Supporting Information for

Transcription

1 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, UMR CNRS 824, Centre d Immunité et d Infection de Lille, F-59 Lille, France. Corresponding author: Dr Oleg Melnyk, oleg.melnyk@ibl.cnrs.fr Dr Vangelis Agouridas, vangelis.agouridas@ibl.cnrs.fr S1

2 Table of Contents 1. General Methods... S3 Reagents and solvents... S3 Analyses... S3 HPLC purification... S3 2. Peptide synthesis... S4 General protocol for automated peptide synthesis... S4 2.1 Synthesis of MPA peptide thioesters... S4 2.2 Synthesis of Cys peptides... S1 3. Reaction of peptide thioester 1a with Cys peptide 2a... S Effect of peptide concentration... S Effect of catalyst... S Effect of MPAA concentration... S Effect of TCEP concentration... S Effect of phosphate buffer concentration... S Effect of added EDT... S Effect of the ph... S Effect of the temperature... S Role of the denaturant at 4 C... S Role of phosphate buffer concentration on peptide thioester epimerization... S Role of phosphate buffer concentration on peptide thioester epimerization in the presence of MPAA... S Analysis of ligation products 3a/4a... S23 4. Total synthesis of human defensin DEFB S One-pot synthesis of DEFB133 polypeptides: protocols... S Analysis of DEFB133a-b after alkylation and trypsin digestion... S35 Synthetic references of the DTYXC(CH 2 CONH 2 )FIMRG (X = S or s) tryptic fragment... S35 DEFB133a, Method A... S37 DEFB133a, Method B... S4 DEFB133a, Method C... S43 DEFB133b, Method C... S45 5. References... S47 S2

3 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-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile- OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Cys(StBu)-OH or Fmoc- Cys(Trt)-OH. Synthesis of bis(2-sulfanylethyl)aminotrityl polystyrene (SEA PS) resin was carried out as described elsewhere. 1 4-mercaptophenylacetic acid (MPAA), 3-mercaptopropionic acid (MPA), tris(2- carboxyethyl)phosphine hydrochloride (TCEP), guanidine hydrochloride (Gn HCl) 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 MS-grade 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 UPLC MS using System Ultimate 3 UPLC (Thermofisher) equipped with a diode array detector, a charged aerosol detector (CAD) and a mass spectrometer (Ion trap LCQfleet). Column: Acquity peptide BEH3C18, 1.7 µm, mm (Waters). MALDI-TOF mass spectra were recorded with a BrukerAutoflex Speed using alpha-cyano-4- hydroxycinnaminic acid, sinapinic acid or 2,5-dihydroxybenzoic acid (DHB) as matrix. The observed corresponded to the monoisotopic ions, unless otherwise stated. The determination of optical purity of the C-terminal amino acid was done by chiral GC-MS following total acid hydrolysis in deuterated aqueous acid (C.A.T. GmbH & Co. Chromatographie und Analysentechnik KG, Heerweg 1, D-727 Tübingen, Germany). 2 HPLC purification Preparative reverse phase HPLC of crude peptides were performed with a preparative HPLC Waters system using a reverse phase column XBridge BEH3 Prep C18 (5 µm, 3 Å, 1 25 mm) and appropriate linear gradient of increasing concentration of eluent B in eluent A (flow rate of 6 ml/min). Selected fractions were then combined and lyophilized. S3

4 2. Peptide synthesis General protocol for automated peptide synthesis Peptide elongation was performed using standard Fmoc/tert-butyl chemistry on an automated peptide synthesizer (.2 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 resin was washed with CH 2 Cl 2, diethylether (3 2 min) and dried in vacuo. 2.1 Synthesis of MPA peptide thioesters Synthesis of SEA off peptide precursors Peptide elongation was performed on SEA PS resin (2 x.1 mmol,.16 mmol/g) using standard Fmoc/tert-butyl chemistry on an automated peptide synthesizer. Typical procedures for the synthesis of SEA off peptide segments were described in previous papers. 1-3 The analytical HPLC and MS analyses of the purified SEA off peptide segments are shown below. Table S 1. Yields for the HPLC purified SEA off peptides Peptide Scale of synthesis (mmol) Mass of crude (mg) Mass of purified peptide (mg) Yield (%) MALDI -TOF [M+H] + calcd. MALDI -TOF [M+H] + found APDTRPAPGSTAPP AHGVTS-SEA off APDTRPAPGSTAPP AHGVTs-SEA off RVKC(StBu)AVKDT YS-SEA off RVKC(StBu)AVKDT Ys-SEA off S4

5 MPA peptide thioesters MPA peptide thioesters were produced from SEA off peptides by exchange in the presence of TCEP and MPA and according to published procedures. 2, 4 Table S 2. Yields for MPA peptide thioesters Peptide Mass of starting SEA off peptide (mg) Mass of purified peptide MPA (mg) Yield (%) epimer MALDI (%) a -TOF [M+H] + calcd. MALDI -TOF [M+H] + found APDTRPAPGSTAPP 1a D-epimer AHGVTS-MPA 1.82 APDTRPAPGSTAPP 1b AHGVTs-MPA RVKCAVKDTYS- 1c D-epimer MPA.37 RVKCAVKDTYs- MPA 1d a Determined by chiral GC-MS analysis after peptide hydrolysis. Analysis performed by CAT GmbH, Germany. S5

6 Intensity (AU, UV 215 nm) APDTRPAPGSTAPPAHGVTS-MPA Intensity (AU, TIC) Figure S 1. LC-MS of peptide 1a. LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. MS trace. = 988. ([M+2H] 2+ ), ([M+3H] 3+ ). Calcd. for M: (average), found: Intensity (AU) APDTRPAPGSTAPPAHGVTS-MPA Intensity (AU) Figure S 2. MALDI-TOF analysis of peptide 1a. Matrix = alpha-cyano-4-hydroxycinnaminic acid, positive detection mode, calcd for [M+H] + (monoisotopic): , found: S6

7 7.15 Intensity (AU, UV 215 nm) Intensity (AU, TIC) APDTRPAPGSTAPPAHGVTs-MPA Figure S 3. LC-MS of peptide 1b. LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. MS trace. = ([M+2H] 2+ ), ([M+3H] 3+ ). Calcd. for M: (average), found: x1 4 6 Intensity (AU) x1 4 Intensity (AU) 6 APDTRPAPGSTAPPAHGVTs-MPA Figure S 4. MALDI-TOF analysis of peptide 1b. Matrix = alpha-cyano-4-hydroxycinnaminic acid, positive detection mode, calcd for [M+H] + (monoisotopic): , found: S7

8 Intensity ( AU, UV 215 nm) 7.69 RVKCAVKDTYS-MPA Intensity (AU, TIC) Figure S 5. LC-MS of peptide 1c. LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. MS trace. = ([M+H] + ), ([M+2H] 2+ ). Calcd. for M: (average), found: x Intensity (AU) x1 4 RVKCAVKDTYS-MPA 2.5 Intensity (AU) Figure S 6. MALDI-TOF analysis of peptide 1c. Matrix = alpha-cyano-4-hydroxycinnaminic acid, positive detection mode, calcd for [M+H] + (monoisotopic): , found: S8

9 Intensity (AU, UV 215 nm) 6.61 Intensity (AU, TIC) RVKCAVKDTYs-MPA Figure S 7. LC-MS of peptide 1d. LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. MS trace. = ([M+H] + ), ([M+2H] 2+ ), ([M+3H] 3+ ). Calcd. for M: (average), found: Intensity (AU) Intensity (AU) RVKCAVKDTYs-MPA Figure S 8. MALDI-TOF analysis of peptide 1d. Matrix = alpha-cyano-4-hydroxycinnaminic acid, positive detection mode, calcd for [M+H] + (monoisotopic): , found: S9

10 2.2 Synthesis of Cys peptides Peptide elongation were performed on Rink-PEG-PS resin (NovaSyn TGR,.25 mmol,.25 mmol/g) or on PAL-Chemmatrix resin (.5 mmol,.18 mmol/g) by using standard Fmoc/tert-butyl chemistry on an automated peptide synthesizer. Peptide 2a has been described elsewhere (see ref 1 ) Table S 3. Yields for Cys peptides Peptide Scale of Mass of Mass of Yield MALDI- MALDIsynthesis (mmol) crude (mg) purified peptide (mg) (%) TOF [M+H] + calcd. TOF [M+H] + found CILKEPVHGV-NH CTKLNANCYM-NH S1

11 HPLC yield for D-epimer (%) HPLC yield for L-epimer (%) 3. Reaction of peptide thioester 1a with Cys peptide 2a 3.1 Effect of peptide concentration A) mm 1 mm.1 mm B) mm 1 mm.1 mm Figure S 9. Effect of peptide concentration. Peptide 1a.1-8 mm, peptide 2a (batch 1) 1.2 equiv, 6 M Gn.HCl, ph 7.2, MPAA 2 mm, TCEP 1 mm, 37 C, under nitrogen. A) Ligation product 3a, HPLC yield; B) Epimerized byproduct 4a, HPLC yield. S11

12 HPLC yield for D-epimer (%) HPLC yield for L-epimer (%) 3.2 Effect of catalyst A) mm MTG 2 mm MPAA 2 mm Thiophenol B) mm MTG 2 mm MPAA 2 mm Thiophenol Figure S 1. Effect of catalyst. Peptide 1a.1 mm, peptide 2a (batch 1) 1.2 equiv, 6 M Gn.HCl, ph 7.2, catalyst 2 mm, TCEP 1 mm, 37 C, under nitrogen. A) Ligation product 3a, HPLC yield; B) Epimerized byproduct 4a, HPLC yield. S12

13 HPLC yield for D-epimer (%) HPLC yield for L-epimer (%) 3.3 Effect of MPAA concentration A) mm MPAA 15 mm MPAA 1 mm MPAA 5 mm MPAA B) mm MPAA 15 mm MPAA 1 mm MPAA 5 mm MPAA Figure S 11. Effect of MPAA concentration. Peptide 1a 1 mm, peptide 2a (batch 1) 1.2 mm, 6 M Gn.HCl, ph 7.2, 37 C, under nitrogen. A) Ligation product 3a, HPLC yield; B) Epimerized byproduct 4a, HPLC yield. S13

14 HPLC yield for D-epimer (%) HPLC yield for L-epimer (%) 3.4 Effect of TCEP concentration A) mm TCEP w/o B) mm TCEP w/o Figure S 12. Effect of TCEP concentration. Peptide 1a 1 mm, peptide 2a (batch 1) 1.2 mm, 6 M Gn.HCl, ph 7.2, MPAA 2 mm, 37 C, under nitrogen. A) Ligation product 3a, HPLC yield; B) Epimerized byproduct 4a, HPLC yield. S14

15 HPLC yield for D-epimer (%) HPLC yield for L-epimer (%) 3.5 Effect of phosphate buffer concentration A) mm phosphate buffer 1 mm phosphate buffer B) mm phosphate buffer 1 mm phosphate buffer Figure S 13. Effect of phosphate buffer concentration. Peptide 1a 1 mm, peptide 2a (batch 2) 1.2 mm, 6 M Gn.HCl, ph 7.2, MPAA 2 mm, TCEP 1 mm, 37 C, under nitrogen. A) Ligation product 3a, HPLC yield; B) Epimerized byproduct 4a, HPLC yield. S15

16 HPLC yield for D-epimer (%) HPLC yield for L-epimer (%) 3.6 Effect of added EDT A) mm EDT w/o B) mm EDT w/o Figure S 14. Effect of added EDT. Peptide 1a 1 mm, peptide 2a (batch 2) 1.2 mm, 6 M Gn.HCl, ph 7.2, MPAA 2 mm, TCEP 1 mm, 37 C, under nitrogen. A) Ligation product 3a, HPLC yield; B) Epimerized byproduct 4a, HPLC yield. S16

17 HPLC yield for D-epimer (%) HPLC yield for L-epimer (%) 3.7 Effect of the ph A) ph 6.4 ph 7.2 ph B) ph 6.4 ph 7.2 ph Figure S 15. Effect of the ph. Peptide 1a 1 mm, peptide 2a (batch 2) 1.2 equiv, 6 M Gn.HCl, ph , MPAA 2 mm, TCEP 1 mm, 37 C, under nitrogen. A) Ligation product 3a, HPLC yield; B) Epimerized byproduct 4a, HPLC yield. S17

18 HPLC yield for D-epimer (%) HPLC yield for L-epimer (%) 3.8 Effect of the temperature A) C 2 C 4 C B) C 2 C 4 C Figure S 16. Effect of the temperature. Peptide 1a 1 mm, peptide 2a (batch 2) 1.2 equiv, 6 M Gn.HCl, ph 7.2, MPAA 2 mm, TCEP 1 mm, 4-37 C, under nitrogen. A) Ligation product 3a, HPLC yield; B) Epimerized byproduct 4a, HPLC yield. S18

19 HPLC yield for D-epimer (%) HPLC yield for L-epimer (%) 3.9 Role of the denaturant at 4 C A) M Gn.HCl 1 mm OG w/o B) 3,5 3 2,5 2 1,5 1 6 M Gn.HCl 1 mm OG w/o, Figure S 17. Effect of the denaturant at 4 C. Peptide 1a 1 mm, peptide 2a (batch 2) 1.2 equiv, 6 M Gn.HCl/1 mm N-octylglycoside (OG), ph 7.2, MPAA 2 mm, TCEP 1 mm, 4 C, under nitrogen. A) Ligation product 3a, HPLC yield; B) Epimerized byproduct 4a, HPLC yield. S19

20 HPLC yield for MPA thioester, D-epimer (%) HPLC yield for MPA thioester, L-epimer (%) 3.1 Role of phosphate buffer concentration on peptide thioester epimerization A) mm phosphate buffer 5 mm phosphate buffer 1 mm phosphate buffer B) mm phosphate buffer 5 mm phosphate buffer 1 mm phosphate buffer S2

21 HPLC yield for hydrolyzed peptide thioester (%) C) mm phosphate buffer 5 mm phosphate buffer 1 mm phosphate buffer Figure S 18. Role of phosphate buffer concentration on peptide thioester 1a epimerization. Peptide 1a 1 mm, 6 M Gn.HCl, ph 7.2, 37 C. A) L-epimer; B) D-epimer; C) Hydrolysis. HPLC yields. S21

22 HPLC yield for MPA thioesters (%) HPLC yield for MPAA thioesters (%) 3.11 Role of phosphate buffer concentration on peptide thioester epimerization in the presence of MPAA A) mm phosphate buffer, L-epimer 1 mm phosphate buffer, L-epimer 5 mm phosphate buffer, L-epimer 2 mm phosphate buffer, D-epimer 1 mm phosphate buffer, D-epimer 5 mm phosphate buffer, D-epimer B) mm phosphate buffer, L-epimer 1 mm phosphate buffer, L-epimer 5 mm phosphate buffer, L-epimer 2 mm phosphate buffer, D-epimer 1 mm phosphate buffer, D-epimer 5 mm phosphate buffer, D-epimer Figure S 19. Role of phosphate buffer concentration on peptide thioester epimerization in the presence of MPAA. Peptide 1a 1 mm, 6 M Gn.HCl, ph 7.2, MPAA 2 mm, 37 C. A) MPAA peptide thioester; B) MPA peptide thioester. HPLC yields. S22

23 3.12 Analysis of ligation products 3a/4a Table S 4. Yield for peptide 3a produced from peptide thioester 1a. Peptide APDTRPAPGSTAP PAHGVTSCILKEP VHGV-NH 2 MPA Method Mass of Yield D- thioester peptide (µmol) of synthesis product (purified, in mg) (%) epimer (%) 2.22 B a 3.8 b 2.9 c MALDI -TOF [M+H] + calcd. MALDI -TOF [M+H] + found a by UV integration of 3a/4a; b By integration of ionic current for ion [M+H] 3+ c Based on the mass of 4a isolated by semi-preparative HPLC: 4a, 121 µg (1.54%). A) B) S23

24 C) Figure S 2. LC-MS analysis of ligation products 3a (A) produced from 1a and 4a (B) produced from 1b. The co-elution of the two products is in (C). Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. S24

25 A) Intensity (AU, TIC) APDTRPAPGSTAPPAHGVTSCILKEPVHGV-NH B) Intensity (AU, TIC) APDTRPAPGSTAPPAHGVTsCILKEPVHGV-NH Figure S 21. ESI-MS spectra of ligation products 3a (A) produced from 1a and 4a (B) produced from 1b. S25

26 A) Intensity(AU) APDTRPAPGSTAPPAHGVTSCILKEPVHGV-NH Intensity(AU) B) 6 Intensity(AU) APDTRPAPGSTAPPAHGVTsCILKEPVHGV-NH Intensity(AU) Figure S 22. MALDI-TOF spectra of: A) ligation products 3a produced from 1a. Matrix = alpha-cyano-4-hydroxycinnaminic acid, positive detection mode, calcd for [M+H] + (monoisotopic): , found: ; B) 4a produced from 1b. Matrix = alpha-cyano- 4-hydroxycinnaminic acid, positive detection mode, calcd for [M+H] + (monoisotopic): , found: S26

27 4. Total synthesis of human defensin DEFB133 Table S 5. Yield for the synthesis of C-terminal fragment of DEFB133. Peptide Scale of Mass of Mass of Yield MALDI- MALDIsynthesis (mmol) crude (mg) purified peptide (mg) (%) TOF [M+H] + calcd. TOF [M+H] + found CTKLNANCYM-NH Table S 6. Yield for the synthesis of segment 5. Peptide C(StBu)FIMRGKC( StBu)RHEC(StBu)H DFEKPIGF-SEA off Scale of synthesi s (mmol) Mass of crude (mg) Mass of purified peptide (mg) Yield (%) MALDI -TOF [M+H] + calcd. MALDI -TOF [M+H] + found S27

28 Intensity (AU, UV 215 nm) Intensity (AU, TIC) C(StBu)FIMRGKC(StBu)RHEC (StBu)HDFEKPIGF-SEA off Figure S 23. LC-MS of peptide 5. LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. MS trace. = ([M+2H] 2+ ), ([M+3H] 3+ ), 735. ([M+4H] 4+ ), ([M+5H] 5+ ). Calcd. for M: (average), found: x1 4 Intensity (AU) C(StBu)FIMRGKC(StBu)RHEC(StBu)H DFEKPIGF-SEA off Figure S 24. MALDI-TOF analysis of peptide 5. Matrix = sinapinic acid, positive detection mode, calcd for [M+H] + (average): , found: S28

29 4.1 One-pot synthesis of DEFB133 polypeptides: protocols method A: One-pot synthesis of DEFB133 at 37 C with pre-incubation of the peptidyl seryl thioester Step 1: To a solution of 6 M Gdn.HCl in.1 M phosphate buffer (2 ml) was added MPAA (67.25 mg, 2 mm) and the ph was adjusted to 7.2 by addition of 6M sodium hydroxide (12 μl). Peptide 1c (3.681 mg) was dissolved in this solution (751 μl, 2.7 mm) and was pre-incubated at 37 C over 15 minutes. Peptide 5 (7.584 mg, 2.7 mm) was then added to the mixture and the reaction was left to proceed at 37 C until consumption of the starting material (i. e. ~2h). Step 2: After 2h, Cys peptide CTKLNANCYM-NH 2 (2.816 mg) was dissolved in a solution of 6M Gdn.HCl in.1 M phosphate buffer containing MPAA (2 mm) and TCEP (1 mm) (259 μl, 1,15 mm). The latter solution was added to the reaction mixture of step 1 and the ph was adjusted to 5.49 by addition of 6M sodium hydroxide (22 μl) (final peptide concentration 2.3 mm). The reaction was monitored by UPLC-MS. After completion, glacial acetic acid (1 μl) was added to the crude mixture, which was then poored into a 1 % acetic acid aqueous solution (4 ml). The aqueous layer was washed with diethyl ether (3 x 1 ml) and was purified by preparative HPLC (Column: XBridge BEH3 Prep C18 (5 µm, 3 Å, 1 25 mm), eluent A: trifluoroacetic acid.1% in water, eluent B 8% acetonitrile trifluoroacetic acid.1%, Gradient -15% B in 5 minutes then 15-35% B in 55 min, 5 C, 6 ml/min, detection at 215 nm). DEFB133 polypeptide was obtained as a white powder after lyophilization (4.65 mg). method B: One-pot synthesis of DEFB133 at 37 C without pre-incubation Step 1: To a solution of 6 M Gdn.HCl in.1 M phosphate buffer (1 ml) was added MPAA (33.64 mg, 2 mm) and the ph was adjusted to 7.2 by addition of 6M sodium hydroxide (64 μl). Cys Peptide 5 (7.669 mg) was first dissolved in this solution (761 μl, 2.7 mm) and the reaction mixture was thermostated at 37 C. Peptidyl seryl thioester 1c (3.69 mg, 2.7 mm) was then added. The reaction was left to proceed at 37 C until consumption of the starting material. Step 2: Cys peptide CTKLNANCYM-NH 2 (2.816 mg) was dissolved in a solution of 6M Gdn.HCl in.1 M phosphate buffer containing MPAA (2 mm) and TCEP (1 mm) (257 μl, 1,2 mm). The latter solution was added to the reaction mixture of step 1 and the ph was adjusted to 5.5 by addition of 6M sodium hydroxide (59 μl) (final peptide concentration 2.3 mm). The reaction was monitored by UPLC-MS. After completion, glacial acetic acid (1 μl) was added to the crude mixture, which was then poored into a 1 % acetic acid aqueous solution (4 ml). The aqueous layer was washed with diethyl ether (3 x 1 ml) and was purified by preparative HPLC (Column: XBridge BEH3 Prep C18 (5 µm, 3 Å, 1 25 mm), eluent A: trifluoroacetic acid.1% in water, eluent B 8% acetonitrile trifluoroacetic acid.1%, Gradient -15% B in 5 minutes then 15-35% B in 55 min, 5 C, 6 ml/min, detection at 215 nm). DEFB133 polypeptide was obtained as a white powder after lyophilization (5.12 mg). method C: One-pot synthesis of DEFB133 at 4 C without pre-incubation Step 1: To a solution of 6 M Gdn.HCl in.1 M phosphate buffer (2 ml) was added MPAA (67.25 mg, 2 mm) and the ph was adjusted to 7.21 by addition of 6M sodium hydroxide (123 μl). Cys Peptide 5 (7.627 mg) was first dissolved in this solution (756 μl, 2.7 mm) and the reaction mixture was S29

30 cooled to 4 C in a jacketed reactor. Peptidyl seryl thioester 1c (3.683 mg, 2.7 mm) was then added. The reaction was left to proceed at 4 C until consumption of the starting material (i. e. ~2h). Step 2: After ~2h, Cys peptide CTKLNANCYM-NH 2 (2.814 mg) was dissolved in a solution of 6M Gdn.HCl in.1 M phosphate buffer containing MPAA (2 mm) and TCEP (1 mm) (254 μl, 1,2 mm). The latter solution was added to the reaction mixture of step 1 and the ph was adjusted to 5.52 by addition of 6M sodium hydroxide (3 μl) (final peptide concentration 2.3 mm). The reaction was monitored by UPLC-MS. After completion, glacial acetic acid (1 μl) was added to the crude mixture, which was then poored into a 1 % acetic acid aqueous solution (4 ml). The aqueous layer was washed with diethyl ether (3 x 1 ml) and was purified by preparative HPLC (Column: XBridge BEH3 Prep C18 (5 µm, 3 Å, 1 25 mm), eluent A: trifluoroacetic acid.1% in water, eluent B 8% acetonitrile trifluoroacetic acid.1%, Gradient -15% B in 5 minutes then 15-35% B in 55 min, 5 C, 6 ml/min, detection at 215 nm). DEFB133 polypeptide was obtained as a white powder after lyophilization (4.1 mg). Table S 7. Yields for DEFB133a-b polypeptides. Peptide RVKCAVKDTYS CFIMRGKCRHE CHDFEKPIGFCT KLNANCYM-NH 2 RVKCAVKDTYS CFIMRGKCRHE CHDFEKPIGFCT KLNANCYM-NH 2 RVKCAVKDTYS CFIMRGKCRHE CHDFEKPIGFCT KLNANCYM-NH 2 RVKCAVKDTYs CFIMRGKCRHE CHDFEKPIGFCT KLNANCYM-NH 2 MPA thioester peptide (µmol) Method Mass of product (purified, in mg) Yield (%) Epimer (%) 2.3 A D- epimer B D- epimer C D- epimer C L- epimer 1.9 MALDI- TOF [M+H] + average calcd. MALDI- TOF [M+H] + found S3

31 Intensity (AU, UV 215nm) 1.52 Intensity (AU, TIC) RVKCAVKDTYSCFIMRGKCRHECHDF EKPIGFCTKLNANCYM-NH 2 Method A Figure S 25. LC-MS analysis of DEFB133a produced using Method A. LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. MS trace. = ([M+3H] 3+ ), ([M+4H] 4+ ), ([M+5H] 5+ ), ([M+6H] 6+ ). Calcd. for M: (average), found: Intensity (AU) RVKCAVKDTYSCFIMRGKCRHEC HDFEKPIGFCTKLNANCYM-NH 2 Method A Figure S 26. MALDI-TOF analysis of DEFB133a produced using Method A. Matrix = sinapinic acid, positive detection mode, calcd for [M+H] + (average): , found: S31

32 Intensity (AU, UV 215 nm) 1.5 Intensity (Au, TIC) RVKCAVKDTYSCFIMRGKCRHEC HDFEKPIGFCTKLNANCYM-NH 2 Method B Figure S 27. LC-MS analysis of DEFB133a produced using Method B. LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. MS trace. = ([M+3H] 3+ ), ([M+4H] 4+ ), 99.5 ([M+5H] 5+ ), ([M+6H] 6+ ). Calcd. for M: (average), found: Intensity (AU) RVKCAVKDTYSCFIMRGKCRHEC HDFEKPIGFCTKLNANCYM-NH 2 Method B Figure 28. MALDI-TOF analysis of DEFB133a produced using Method B. Matrix = sinapinic acid, positive detection mode, calcd for [M+H] + (average): , found: S32

33 Intensity (AU, UV 215 nm) 1.5 Intensity (Au, TIC) RVKCAVKDTYSCFIMRGKCRHEC HDFEKPIGFCTKLNANCYM-NH 2 Method C Figure S 29. LC-MS analysis of DEFB133a produced using Method C. LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. MS trace. = ([M+3H] 3+ ), ([M+4H] 4+ ), 99.5 ([M+5H] 5+ ), ([M+6H] 6+ ). Calcd. for M: (average), found: Intensity (AU) RVKCAVKDTYSCFIMRGKCRHEC HDFEKPIGFCTKLNANCYM-NH 2 Method C Figure S 3. MALDI-TOF analysis of DEFB133a produced using Method C. Matrix = sinapinic acid, positive detection mode, calcd for [M+H] + (average): , found: S33

34 Intensity (AU, UV 215 nm ) 1.44 Intensity (AU, TIC) RVKCAVKDTYsCFIMRGKCRHEC HDFEKPIGFCTKLNANCYM-NH 2 Method C Figure S 31. LC-MS analysis of DEFB133b produced using Method C. LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. MS trace. = ([M+3H] 3+ ), ([M+4H] 4+ ), ([M+5H] 5+ ), ([M+6H] 6+ ). Calcd. for M: (average), found: Intensity (AU) RVKCAVKDTYsCFIMRGKCRHEC HDFEKPIGFCTKLNANCYM-NH 2 Method C Figure S 32. MALDI-TOF analysis of DEFB133b produced using Method C. Matrix = sinapinic acid, positive detection mode, calcd for [M+H] + (average): , found: S34

35 4.2 Analysis of DEFB133a-b after alkylation and trypsin digestion General procedure: The peptide to be analyzed (~1 µg) is diluted with dithiothreitol (1 mg/ml in.25 M ammonium bicarbonate, 1 µl) and treated with iodoacetamide (1 mg/ml in.25 M ammonium bicarbonate, 1 µl) for 1 min. The alkylation step is monitored by MALDI-TOF mass spectrometry. Then, trypsin (.1 mg/ml, 1 µl) is added to the mixture to cleave the alkylated peptide. The crude tryptic digests are analyzed with a System Ultimate 3 UPLC (Thermofisher) equipped with DAD (diode array detector), CAD (charged aerosol detector) and Ion trap LCQfleet mass spectrometer (Thermofisher). Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min. Synthetic references of the DTYXC(CH 2 CONH 2 )FIMRG (X = S or s) tryptic fragment A) Intensity (AU, UV 215 nm) 9.24 DTYSC(CH 2 CONH 2 )FIMR-OH Intensity (AU, TIC) 597. DTYSC(CH 2 CONH 2 )FIMR-OH B) Intensity (AU, UV 215 nm) 9.1 DTYsC(CH 2 CONH 2 )FIMR-OH S35

36 Intensity (AU, TIC) DTYsC(CH 2 CONH 2 )FIMR-OH C) Intensity (AU, UV 215 nm) DTYSC(CH 2 CONH 2 )FIMR-OH DTYsC(CH 2 CONH 2 )FIMR-OH Figure S 33. A) LC-MS analysis of a synthetic reference of tryptic fragment DTYSC(CH 2 CONH 2 )FIMRG-OH. LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. MS trace. = ([M+H] + ), 597. ([M+2H] 2+ ). Calcd. for M: (average), found: B) LC-MS analysis of a synthetic reference of tryptic fragment DTYsC(CH 2 CONH 2 )FIMRG-OH. LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. MS trace. = ([M+H] + ), ([M+2H] 2+ ). Calcd. for M: (average), found: C) Coelution control of synthetic references DTYXC(CH 2 CONH 2 )FIMRG-OH (X = S and X = s). S36

37 DEFB133a, Method A A) Intensity (AU, UV 215 nm) 8.38 RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGFCTKLNANCYM-NH 2 A method, trypsic fragments B) Intensity (AU, TIC) RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGFCTKLNANCYM-NH 2 A method, trypsic fragments S37

38 C) Intensity (AU, UV 215 nm) 8.36 RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGFCTKLNANCYM-NH 2 A method, trypsic peptides and synthetic DTYsCFIMR-OH D) Intensity (AU, UV 215 nm) 8.38 RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGFCTKLNANCYM-NH 2 A method, trypsic peptides RT: 9.22 MA: RT: 9.4 MA: S38

39 E) Intensity (AU, mass range 597 Da) RT: 9.45 MA: 1721 RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGFCTKLNANCYM-NH 2 A method, trypsic peptides RT: 9.23 MA: Figure S 34. LC-MS analysis of tryptic digest of peptide DEFB133a obtained by method A. A) LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. B) MS trace of the peak at 9.22 min. = ([M+H] + ), ([M+2H] 2+ ). Calcd. for M: (average), found: C) Coelution control with a synthetic reference peptide containing a D-Ser residue. D) UV quantification of the D-epimer (8.8 %). E) Ionic current quantification of the D-epimer (7.3 %). S39

40 DEFB133a, Method B A) Intensity (AU, UV 215 nm) RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGFCTKLNA NCYM-NH 2 B method, trypsic peptides B) Intensity (AU, TIC) 597. RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGFCTKLNANCYM-NH 2 B method, trypsic peptides S4

41 C) Intensity (AU, UV 215 nm) RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGF CTKLNANCYM-NH 2 B method, trypsic peptides co-eluted with DTYsCFIMR-OH D) Intensity ( AU, UV 215 nm) 8.38 RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGFCTKLNANCYM-NH 2 B method, trypsic peptides RT: 9.24 MA: RT: 9.5 MA: S41

42 E) Intensity (AU, mass range 597 Da) RT: 9.41 MA: RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGFCTKLNANCYM-NH 2 B method, trypsic peptides RT: 9.25 MA: Figure S 35. LC-MS analysis of tryptic digest of peptide DEFB133a obtained by method B. A) LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. B) MS trace of the peak at 9.24 min. = ([M+H] + ), 597. ([M+2H] 2+ ). Calcd. for M: (average), found: C) Coelution control with a synthetic reference peptide containing a D-Ser residue. D) UV quantification of the D-epimer (3.42 %). E) Ionic current quantification of the D-epimer (2.9 %). S42

43 DEFB133a, Method C A) Intensity (AU, UV 215 nm) RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGF CTKLNANCYM-NH 2 C method, trypsic peptides B) Intensity (AU, TIC) RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGFCTKLNA NCYM-NH 2 C method, trypsic peptides S43

44 C) Intensity (AU, UV 215 nm) RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGFCT KLNANCYM-NH 2 C method, trypsic peptides and DTYsCFIMR-OH D) Intensity ( AU, mass range 597 Da) RT: 9.31 MA: RVKCAVKDTYSCFIMRGKCRHECHDFEKPIGFCTKLNANCYM- NH 2 C method, trypsic peptides RT: 9.15 MA: Figure S 36. LC-MS analysis of tryptic digest of peptide DEFB133a obtained by method C. A) LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. B) MS trace of the peak at 9.24 min. = ([M+H] + ), ([M+2H] 2+ ). Calcd. for M: (average), found: C) Coelution control with a synthetic reference peptide containing a D-Ser residue. D) Ionic current quantification of the D-epimer (.73 %). S44

45 DEFB133b, Method C A) Intensity ( AU, UV 215 nm) 8.38 RVKCAVKDTYsCFIMRGKCRHECHDFEKPIGF CTKLNANCYM-NH 2 C method, trypsic peptides B) Intensity (AU, TIC) RVKCAVKDTYsCFIMRGKCRHECHDFEKPIGFCTKLNANCYM-NH 2 C method, trypsic peptides S45

46 C) Intensity (AU, UV 215 nm) 8.38 RVKCAVKDTYsCFIMRGKCRHECHDFEKPIGFCTK LNANCYM-NH 2 C method, trypsic peptides and DTYSCFIMR-OH D) Intensity (AU, UV 215 nm) 8.38 RVKCAVKDTYsCFIMRGKCRHECHDFEKPIGFCTK LNANCYM-NH 2 C method, trypsic peptides RT: 9.3 MA: RT: 9.25 MA: S46

47 E) Intensity (AU, mass range 597 Da) RT: 9.21 MA: 5752 RVKCAVKDTYsCFIMRGKCRHECHDFEKPIGFCTKLNANCYM-NH 2 C method, trypsic peptides RT: 9.49 MA: Figure S 37. LC-MS analysis of tryptic digest of peptide DEFB133b obtained by method C. A) LC trace. Chromatography conditions: Column: Acquity peptide BEH3C18, 1.7µ, 2.1*1mm (Waters), eluent from trifluoroacetic acid.1% in water to 7% acetonitrile trifluoroacetic acid.1% in 2 min., 5 C,.4 ml/min, detection at 215 nm. B) MS trace of the peak at 9.3 min. = ([M+H] + ), ([M+2H] 2+ ). Calcd. for M: (average), found: C) Coelution control with a synthetic reference peptide containing a L-Ser residue. D) UV quantification of the L-epimer (4.1 %). E) Ionic current quantification of the L-epimer (1.9 %). 5. References 1. Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.; Melnyk, O., Bis(2-sulfanylethyl)amino native peptide ligation. Org. Lett. 21, 12, Boll, E.; Drobecq, H.; Ollivier, N.; Blanpain, A.; Raibaut, L.; Desmet, R.; Vicogne, J.; Melnyk, O., One-pot chemical synthesis of small ubiquitin-like modifier (SUMO) protein-peptide conjugates using bis(2-sulfanylethyl)amido peptide latent thioester surrogates Nat. Protoc. 215, 1, Ollivier, N.; Raibaut, L.; Blanpain, A.; Desmet, R.; Dheur, J.; Mhidia, R.; Boll, E.; Drobecq, H.; Pira, S. L.; Melnyk, O., Tidbits for the synthesis of bis(2-sulfanylethyl)amido (SEA) polystyrene resin, SEA peptides and peptide thioesters. J. Pept. Sci. 214, 2, S47

48 4. Dheur, J.; Ollivier, N.; Vallin, A.; Melnyk, O., Synthesis of peptide alkylthioesters using the intramolecular N,S-acyl shift properties of bis(2-sulfanylethyl)amido peptides. J. Org. Chem. 211, 76, S48