000 2500 3000 3500m/z

1477.8

0

500

1000

1500

2000

1472 1476 1480 1484 1488m/z

S40

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 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). Guanidinium hydrochloride (Gdn.HCl) (4.01 g, 42,0 mmol), MPAA (235.5 mg, 1.400 mmol) and TCEP (401.3 mg, 1.400 mmol) were dissolved in sodium phosphate buffer (0.1 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, 21.27 µmol) was dissolved in the above solution (6.076 mL). The mixture was stirred at 37 °C for 48 h. Then, Cys peptide 15 (54.41 mg, 31.90 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 0.1% TFA (6 mL). The mixture was acidified to pH 3 by adding aqueous TFA (10% by vol, 2 µL) and then extracted with Et2O (3 ×10 mL) and heptane (1 × 1 mL). The solution was filtered and purified by RP-HPLC (Vydac C18 column, eluent A : water containing 0.1% TFA by vol, eluent B: water/acetonitrile : ¼ by vol containing 0.1% 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 19.45 mg (32%) of cyclic and branched peptide 16a.

S41

Characterization of peptide 16a

Figure S50. LC-MS analysis of peptide 16a. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: M calcd (mean) 2545.0, found after deconvolution 2544.5

Time (min)12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0

0.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0


m/z500 700 900 1100 1300 1500 1700 1900

0

100 849.3

1273.5

[M+3H]3+

Intensity (AU)

[M+2H]2+

16a

16a

S42

Figure S51. MALDI-TOF analysis of peptide 16a. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H]+

m/z calcd (monoisotopic) 2544.4, found 2544.4.

2544.4

1273.20

2000

4000

6000

1000 1500 2000 2500 3000m/z

2544.4

0

2000

4000

6000

2540 2544 2548 2552 2556m/z

S43

Characterization of peptide 16b

Figure S52. LC-MS analysis of peptide 16b. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: M calcd (mean) 2561.06, found after deconvolution 2561.9

Time (min)0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00

0.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

1600.0


m/z400 800 1200 1600 2000

0

100855.0

1281.6

[M+3H]3+

Intensity (AU)

[M+2H]2+

16b

16b

S44

Figure S53. MALDI-TOF analysis of peptide 16b. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H]+ m/z calcd (monoisotopic) 2560.37, found 2560.19.

2560.19

0

2

4

6

4x10

1000 1500 2000 2500 3000m/z

2560.19

0

2

4

6

8

2556 2560 2564 2568 2572m/z

S45

Characterization of peptide 16c

Figure S54. LC-MS analysis of peptide 16c. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: M calcd (mean) 2587.15, found after deconvolution 2586.99

Time (min)0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00

0.0

100.0

200.0

300.0

400.0

500.0

600.0

700.0

800.0

900.0

1000.0


m/z800 1000 1200 1400 1600 1800 2000

0

100 863.3

1294.5

[M+3H]3+

Intensity(AU)

[M+2H]2+

16c

16c

S46

Figure S55. MALDI-TOF analysis of peptide 16c. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H]+


2586.49

1294.260

2

4

6

8x104

1500 2000 2500 3000 3500m/z

2586.49

0

2

4

6

8

2584 2588 2592 2596 2600m/z

16c

S47

Characterization of peptide 16d

Figure S56. LC-MS analysis of peptide 16d. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: M calcd (mean) 2559.09, found after deconvolution 2559.43

,,,,,

Time (min)

8.00 12.00 16.00 20.00 24.00 28.00

0.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

1600.0

1800.0 Intensity, light scattering (AU)

m/z600 1000 1400 1800

0

100854.1

1280.5

[M+3H]3+

Intensity (AU)

[M+2H]2+

16d

16d

S48

Figure S57. MALDI-TOF analysis of peptide 16d. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H]+ m/z calcd (monoisotopic) 2558.39, found 2558.6.

2558.6

0

2000

4000

6000

8000

1500 2000 2500 3000 3500 4000

m/z

2558.6

0

2000

4000

6000

8000

2554 2558 2562 2566 2570m/z

16d

S49

Characterization of peptide 16e

Figure S58. LC-MS analysis of peptide 16e. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: M calcd (mean) 2575.09, found after deconvolution 2574.92.

Time (min)2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00

0.0

100.0

200.0

300.0

400.0

500.0

600.0

700.0


m/z400 600 800 1000 1200 1400 1600 1800 2000

0

100 859.3

1288.3

[M+3H]3+

Intensity (AU)

[M+2H]2+

16e

16e

S50

Figure S59. MALDI-TOF analysis of peptide 16e. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H]+


Characterization of peptide 16g

2574.6

0.0

0.2

0.4

0.6

0.8

1.0

x104

1000 1500 2000 2500 3000m/z

Time (min)2.00 6.00 10.00 14.00 18.00 22.00 26.00 30.00

0.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0


16e

16g

S51

Figure S60. LC-MS analysis of peptide 16g. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 × 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: M calcd (mean) 1188.4, found after deconvolution 1188.9.

m/z200 600 1000 1400 1800

0

100 595.0

1188.9

[M+2H]2+

Intensity (AU)

[M+H]+

16g

S52

Figure S61. MALDI-TOF analysis of peptide 16g. Matrix : α-cyano-4-hydroxycinnamic acid, [M+H]+


1188.59

0

250

500

750

1000

1250

600 800 1000 1200 1400 1600m/z

1188.59

0

250

500

750

1000

1250

1180 1184 1188 1192 1196 1200m/z

16g

16g

S53

NMR analysis of peptide 16g

Figure S62. 1H NMR spectrum of peptide 16g.

Figure S63. 1H-1H NOESY spectrum of peptide 16g

-1.5-0.50.51.52.53.54.55.56.57.58.59.510.511.5f1 (ppm)

-5.0E+07

0.0E+00

5.0E+07

1.0E+08

1.5E+08

2.0E+08

2.5E+08

3.0E+08

3.5E+08

0.00

0.84

0.85

0.87

0.89

0.90

0.90

0.91

0.91

0.92

0.93

0.94

1.40

1.41

1.59

1.60

1.61

1.62

1.66

1.67

1.68

1.70

1.75

1.77

1.86

1.86

1.87

2.06

2.08

2.42

2.44

2.45

2.85

2.87

2.91

2.92

2.93

2.94

2.95

2.96

2.97

2.98

3.21

3.22

3.23

7.00

7.09

7.17

7.21

8.08

8.08

8.09

8.17

8.19

8.19

8.21

8.34

8.36

8.39

8.40

8.43

8.47

8.48

8.64

8.65

-2-101234567891011f2 (ppm)

0

1

2

3

4

5

6

7

8

9

f1 (p

pm)

16g

16g

S54

Figure S64. 1H-1H TOCSY spectrum of peptide 16g

Figure S65. 1H-1H TOCSY-NOESY spectrum of peptide 16g

-2-101234567891011f2 (ppm)

0

1

2

3

4

5

6

7

8

9

f1 (p

pm)

S55

Proteomic analysis

General procedure illustrated with peptide 16a Peptide 16a (140 µg) was dissolved in ammonium bicarbonate buffer (25 mM, 140 µL) containing DTT (0,1 mg/mL final concentration). An aliquot of this solution corresponding to 50 µg of peptide 16a was alkylated with iodoacetamide (10 mg/mL in 25 mM ammonium bicarbonate buffer, 5 µL) for 30 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

2544.4

0.0

0.5

1.0

1.5

4x10

1000 1500 2000 2500 3000m/z

calcd. for [M+H]+ (monoisotopic) 2544.4, found 2544.4

S56

B) After alkylation

C) After trypsin digestion

2658.3

0.00

0.25

0.50

0.75

1.00

1.25

4x10

1000 1500 2000 2500 3000m/z

2034.0

661.3 2676.41017.5

0

2

4

6

5x10

1000 1500 2000 2500 3000 m/z




S57

D) In source fragmentation of ion at m/z 661.3

E) In source fragmentation of ion at m/z 2034.0

S58

F) LC-MS analysis of the trypsin digest, LC trace

G) LC-MS analysis of the trypsin digest, MS trace

Time (min)0.00 5.00 10.00 15.00 20.00 25.00 30.00

0.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

1600.0

m/z640 650 660 670 680 690 700

%

0

100 661.4

662.4

663.3

[M+H]+m/z calcd.661.3, obs 661.4

Intensity (Ligth scattering, AU)

Intensity (AU)

S59

H) LC-MS analysis of the trypsin digest, MS trace

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 m/z 661.3. E) In source fragmentation of ion at m/z 2034.0. F, G, H) LC-MS analysis of the trypsin digest.

m/z400 600 800 1000 1200 1400 1600 1800 2000

0

100679.1

509.6

1018.2

[M+3H]3+

Intensity (AU)

[M+2H]2+

[M+4H]4+

[M+H]+m/z calcd.2034.07, obs 2034.36

S60

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

B) After alkylation

2544.4

0.0

0.5

1.0

1.5

4x10

1000 1500 2000 2500 3000m/z

Peptide 17a, calcd. for [M+H]+ (monoisotopic) 2544.37, found 2544.4

S61


2658.2

1330.20.0

0.5

1.0

1.5

2.0

4x10

1000 1500 2000 2500 3000 m/z

1014.5

2034.1

1662.9

661.4

0

1

2

3

5x10

500 1000 1500 2000 2500 3000m/z

calcd.for [M+H]+ (monoisotopic)

2658.41, found 2658.2

calcd.for [M+H]+ (monoisotopic) 1662.89 found 1662.9

calcd.for [M+H]+ (monoisotopic) 1014.53, found 1014.5

Contamination by 16a

S62

D) In source fragmentation of ion at m/z 1662.9.

E) In source fragmentation of ion at m/z 1014.5.

F) LC-MS analysis of the trypsin digest

S63

Time (min)0.00 5.00 10.00 15.00 20.00 25.00 30.00

0.0

100.0

200.0

300.0

400.0

500.0

600.0

700.0


16a (contamination)

S64



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 m/z 1662.9. E) In source fragmentation of ion at m/z 1014.5. F, G, H) LC-MS analysis of the trypsin digest.

m/z400 600 800 1000 1200 1400 1600 1800 2000

0

100507.8

1014.7[M+H]+

[M+2H]2+In

tens

ity(A

U)

m/z400 500 600 700 800 900 1000 1100 1200 1300 1400

0

100832.5

555.3[M+3H]3+

Intensity (AU)

[M+2H]2+

[M+H]+m/z calcd.1662.89, obs 1662.82

[M+H]+m/z calcd.1014.53, obs 1014.7

S65

Peptide 16b A) Before alkylation

B) After alkylation

2560.19

0

2

4

6

4x10

1000 1500 2000 2500 3000m/z

2674.21

0

1

2

3

4x10

1000 1500 2000 2500 3000

m/z



S66



2034.09

1017.55 1400.68 2692.450

1

2

3

4x10

1000 1500 2000 2500 3000m/z


S67

E) LC-MS analysis of the trypsin digest, LC trace

F) LC-MS analysis of the trypsin digest, MS trace

Time (min)

0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00

0.0

100.0

200.0

300.0

400.0

500.0

600.0

700.0

800.0

900.0


m/z400 800 1200 1600 2000

0

100679.0

509.5

1018.1

[M+4H]4+

Intensity (AU)

[M+2H]2+

[M+3H]3+


S68


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 m/z 2034.0. E, F, G) LC-MS analysis of the trypsin digest.

m/z400 800 1200 1600 20000

100677.4[M+H]+


Intensity (AU)

S69

Peptide 16c A) Before alkylation

B) After alkylation

2586.49

1294.260

2

4

6

84x10

1500 2000 2500 3000 3500m/z

2700.51

1351.260

500

1000

1500

2000

2500

1000 2000 3000 4000m/z



S70



2034.10

703.42

0

2

4

6

4x10

1000 1500 2000 2500 3000m/z

m/z200 400 600

0

1

2

3

4

5

Abs. Int. * 1000b C I Ly K L I C L G

y 1

b 2 y 2 b 3

y 3b 4

y 4

b 5 y 5

y 6



S71



,,,

Time (min)2.00 6.00 10.00 14.00 18.00 22.00 26.00 30.00

0.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

1600.0


S72



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 m/z 703.42. E) In source fragmentation of ion at m/z 2034.10. F, G, H) LC-MS analysis of the trypsin digest.

m/z600 1000 1400 1800

0

100703.6

Intensity (AU)

[M+H]+

m/z400 800 1200 1600 2000

0

100679.2

509.7

1018.4

1357.7

Intensity (AU)

[M+2H]2+

[M+3H]3+



S73

Peptide 16d A) Before alkylation

B) After alkylation

2558.6

0

2000

4000

6000

8000

1500 2000 2500 3000 3500 4000m/z

2672.47

0.0

0.2

0.4

0.6

0.8

1.0

4x10

1000 1500 2000 2500 3000

m/z



S74



2048.11

661.410

2000

4000

6000

750 1250 1750 2250 2750

m/z

m/z100 200 300 400 500 600

0

2

4

6

8

10

Abs. Int. * 1000b C I Ly K L I C A G

y 1

b 2y 2

b 3

y 3

b 4

y 4a 5

b 5

y 5

a 6

y 6



S75



m/z500 1000 1500

0

1

2

3

Abs. Int. * 1000b C* G G T L A L L Ty H V T L L A L P S P G G C*

a 1

b 1y 1

a 2

b 2

y 2

a 3

b 3y 3 b 4

y 4 a 5

b 5

y 5

y 6

a 7

b 7

y 7

y 8

a 9

b 9

y 9 b 10

y 10

a 11

b 11

a 12

b 12y 12

b 13

y 13a 14

y 14

y 15

Time (min)0.00 5.00 10.00 15.00 20.00 25.00 30.00

0.0

200.0

400.0

600.0

800.0

1000.0


C I L KG A

NH2

O

H OH

S76



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 m/z 661.41. E) In source fragmentation of ion at m/z 2048.11. F, G, H) LC-MS analysis of the trypsin digest.

m/z450 550 650 750 850 950 1050 1150

0

100661.4

533.3

Intensity (AU)

[M+H]+

m/z400 500 600 700 800 900 1000 1100

0

100683.7

513.0

1025.1

Intensity (AU)

[M+2H]2+

[M+3H]3+

[M+4H]4+



S77

Peptide 16e A) Before alkylation

B) After alkylation

2574.6

0.0

0.2

0.4

0.6

0.8

1.0

4x10

1000 1500 2000 2500 3000

m/z

2688.9

0

1000

2000

3000

4000

5000

6000

1000 1500 2000 2500 3000m/z



S78




2048.2

677.50

2

4

6

8

4x10

1000 1500 2000 2500 3000

m/z

m/z100 200 300 400 500 600 700

0

1

2

3

4

5

Abs . Int. * 1000b I L Ky K L I C

y 1

y 2

b 3

y 3

b 4

y 4b 5

b 6

y 6

m/z250 500 750 1000 1250 1500 1750

0

10

20

30

40

50

Abs. Int. * 1000b C* G G T L A Ly H V T L L A L P S P L T G G C*

b 1

y 1

b 2y 2

b 3

y 3

b 4

y 4

b 5y 5

y 6y 7

y 8

b 9y 9 b 10

y 10

b 11

y 11

y 12y 13y 14

y 15



S79



Time (min)6.00 10.00 14.00 18.00 22.00 26.00 30.00

0.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

1600.0


m/z450 550 650 750 850 950 1050

0

100 677.4

533.4

Intensity (AU)

[M+H]+


S80


Figure S70. 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 m/z 677.5. E) In source fragmentation of ion at m/z 2048.09. F, G, H) LC-MS analysis of the trypsin digest.

m/z500 700 900 1100 1300 1500

0

1001025.1

683.7

Intensity (AU)

[M+2H]2+

[M+3H]3+


S81

References (1) Boll, E.; Dheur, J.; Drobecq, H.; Melnyk, O. Org. Lett. 2012, 14, 2222-2225. (2) Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.; Melnyk, O. Org. Lett. 2010, 12, 5238-5241. (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. 2014, 20, 92–97.

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