Supporting Information

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1 Supporting Information Palladium Mediated Rapid Deprotection of N-Terminal Cysteine under Native Chemical Ligation Conditions for the Efficient Preparation of Synthetically Challenging Proteins Muhammad Jbara, Suman Kumar Maity, Mallikanti Seenaiah and Ashraf Brik* Schulich Faculty of Chemistry, Technion-Israel Institute of Technology Haifa, (Israel) These authors contributed equally Table of Contents General methods: List of the protected amino acids used in peptides synthesis: Synthesis of Proc-Cys(Trt)-OH: S2 S3 S4 Synthesis of model peptides bearing Proc-Cys, Thz, D- and L- Cys residues at the N-termini: Racemization study during removal of Proc with model peptide: Racemization study during Thz opening with model peptide: S5-S7 S8 S9 Synthesis of model peptides bearing Proc-Cys and Thz at the N-termini and thioester at the C-termini: Proc deprotection by palladium chloride [Pd(Cl) 2 ]: S9-S11 S12 Synthesis of model peptide containing tryptophan to check the compatibility of palladium reagents against all 20 amino acids: Syntheses of fragment 5: Synthesis of fragment 9: Synthesis of fragment 10: Synthesis of fragment 11: S12-S14 S14 S16 S17 S19 S1

2 One-pot ligation and Proc removal: Synthesis of fragment 12: Synthesis of neddylated cullin, 13: Evaluation of UCH-L3 activity on neddylated cullin, 14: Circular dichroism spectroscopy: Figure S16, S17 and S18: Synthesis of eleven-mer model peptides, Proc-Cys (LYRAGLYRAX, X=G,A,R,T,F)-NHNH 2 : Switching of Proc-Cys(LYRAGLYRAX)-NHNH 2 to MPAA thioester One-pot switching of Proc-Cys(LYRAGLYRAR)-NHNH 2 and ligation The effect of the DTT on Pd chelation to the peptide S21 S22 S23 S24 S24 S25-S26 S27 S27-S31 S32 S34 Experimental Section Materials and methods General methods SPPS was carried out manually in syringes, equipped with teflon filters, purchased from Torviq or by using an automated peptide synthesizer (CS336X, CSBIO). If not differently described, all reactions were carried out at room temperature. Analytical grade DMF was purchased from Biotech. Commercial reagents were used without further purification. Resins were purchased from Creosalus, protected amino acids were purchased from GL Biochem and activating reagents [(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), hydroxybenzotriazole (HOBt), [(6-chlorobenzotriazol-1- yl)oxy-(dimethylamino)methylidene]-dimethylazanium hexafluorophosphate (HCTU), 1- [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU)] were purchased from Luxembourg Bio Technologies. S2

3 Analytical HPLC was performed on a Thermo instrument (Dionex Ultimate 3000) using analytical columns Xbridge (waters, BEH300 C4, 3.5µm, mm) and XSelect (waters, CSH C18, 3.5 µm, mm) at flow rate of 1.2 ml/min. Preparative HPLC was performed on a Waters instrument using XSelect C18 10µm mm and semi preparative HPLC was performed on a Thermo Scientific instrument (Spectra System SCM1000) using Jupiter C4 10 µm, 300 Å, mm column, at flow rate of 15 and 4 ml/min respectively. All synthetic products were purified by HPLC and characterized by mass spectrometry using LCQ Fleet Ion Trap (Thermo Scientific). All calculated masses have been reported as an average isotope composition. 1 H and 13 C NMR spectra were recorded using CDCl 3 as a solvent. Chemical shifts were reported in δ units (ppm) with reference to TMS as an internal standard and J values are given in Hz. 1 H and 13 C-NMR spectra were recorded on a Bruker AMX-400 MHz. Flash column chromatography was carried out with silica gel ( mesh). Chemicals were purchased from Aldrich, Strem Chemicals and Alfa Aesar. The reactions were carried out in oven-dried glassware under nitrogen. Analytical thin-layer chromatography (TLC) was performed on pre-coated plates (0.25 mm, silica gel 60 F254). Compound spots were stained with ninhydrin solution. Buffer A: 0.1% TFA in water; buffer B: 0.1% TFA in acetonitrile. List of the protected amino acids used in peptides synthesis: Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe- OH, Fmoc-Pro-OH, Fmoc-His(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc- Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc- S3

4 Thr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Met-OH, Boc-Cys(Trt)-OH, Boc-Thz-OH, Proc-Cys(Trt)-OH, Boc-Nle-OH, Boc-Ile-OH, Fmoc-Leu-Thr(ψMe,MePro)-OH, Fmoc-Gly-Ser(ψMe,MePro)-OH, Fmoc-Tyr(tBu)- Ser(ψMe,MePro)-OH. Synthesis of Proc-Cys(Trt)-OH: To a stirred solution of Fmoc-Cys(Trt)-OH (1g) in 6 ml dichloromethane (DCM) diethylamine was added (6 ml) and stirred at room temperature for 4 h and the progres of the reaction was monitored by TLC. After completion of the reaction, DCM and diethylamine was removed by rotary evaporator and dried under high vacuum. The crude product, H 2 N-Cys(Trt)-OH, was used for further reaction without purification. H 2 N- Cys(Trt)-OH (500 mg, 1.37 mmol) was dissolved in 25 ml 2N NaOH solution and the ph was adjusted to 8.5. To the above stirred solution, propergyl chloroformate (0.68 ml, 6.8 mmol) was added dropwise and stirring was continued for additional 5 h. Ethyl acetate was added to the reaction mixture, which was then acidified with saturated citric acid solution and extracted with ethyl acetate. The combined organic layer was dried over anhydrous Na 2 SO 4, filtered and the solvent was removed under reduced pressure. The resulting crude material was purified by flash column chromatography (10% MeOH: CHCl 3 ) to afford Proc-Cys(Trt)-OH as a white solid (~85% yield). 1 H NMR (400 MHz, CDCl 3, ppm): (m, 15H), 5.26 (d, J = 8 Hz, 1H), 4.69 (dd, J = 6.2, 2.1 Hz, S4

5 2H), 4.30 (dd, J = 12.8, 5.6 Hz, 1H), (m, 2H), 2.51 (s, 1H). 13 C NMR (100 MHz, CDCl 3, ppm): 174.6, 154.9, 144.0, 129.4, 128.1, 127.0, 74.9, 67.3, 52.9, 52.7, 33.5, Calcd for C 26 H 23 NO 4 S: Da, Observed: Da [M+Na]. Synthesis of eleven-mer model peptides (LYRAGLYRAG) bearing Proc-Cys, Thiazolidine (Thz), D- and L- Cys residues at the N-termini: The synthesis was carried out using Fmoc-SPPS on Rink amide resin (0.43 mmol/g, 0.2 mmol scale). Peptide synthesis was performed on peptide synthesizer in presence of 4 equiv of amino acid, HCTU and 8 equiv of N,N'-diisopropylethylamine (DIEA). For the synthesis of the model peptides, the pre-swollen resin was treated with 20% piperidine in DMF containing 0.1 mmol HOBt (3-5-3 min) to remove the Fmoc-protecting group. The amino acids were coupled on an automated peptide synthesizer. After coupling of the ten amino acids, the resin was divided into four parts and Proc-Cys(Trt)-OH, Boc-Thz-OH, Boc-D-Cys(Trt)-OH, Boc-L-Cys(Trt)-OH were coupled on the separated resins manually. Deprotection and cleavage from the resin: The resin was washed with DMF, MeOH, DCM and dried. The peptide was cleaved using TFA:triisopropylsilane (TIS):water (95:2.5:2.5) cocktail for 2 h. The cleavage mixture was filtered and the combined filtrate was added dropwise to a 10-fold volume of cold ether and centrifuged. The precipitated crude peptide was dissolved in acetonitrile-water (1:1) and was further diluted to ~30% with water and lyophilized. The HPLC analysis was carried out on a C18 analytical column using a gradient of 0-60% B over 30 min. For preparative HPLC, the same gradient was used to purify the model peptides to the desired product in ~60% yield. S5

6 Figure S1: Analytical HPLC and mass data of the purified Proc-Cys-LYRAGLYRAG. Observed mass ±0.1 (calcd ). Figure S2: Analytical HPLC and mass data of the purified Thz-LYRAGLYRAG. Observed mass ±0.2 (calcd ). S6

7 Figure S3: Analytical HPLC and mass data of the purified D-Cys-LYRAGLYRAG. Observed mass ±0.2 (calcd ). Figure S4: Analytical HPLC and mass data of the purified L-Cys-LYRAGLYRAG- NH2. Observed mass ±0.2 (calcd ). S7

8 Racemization study during removal of Proc from model peptide: Figure S5: Analytical HPLC traces of (A) D-Cys-LYRAGLYRAG, (B) L-Cys- LYRAGLYRAG, (C) Proc-Cys-LYRAGLYRAG, and (D) after proc removal. S8

9 Racemization study during Thz opening from model peptide: Figure S6: Analytical HPLC traces of (A) D-Cys-LYRAGLYRAG, (B) L-Cys- LYRAGLYRAG, (C) Thz-LYRAGLYRAG, and (D) after Thz opening. # is 4- mercaptophenylacetic acid (MPAA). Synthesis of model peptides (LYRAGLYRG) bearing Proc-Cys and Thz at the N- termini and thioester at the C-termini: Peptide thioesters were prepared by N-acylurea methodology using recently reported second generation linker o-amino(methyl)aniline (MeDbz). Fmoc protected MeDbz was coupled manually on pre-swollen Rink amide resin (0.2 mmol) using 4 equiv amino acid, HATU and 8 equiv DIEA for 1.5 h. The remaining amino acids were coupled on an S9

10 automated peptide synthesizer using 4 equiv amino acid, HCTU and 8 equiv of DIEA. After coupling of the ten amino acids the resin was divided and Proc-Cys(Trt)-OH and Boc-Thz-OH were coupled separately on 0.1 mmol resin. MeDbz cyclization: The resin was washed with DCM and a solution of p-nitrophenyl chloroformate (120 mg, for 0.1 mmol scale) in 4 ml DCM was added and shaken for 30 min at room temperature and washed with DCM (3 5 ml). This step was repeated twice. Following this, the resin was treated with a solution of DIEA (0.45mL) in DMF (4 ml) and was shaken for 10 min for a complete cyclization and washed with DMF ( 2). The peptides were cleaved from resin as described above. Thioesterification: The crude peptide-n-acyl-n'-methylacylurea (MeNbz) (25 mg) was dissolved in 1 ml 6 M Gn HCl buffer (ph ~7). This mixture was treated with 100 equiv of methyl-3- was monitored using analytical HPLC C18 column and a gradient of 0-60 %B over 30 min. For preparative HPLC, a similar gradient was used to afford the corresponding peptide in ~60 % yield. S10

11 Figure S7: Analytical HPLC and mass data of the purified Proc-Cys-LYRAGLYRAG- MMP thioester. Observed mass ±0.2 (calcd ). Figure S8: Analytical HPLC and mass data of the purified Thz-LYRAGLYRAG-MMP thioester. Observed mass ±0.2 (calcd ). S11

12 Proc deprotection by palladium chloride [Pd(Cl) 2 ]: Figure S9: Analytical HPLC and mass data of (A) ligation reaction between Proc-Cys- LYRAGLYRAG-MMP and Cys-LYRAGLYRAG, major peak corresponds to the ligation product with the observed mass Da ± 0.6 Da (calcd Da); (B) Proc removal by Pd(Cl) 2 for 5 min with the observed mass Da ± 0.1 Da (calcd Da). Synthesis of eleven-mer model peptide (Cys-LWRAGMYRAG) containing tryptophan to check the compatibility of palladium reagents against all 20 amino acids: The sequences of H2B(22-125), fragment 6 and cullin fragment 10, which we treated with palladium reagents, contained all the canonical amino acids except Trp. Therefore S12

13 we prepared an eleven-mer model peptide containing tryptophan as well as Cys at the N- terminal to check the compatibility of the palladium reagents against all the amino acids. Figure S10: Analytical HPLC and mass data of the purified Cys-LWRAGMYRAG-NH 2. Observed mass ±0.1 (calcd ). We then ligated this peptide (1mg) with Thz-LYRAGLYRAG-MMP (1.05 mg) in presence of 20 equiv MPAA, 10 equiv TCEP in 6M Gn HCl, 200 mm phosphate buffer (390 μl) at 2 mm concentration and subsequently opened the Thz using 60 equiv allylpalladium chloride dimer ([Pd(allyl)Cl] 2 ). The reaction was complete after 15 min as evidenced by the analyitical HPLC and mass data and no any side reaction was observed due to the presence of Trp. S13

14 Figure S11: Analytical HPLC and mass data of (A) the ligation reaction between Thz- LYRAGLYRAG-MMP and Cys-LWRAGMYRAG-NH 2. Peak a corresponds to the starting Cys fragment and peak b corresponds to the ligation product having overserved mass ±0.2 (calculated ). (B) Thz opening reaction after 15min. Peak c corresponds to the Thz opened product having observed mass ±0.2 (calcd ). Syntheses of fragment 5: The synthesis was carried out using the N-acylurea method on Rink amide resin (0.43 mmol/g, 0.1 mmol scale). For the synthesis of the fragment, the pre-swollen resin was treated with 20% piperidine in DMF containing 0.1 mmol HOBt (3-5-3 min) for Fmoc removal. Fmoc-3,4-diaminobenzoic acid (Fmoc-Dbz) was coupled to the resin using S14

15 HBTU/HOBt for 1 h, (2 cycles). Peptide synthesis was performed on peptide synthesizer in presence of 4 equiv of amino acid, 4 equiv HCTU and 8 equiv of DIEA. The Thz protected δ-mercaptolysine was manually coupled for 1.5 h at the position Lys120 using HATU/DIEA. The Nvoc protected Lys was manually coupled for 1.5 h at the positions Lys57 using HATU/DIEA. Proc-Cys(Trt)-OH was manually coupled at the N-terminal of the peptide using 2.5 equiv amino acid, 2.5 equiv HATU and 5 equiv DIEA for 1.5 h. The remaining amino acids were coupled using peptide synthesizer as described above. Analytical HPLC analysis was performed to ensure complete reactions. Dbz cyclization and cleavage: Dbz cyclization followed by deprotection, cleavage from resin and purification was performed as described above to afford the purified peptide in 40-50% yield. S15

16 Figure S12: Analytical HPLC and mass data of (A) crude and (B) purified fragment 5 with the observed mass ±0.4 (calcd ). Synthesis of fragment 9: The synthesis was carried out according to the following scheme: Fragment 9 i.e. NEDD(1-56)-thioester was prepared by N-acylurea methodology using second generation linker o-amino(methyl)aniline (MeDbz). Fmoc protected MeDbz was coupled manually on pre-swollen Rink amide resin (0.1 mmol) using 4 equiv amino acid, HATU and 8 equiv DIEA for 1.5 h. The remaining amino acids were coupled on an automated peptide synthesizer using 4 equiv amino acid, HCTU and 8 equiv of DIEA. The first amino acid Ala and sterically hindered amino acids such as Val, Pro, Ile, Thr, Arg, Gln were doubly coupled. Pseudoproline dipeptides Fmoc-Leu-Thr(ψMe,MePro)- OH and Fmoc-Tyr(tBu)-Ser(ψMe,MePro)-OH were coupled manually at positions Leu 8 - Thr 9 and Tyr 45 -Ser 46 by using 2.5 equiv of dipeptides, 2.5 equiv of HATU and 5 equiv of DIEA for 1.5 h. Analytical cleavage and HPLC analysis were performed to ensure complete reactions. MeDbz cyclization: MeDbz cyclization was carried out similarly as described for model peptide thioesters. The peptide was cleaved similarly as described above. S16

17 Thioesterification: Thioesterification was carried out similarly as described for model peptide thioesters. After switching to MMP thioester, the peptide was purified on a preparative C18 column using a gradient 20-60% B over 60 min to afford fragment 9 in ~ 40 % yield. Figure S13: Analytical HPLC and mass data of (A) crude NEDD(1-56)-MeNbz and (B) purified fragment 9 with the observed mass ±0.5 (calcd ). Synthesis of fragment 10: The synthesis of fragment 10 was carried out using Rink amide resin (0.27 mmol/g, 0.1 mmol scale). The pre-swelled resin was treated with 20% piperidine in DMF containing 0.1 mmol HOBt (3-5-3 min) to remove the Fmoc protecting group. Unless mentioned otherwise, all amino acids were coupled in an automated peptide synthesizer using 4 equiv of amino acid, 8 equiv of DIEA and 4 equiv S17

18 of HCTU to the initial loading of the resin. N α -Fmoc-N ε -Alloc-Lys-OH was coupled at position Lys720 of the cullin1 sequence. Ala714 was mutated to masked Cys in the Thz form to enable native chemical ligation after Thz opening. The Alloc protecting group was removed using 0.2 equiv tetrakis(triphenylphosphine)palladium(0) (Pd[P(C 6 H 5 ) 3 ] 4 ) and 20 equiv phenylsilane in dry DCM. Then other 20 amino acids from NEDD8 sequence were coupled where Ala57 was mutated to Cys. Pseudoproline dipeptide Fmoc- Gly-Ser(ψ Me, Me pro)-oh was coupled manually at positions Gly 64 -Ser 65 by using 2.5 equiv of dipeptides, 2.5 equiv of HATU and 5 equiv of DIEA for 1.5 h. Analytical cleavage and HPLC analysis were performed to ensure complete reactions. Deprotection and cleavage from the resin: The peptide was deprotected and cleaved from resin as described above for model peptides and purified on a preparative C18 column using a gradient 20-60% B over 60 min to afford fragment 10 in ~ 50% yield. S18

19 Figure S14: Analytical HPLC and mass data of (A) crude and (B) purified fragment 10 with observed mass ±0.3 (calcd ). Synthesis of fragment 11: Fragment 11, cullin( )-menbz, was synthesized on Rink amide resin (0.27 mmol/g, 0.1 mmol scale) using MeDbz as a linker, similarly as described for fragment 9 in ~50% yield. In this case, cullin( )-menbz was directly used in the NCL reaction. S19

20 Figure S15: Analytical HPLC and mass data of fragment 11. (A) HPLC of the crude peptide, where peak a corresponds to desired product and b corresponds to a deletion of 217 Da mass from the desired product. (B) HPLC of the purified fragment 11 with the observed mass ±0.1 (calcd ). S20

21 One-pot ligation between Ub-thioester and H2B fragment 6 followed by Proc removal: Fragment, 6, (3 mg, mmol) and Ub-MPA, (2.5 mg, mmol), were dissolved in argon purged 6 M Gn HCl, 200 mm Na 2 HPO 4 buffer (125 μl, 2 mm) containing 20 equiv of MPAA and 10 equiv of TCEP, ph ~7.3. The reaction was incubated at 37 C for 3 h. followed by addition of 4.5 mg (50 equiv) PdCl 2 solution in argon purged 6 M Gn HCl, 200 mm Na 2 HPO 4 buffer (PdCl 2 takes ~30 min to dissolve) ph ~7.3. The reaction was incubated at 37 C for (5-10min). After that the reaction mixture was treated with 19 mg (500 equiv) ditiothreitol (DTT) to quench and precipitate the palladium from the reaction mixture. The reaction mixture was centrifuged and supernatant solution was collected. The precipitate was washed twice with 50% acetonitrile/h 2 O, centrifuged and supernatant solutions were combined followed by purification. Progress of the reaction was monitored by analytical HPLC using C4 column with a gradient of 0-60% buffer B over 30 min. For semi-preparative HPLC, the same gradient was used to isolate the Cys-H2B(22-125)K34Ub in 40% yield (~ 2 mg). S21

22 Synthesis of neddylated Cullin( ) fragment 12: In a typical ligation, fragment 10 (2 mg, mmol) and fragment 9 (3.46 mg, mmol) were dissolved in argon purged 6 M Gn HCl, 200 mm phosphate buffer (336 μl, 1.5 mm) containing 50 equiv of MPAA and 25 equiv of TCEP at ph 7.2. The reaction mixture was incubated at 37 C for 2 h. Progress of the reaction was monitored by using analytical HPLC C4 column with a gradient of 0-60% B over 30 min. After completion of the reaction, Thz deprotection was carried out by adding mg (100 equiv) of [Pd(allyl)Cl] 2 dissolved in argon purged 6 M Gn HCl, 200 mm phosphate buffer (100 μl) and incubated at 37 C for 15 min. After that the reaction mixture was treated with 75 mg (1000 equiv) ditiothreitol (DTT) to quench and precipitate the palladium from the reaction mixture. The reaction mixture was centrifuged and supernatant solution was collected. The precipitate was washed twice with 50% acetonitrile/h 2 O, centrifuged and supernatant solutions were combined followed by purification. For semi-preparative HPLC, a gradient of 0-60% B in 30 min was used to isolate the product in ~45% yield (2.3 mg). S22

23 Synthesis of neddylated cullin( ), 13: In a typical ligation, fragment 12 (1 mg, mmol) and fragment 11 (0.21 mg, mmol) were dissolved in argon purged 6 M Gn HCl, 200 mm phosphate buffer (95 μl, 1 mm) containing 50 equiv of MPAA and 25 equiv of TCEP at ph 7.2. The reaction mixture was incubated at 37 C for 6 h. Progress of the reaction was monitored by using analytical HPLC C4 column with a gradient of 0-60% B over 30 min. After completion of the reaction, the reaction mixture was dialysed in a Slide-A-Lyzer 3.5K dialysis cassette (Thermo scientific, ml) in 6 M Gn HCl, 200 mm phosphate buffer (500 ml) for overnight and then subjected to radical induced desulfurization by adding TCEP (10.7 mg, 250 mmol), VA-044 (3.1 mg, 100 eqiv) and tert-butyl thiol (15 μl) and incubated at 37 C for 6 h. Progress of the reaction was monitored by analytical HPLC C4 column with a gradient of 0-60% B over 30 min. For semi-preparative HPLC, the same gradient was used to isolate the product in ~30% yield. S23

24 Evaluation of UCH-L3 activity on neddylated cullin substrate: HS O O Nedd8(1-76) NH Nedd8(1-76) NH HS H 2 N O H N K 720 Cullin ( ) 12 VA-044, TCEP, tbush K 720 Cullin ( ) 14 The intermediate 12 was desulfurized and the product 14 was used as a substrate of UCHL3 to test the activity of the enzyme on it. The protein 14 was dissolved in 6 M urea solution to 5% of the total volume and then further diluted with the assay buffer (50 mm HEPES, 0.5 mm ethylenediamine tetracarboxylic acid (EDTA), ph 8). The exact concentration of the protein solution was determined using Pierce BCA Protein Assay Kit (Thermo scientific). Stock solution of 1 µm recombinant human UCH-L3 (BostonBiochem) was prepared by diluting 33.3 µm of the enzyme with enzyme buffer (50 mm HEPES buffer, 0.5 mm EDTA, 1 mm DTT, 0.5 mg/ml BSA, ph 8.0). The enzymatic reaction was carried out by incubating the substrate (20 µm, 50 µl) with UCH-L3 (48.2 nm, 50 µl) for 30 mins at 37 C. Enzyme activity was detected by analytical HPLC using C4 column, 0-60% B over 45 min. Circular dichroism spectroscopy: The protein 14 was dissolved in 6M urea solution (5% of the total volume) and then further diluted with 20 mm Tris buffer solution and ph was adjusted to 7.3. The exact concentration of the protein solution was determined to be 51.2 μm (Pierce BCA Protein Assay Kit, Thermo scientific). With this solution circular dichroism spectrum was recorded in a Chirascan (Applied Photophysics) instrument. S24

25 Figure S16: 1 H-spectrum of Proc-Cys(Trt)-OH in CDCl 3 (400 MHz). Figure S17: 13 C-spectrum of Proc-Cys(Trt)-OH in CDCl 3 (100 MHz). S25

26 Figure S18: ESI-MS data of Proc-Cys(Trt)-OH. S26

27 Synthesis of eleven-mer model peptides, Proc-Cys(LYRAGLYRAX)-NHNH 2 (X=G,A,R,T,F): The synthesis of the five peptides was carried out using standard Fmoc-SPPS, followed by cyclization as described above, to form peptide (LYRAGLYRAX,)-MeNbz. Each one of the crude peptides was switched to C-terminal hydrazide via the addition of solution of 100 equiv of hydrazine in 1 ml of 6 M Gn HCl buffer (ph ~7) and left to react for 1h at room temperature. The reaction was monitored using analytical HPLC C4 column and a gradient of 0-60 %B over 30 min. For preparative HPLC, a similar gradient was used to afford the corresponding peptide in ~50-60% yield. Switching of Proc-Cys(LYRAGLYRAX)-NHNH 2 to MPAA thioester: The peptide-hydrazide was dissolved in 6 M Gn HCl buffer, ph 3. The solution was cooled in a freezing mixture (-18 o C) for 5 min, then, 20 equiv of sodium nitrite solution were added and left to react for 25 min. Then, 100 equiv of MPAA in 6 M Gn HCl buffer was added. The ph was adjusted to 7 and left to react for 15 min, then 50 equiv of TCEP was added. The progress of the reaction was monitored by analytical HPLC using C4 column with a gradient of 0-60% B over 30 min. S27

28 Figure S19: Analytical HPLC and mass data of (A) purified Proc-CysLYRAGLYRAG- NHNH 2, observed mass , (calcd ). (B) Switching reaction, Peak b corresponds to the cyclized peptide Proc-CysLYRAGLYRAG, observed mass , (calcd ). Peak c corresponds to the switched peptide Proc-CysLYRAGLYRAG- MPAA, observed mass , (calcd ). Switching of Proc-Cys(LYRAGLYRAG)-NHNH 2 to MPAA thioester: S28

29 Figure S20: Analytical HPLC and mass data of (A) purified Proc-CysLYRAGLYRAA- NHNH 2, observed mass , (calcd ). (B) Switching reaction, Peak b corresponds to the cyclized peptide Proc-CysLYRAGLYRAA, observed mass , (calcd ). Peak c corresponds to the switched peptide Proc-CysLYRAGLYRAA- MPAA, observed mass , (calcd ). Switching of Proc-Cys(LYRAGLYRAA)-NHNH 2 to MPAA thioester: S29

30 Figure S21: Analytical HPLC and mass data of (A) purified Proc-CysLYRAGLYRAF- NHNH 2, observed mass , (calcd ). (B) Switching reaction, Peak b corresponds to the cyclized peptide Proc-CysLYRAGLYRAF, observed mass , (calcd ). Peak c corresponds to the switched peptide Proc-CysLYRAGLYRAF- MPAA, observed mass , (calcd ). Switching of Proc-Cys(LYRAGLYRAF)-NHNH 2 to MPAA thioester: S30

31 Figure S22: Analytical HPLC and mass data of (A) purified Proc-CysLYRAGLYRAT- NHNH 2, observed mass , (calcd ). (B) Switching reaction, Peak b corresponds to the cyclized peptide Proc-CysLYRAGLYRAT, observed mass , (calcd ). Peak c corresponds to the switched peptide Proc-CysLYRAGLYRAT- MPAA, observed mass , (calcd ). Switching of Proc-Cys(LYRAGLYRAT)-NHNH 2 to MPAA thioester: S31

32 One-pot switching of Proc-Cys(LYRAGLYRAR)-NHNH 2 to MPAA thioester and ligation with Cys-LYRAGLYRAG: To examine the reactivity of the switched and cyclized product, we performed switching of the fifth model peptide Proc-Cys(LYRAGLYRAR)-NHNH 2 to MPAA-thioester as described before, and then ligated with Cys-LYRAGLYRAG by adding 1.3 equiv of this peptide to the switching mixture. This gave the expected ligation product within 30 min where both the switched and cyclized products were ligated. The progress of the reaction was monitored by analytical HPLC using C4 column with a gradient of 0-60% B over 30 min (Figure S23). Switching of Proc-Cys(LYRAGLYRAR)-NHNH 2 to MPAA thioester and One-pot ligation with Cys-LYRAGLYRAG: S32

33 Figure S23: Analytical HPLC and mass data of (A) purified Proc-CysLYRAGLYRAR- NHNH 2, observed mass Da, (calcd Da); (B) Switching reaction, Peak b corresponds to the cyclized peptide Proc-CysLYRAGLYRAR, observed mass Da, (calcd Da). Peak c corresponds to the switched peptide Proc- CysLYRAGLYRAR-MPAA, observed mass Da, (calcd Da); (C) Ligation of switched and cyclized product with Cys-LYRAGLYRAG: peak d corresponds to Cys- LYRAGLYRAG with the observed mass Da (calcd Da) and peak e corresponds to the ligation product with the observed mass Da, (calcd Da) S33

34 The effect of the DTT on Pd chelation to the peptide Figure S24: Analytical HPLC traces of (A) Thz-LYRAGLYRAG, (B) Thz- LYRAGLYRAG, after [Pd(allyl)Cl] 2 addition, (C) Cys-LYRAGLYRAG, after DTT addition. S34