A straightforward method for automated Fmoc-based synthesis of bio-inspired peptide crypto-thioesters† †Electronic supplementary information (ESI) available: Detailed synthetic procedures, characterization and kinetics studies. See DOI: 10.1039/c5sc02630j

A bio-inspired method for the synthesis of peptide thioester surrogates for native chemical ligation was developed. The process can be fully automated and does not require postsynthetic steps.


Supporting Information
1) General information S2 2) General procedures for solid phase peptide synthesis S3 3) Optimization of the benzyl group to maximize the N-acylation yield S4 a-Synthesis of cysteinyl peptide resin 1 S4 b-Synthesis of peptide resins 2a-d S5 c-Study on the solid-supported N-acylation of 2a-d S9 4) Evaluation of the phenol pKa of an N-acyl-N-(Hnb)Cys compound S3 S12 5) Characterization of a model N-acyl-N-(Hnb)Cys compound S5 S14 a-NMR of S5 S14 b-Molar extinction coefficient of the Hnb group of S5 S24 6) Optimization of the cysteine thiol protecting group S24 a-Synthesis and stability of (S-Trt) model peptide S7 S24 b-Synthesis and stability of (S-StBu) model peptide 7 S28 7) Optimization of the spacer between the cysteine and the resin S30 a-Direct attachment to Rink linkeramide hydrolysis co-product S30 b-Direct attachment to PHB Tentagel resin (Wang type linker) S31 c-Introduction of a Gly residue as a spacer S32 8) N-Acylation with the 20 different proteogenic amino acids S34 9) Kinetics studies of the NCL with model Ac-LYRAA(Hnb)C peptides S57 a-Synthesis of model cysteinyl peptide 6 S57 b-Influence of the nature of the cysteine C-terminus S59 c-Study of the hydrolysis of the thioester precursor 9 and epimerization S62 d-Influence of the buffer S64 e-Influence of masking phenol or thiol groups S65 f-Influence of the pH S71 10) Influence of the C-terminal aa: NCL with model Ac-LYRAX(Hnb)C peptides S73 a-Synthesis of peptide Ac-LYRAS-(Hnb)C(StBu)G-NH 2 10 S73 b-NCL with peptide Ac-LYRAS-(Hnb)C(StBu)G-NH 2 10 S75 c-Synthesis of peptide Ac-LYRAV-(Hnb)C(StBu)G-NH 2 11 S76 d-NCL with peptide Ac-LYRAV-(Hnb)C(StBu)G-NH 2 11 S78 e-NCL with peptide 11 under optimized conditions S78 f-Determination of apparent second order kinetic constants S79 11) Application to MT7 S83 Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2015 a-Synthesis of cysteinyl peptide 19 S83 b-Synthesis of crypto-thioester peptide 18 S86 c-NCL S89 12) Application to Cg-Bigdef1 S91 a-Synthesis of cysteinyl peptide 22 S91 b-Synthesis of crypto-thioester peptide 21 S93 c-NCL S96

1-General information
All reagents and solvents were used without further purification. Protected amino acids, Fmoc-Gly-Wang resin, Fmoc-Rink polystyrene resin, Rink linker, HBTU and HCTU were purchased from Merck Biosciences (Nottingham, UK). Aminomethyl TentaGel R resin and pre-loaded Fmoc-Cys(StBu)-PHB (Wang type) Tentagel resin were purchased from Rapp polymers (Tuebingen, Germany). Fmoc-K(Boc)-Mppa-OH was purchased from Polypeptide laboratories (Strasbour, France). Peptide synthesis grade DMF and HATU were obtained from Applied Biosystems (Courtaboeuf, France). Ultrapure water was obtained using a Milli-Q water system from Millipore (Molsheim, France). All other chemicals were from Sigma Aldrich (St-Quentin-Fallavier, France) and solvents from SDS-Carlo Erba (Val de Reuil, France). 1 H and 13 C NMR spectra were recorded on a Bruker AVANCE III 600 instrument, at a constant temperature of 25°C. Chemical shifts are reported in parts per million from low to high field and referenced to tetramethylsilane (TMS). Coupling constants (J) are reported in hertz (Hz). Standard abbreviations indicating multiplicity were used as follows: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, m = multiplet, b= broad signal.
High resolution ESI-MS analyses were performed on a maXis ultra-high-resolution Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany), using the positive mode. MALDI-TOF analyses were performed on an Ultraflex instrument (Bruker Daltonics, Bremen, Germany) equipped with a 337-nm nitrogen laser and a gridless delayed extraction ion source. The sample was co-crystallized with a solution of αcyano-4-hydroxy-cinnamic acid (HCCA) as a matrix. The reported m/z values correspond to the monoisotopic ions if not specified otherwise. Peptides incorporating an N-(2-hydroxy-5-nitrobenzyl) group displayed a typical MALDI fragmentation pattern, consisting in -16 Da and -34 Da minor peaks in addition to the [MH] + peak.
HPLC analyses and semi-preparative purifications were carried out on a LaChrom Elite system equipped with a Hitachi L-2130 pump, a Hitachi L-2455 diode array detector and a Hitachi L-2200 autosampler. Nucleosil C18 (300 Å, 5 μm, 250 × 4.6 mm, 1 mL/min flow rate) or Chromolith HighResolution RP-18e (150 Å, 10 × 4.6 mm, 3 mL/min flow rate) columns were used for analysis and Nucleosil C18 (300 Å, 5 μm, 250 × 10 mm, 3 mL/min flow rate) for purification. Solvents A and B are 0.1 % TFA in H 2 O and 0.1 % TFA in MeCN, respectively. Each gradient was followed by a washing step (95% B/A over 0.5 min for the HR Chromolith; over 1 min for the Nucleosil C18 column) to identify eventual co-products not eluted during the gradient.

2-General procedures for solid phase peptide synthesis
Fmoc-based solid phase peptide syntheses (SPPS) were carried out on either a Prelude synthesizer from Protein Technologies or a 433A synthesizer from Applied Biosystems. Microwave-assisted Fmoc-SPPS syntheses were carried out on an Initiator+ SP Wave synthesizer from Biotage.
Syntheses starting from Fmoc-Gly-Wang or Rink polystyrene resins were performed on the 433A synthesizer, using the 0.1 mmol scale Fastmoc program purchased from the manufacturer, with a single coupling with HCTU followed by capping.
The crude peptide was deprotected and cleaved from the resin through a treatment with TFA/H 2 O/iPr 3 SiH/phenol, 88/5/2/5 for 2 h, and the peptide was precipitated by dilution into an ice-cold 1:1 diethyl ether/petroleum ether mixture, recovered by centrifugation and washed twice with diethyl ether.

3-Optimization of the benzyl group to maximize the N-acylation yield
3a-Synthesis of cysteinyl peptide resin 1 Supplementary scheme S1: Synthesis of peptide resin 1.
Peptide resin 1 was obtained through automated SPPS (protocol p S3) starting from Fmoc-Gly-Wang resin (130 mg, 0.79 mmol/g, 0.1 mmol). An aliquot of the resin was cleaved (protocol p S3) in order to characterize the corresponding peptide S1. Supplementary figure S1: HPLC trace of crude S1.
Peptide resin 1 (10 µmol) was introduced into a polypropylene syringe fitted with a polypropylene frit and a PTFE stopcock, then swollen in a DMF/MeOH/AcOH (9:9:2) mixture for 5 min. The syringe was drained and the resin was washed with a DMF/MeOH (1:1)

General procedure for solid-supported N-acylation
Peptide resin (2a-d) was introduced into a polypropylene syringe fitted with a polypropylene frit and a PTFE stopcock and swollen with NMP. Then, Fmoc-Gly-OH (10 equiv.), HBTU (9.5 equiv.) and HOBt (9.5 equiv.) were dissolved in NMP (0.1 M final concentration in amino acid) prior to addition of iPr 2 NEt (20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 2 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. An aliquot resin was cleaved using the standard procedure (p S3) to determine the coupling yields and characterize the corresponding N-acylated peptides.  N-acylation of peptide resin 2d using HBTU/HOBt followed by deprotection of the Fmoc group then cleavage of the resin did not show any detectable amount of the expected N-acylated peptide 3d. For characterization purpose, the N-acylation was performed using HATU as the coupling reagent, yielding to a small amount of 3d together with the non-acylated peptide S2d.
Supplementary scheme S5: N-acylation of peptide resin 2d using HATU as the coupling reagent.

Synthesis of S-alkylated compound S3
In order to prevent any N-S shift that would make uncertain the determination of the pKa of the phenol group, we synthesized a model dipeptide S3 S-alkylated with an acetamidomethyl (Acm) protective group.
Supplementary scheme S6: Synthesis of the model dipeptide S3.

Determination of the phenol pKa of S-alkylated compound S3
The pka of the phenol group was determined by acid-base titration of a dilute (10 µM) solution of compound S3 in Milli-Q water. The pH was adjusted with 1M HCl and NaOH solutions. The titration was followed by UV spectrophotometry on an Uvikon 923 double beam UV/VIS spectrophotometer. The results were plotted at the  max of the phenol and phenolate species (determined to be 320 and 407 nm, respectively) against pH.
Supplementary figure S11: UV absorbance of compound S3 as a function of pH.

5a-1 H and 13 C NMR of S5 and comparison with 1 H NMR of O-methylated S6
Supplementary scheme S7: Synthesis of the compound S5.
Fmoc-protected Rink polystyrene resin (63.3 mg, 0.79 mmol/g, 0.05 mmol) was introduced into a polypropylene syringe fitted with a polypropylene frit and a PTFE stopcock and swollen with NMP. Fmoc group was deprotected by three successive treatments with 20% piperidine in NMP (5 mL) for 3 min. Then, Fmoc-Cys(StBu)-OH (216 mg, 0.5 mmol, 10 equiv.) and HCTU (186 mg, 0.45 mmol, 9 equiv.) were dissolved in NMP (1 mL) prior to addition of iPr 2 NEt (175 µL, 1 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (5 mL) for 3 min to give resin S4. The 2-hydroxy-5-nitrobenzyl linker was then introduced following the general procedure (p S5 for 0.05 mmol). Then, acetic acid (30 µL, 0.5 mmol, 10 equiv.) and HCTU (186 mg, 0.45 mmol, 9 equiv.) were dissolved in NMP (1 mL) prior to addition of iPr 2 NEt (175 µL, 1 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Amino acid-resin was then treated with 20% piperidine in NMP (5 mL, 3 × 3 min), washed with NMP then CH 2 Cl 2 . Finally, the compound S5 was obtained after treatment with TFA/H 2 O/iPr 3 SiH, 93/5/2 (4 mL) for 2 h and characterized. Note that the hydrolysis of the C-terminal amide into the acid carboxylic was quantitative under the cleavage conditions. The product was purified by flash reverse phase chromatography (45 mg crude in 1 mL MeCN, column: RP18 25-40 µm -20 g, Götec-Labortechnik GmbH, gradient: 30-50% B/A over 20 min, 35 mL/min). Pure S5 has been analyzed by 1H NMR in DMSO-d 6 . Two conformers are observed: trans and cis isomers (evaluated ratio 73:27). On the supplementary figure S14, arrows indicate observed NOE on a ROESY 2D spectrum (mixing time = 200 ms) used to characterize the two conformers.
Supplementary scheme S8: Synthesis of the compound S6.
Fmoc-protected Rink polystyrene resin (63.3 mg, 0.79 mmol/g, 0.05 mmol) was introduced into a polypropylene syringe fitted with a polypropylene frit and a PTFE stopcock and swollen with NMP. Fmoc group was deprotected by three successive treatments with 20% piperidine in NMP (5 mL) for 3 min. Then, Fmoc-Cys(StBu)-OH (216 mg, 0.5 mmol, 10 equiv.) and HCTU (186 mg, 0.45 mmol, 9 equiv.) were dissolved in NMP (1 mL) prior to addition of iPr 2 NEt (175 µL, 1 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (5 mL) for 3 min to give resin S4. The 2-hydroxy-5-nitrobenzyl linker was then introduced following the general procedure (p S5 for 0.05 mmol). Then, acetic acid (30 µL, 0.5 mmol, 10 equiv.) and HCTU (186 mg, 0.45 mmol, 9 equiv.) were dissolved in NMP (1 mL) prior to addition of iPr 2 NEt (175 µL, 1 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Amino acid-resin was then treated with 20% piperidine in NMP (5 mL, 3 × 3 min), washed with NMP then CH 2 Cl 2. Methylation of the phenol group was performed using a solution of MeI (50 equiv.), iPr 2 NEt (50 equiv.) in DMF for 4 h and the resin was washed with NMP and CH 2 Cl 2 . Finally, the compound S6 was obtained after treatment with TFA/H 2 O/iPr 3 SiH, 93/5/2 (4 mL) for 2 h and characterized. Note that the hydrolysis of the C-terminal amide into the acid carboxylic was quantitative under the cleavage conditions. The product was purified by semi-preparative HPLC (Nucleosil, 40-55 % B/A over 15 min).
Pure S6 has been analyzed by 1 H NMR in DMSO-d 6 for comparison of the cis-trans ratio with S5. Two conformers are observed: trans and cis isomers (evaluated ratio 72:28, nearly identical with S5 ratio). On the supplementary figure S19, arrows indicate observed NOE on a ROESY 2D spectrum (mixing time = 200 ms) used to characterize the two conformers.
Supplementary figure S19: Cis and trans isomers of S6 and observed ROESY connectivities. Supplementary table S1: Molar extinction coefficient of S5.

6-Optimization of the cysteine thiol protecting group
6a-Synthesis and stability of (S-Trt) model peptide S7 Supplementary scheme S9: Synthesis of the model peptide S7 showing hydrolysis co-product S8.
Fmoc-protected Rink polystyrene resin (126.6 mg, 0.79 mmol/g, 0.1 mmol) was introduced into a polypropylene syringe fitted with a polypropylene frit and a PTFE stopcock and swollen with NMP. Fmoc group was deprotected by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Then, Fmoc-Cys(Trt)-OH (586 mg, 1 mmol, 10 equiv.) and HCTU (372 mg, 0.90 mmol, 9 equiv.) were dissolved in NMP (2 mL) prior to addition of iPr 2 NEt (348 µL, 2 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. The 2-hydroxy-5-nitrobenzyl linker was then introduced following the general procedure (p S5). The Ac-LYRAA sequence was installed by standard SPPS procedure (protocol p S3), an extended double coupling (2 × 2h) was performed for the first alanine and a normal double coupling (2 × 30 min) was performed for the arginine. Peptide resin was then treated with 20% piperidine in NMP (10 mL, 3 × 3 min), washed with NMP then CH 2 Cl 2 , and finally cleaved following the general procedure p S3 to give compound S7 and side product S8 (89:11) (Measured by HPLC at 275nm). Peptides were obtained as a mixture of C-terminal amide and acid arising from hydrolysis of the amide under the TFA cleavage conditions. 2 Their corresponding thioester forms arising from a premature N-S shift was also observed (compounds S7' and S8').   6b-Synthesis and stability of (S-StBu) model peptide 7
Fmoc-protected Rink polystyrene resin (126.6 mg, 0.79 mmol/g, 0.1 mmol) was introduced into a polypropylene syringe fitted with a polypropylene frit and a PTFE stopcock and swollen with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Then, Fmoc-Cys(StBu)-OH (432 mg, 1 mmol, 10 equiv.) and HCTU (372 mg, 0.90 mmol, 9 equiv.) were dissolved in NMP (2 mL) prior to addition of iPr 2 NEt (348 µL, 2 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. The 2-hydroxy-5-nitrobenzyl linker was then introduced following the general procedure (p S5). The Ac-LYRAA sequence was installed by standard SPPS procedure (protocol p S3), an extended double coupling (2 × 2h) was performed for the first alanine and a normal double coupling (2 × 30 min) was performed for the arginine. Peptide resin was then treated with 20% piperidine in NMP (10 mL, 3 × 3 min), washed with NMP then CH 2 Cl 2 , and finally cleaved following the general procedure p S3 to give compound 7. Peptide was obtained as a mixture of C-terminal amide and acid arising from hydrolysis of the amide under the TFA cleavage conditions.

7-Optimization of the spacer between the cysteine and the resin
7a-Direct attachment to Rink linkeramide hydrolysis co-product 2 Supplementary scheme S11: Synthesis of the model peptide 7.
During the cleavage step (using procedure p S3) in order to obtain peptide 7, the formation of peptide 8 was also observed (12 % determined by HPLC integration at 275 nm) from the hydrolysis of the C-terminal amide function into a C-terminal carboxylic acid. Pre-loaded Fmoc-Cys(StBu)-PHB Tentagel resin (527 mg, 0.19 mmol/g, 0.1 mmol) was introduced into a polypropylene syringe fitted with a polypropylene frit and a PTFE stopcock and swollen with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. The 2-hydroxy-5-nitrobenzyl linker was then introduced following the general procedure (p S5). For a 10 µmol aliquot, acetic acid (6 µL, 0.1 mmol, 10 equiv.) and HCTU (37.2 mg, 0.09 mmol, 9 equiv.) were dissolved in NMP (0.2 mL) prior to addition of iPr 2 NEt (35 µL, 0.2 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Amino acid-resin was then treated with 20% piperidine in NMP (1 mL, 3 × 3 min), washed with NMP then CH 2 Cl 2. Finally, the product was cleaved using TFA / H 2 O (95:5) for 2h. The solvents were removed by evaporation and analysis showed the complete formation of the C-terminal piperidine adduct S9 and no trace of the expected Cys(StBu) compound.

7c-Introduction of a Gly residue as a spacer
Supplementary scheme S13: Synthesis of the model peptide 9.
Tentagel R resin (476 mg, 0.21 mmol/g, 0.1 mmol) was introduced into a polypropylene syringe fitted with a polypropylene frit and a PTFE stopcock and swollen with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Then, Fmoc-Rink-OH (270 mg, 0.5 mmol, 5 equiv.) and HATU (190 mg, 0.5 mmol, 5 equiv.) were dissolved in NMP (2 mL) prior to addition of iPr 2 NEt (175 µL, 1 mmol, 10 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1.5 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Fmoc-Gly-OH (297 mg, 1 mmol, 10 equiv.) and HCTU (372 mg, 0.90 mmol, 9 equiv.) were dissolved in NMP (2 mL) prior to addition of iPr 2 NEt (348 µL, 2 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Then, Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Fmoc-Cys(StBu)-OH (432 mg, 1 mmol, 10 equiv.) and HCTU (372 mg, 0.90 mmol, 9 equiv.) were dissolved in NMP (10 mL) prior to addition of iPr 2 NEt (348 µL, 2 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1.5 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. The 2-hydroxy-5-nitrobenzyl linker was then introduced following the general procedure (p S5). The Ac-LYRAA sequence was installed by standard SPPS procedure (protocol p S3), an extended double coupling (2 × 2h) was performed for the first alanine and a normal double coupling (2 × 30 min) was performed for the arginine. Peptide resin was then treated with 20% piperidine in NMP (10 mL, 3 × 3 min), washed with NMP then CH 2 Cl 2 , and finally cleaved following the general procedure p S3 to give compound 9. The product was purified by HPLC (Nucleosil, 40-45 % B/A over 10 min).
The 20 different proteogenic amino acids were then coupled on 4 using a single standard automated coupling on the prelude (protocol p S3), after that the peptide resins were treated with 20 % piperidine in NMP (3 × 3 min); finally, an aliquot (5 µmol) of each resins was cleaved using 1mL of a TFA/H 2 O/iPr 3 SiH (93:5:2) solution for 2 h. After evaporation of TFA, the samples were analyzed by HPLC; the Nacylation yields were quantified by integration at 315 nm, not taking into account eventual differences in molar extinction coefficient of the products. In the case of compound 5'e, a minor peak was observed showing the same m/z as expected for compound 5'e. It was attributed to epimerization of Fmoc-Cys(Trt) during coupling, yielding compound S12 after TFA cleavage (ratio: L-Cys / D-Cys 93:7). D-Cys -S12: In the case of compound 5'j, concomitant formation of pyroglutamate (S13) was observed during the TFA treatment.

9-Kinetics studies of the NCL with model Ac-LYRAA(Hnb)C peptides
9a-Synthesis of the model cysteinyl peptide 6 Supplementary scheme S16: Synthesis of the model cysteinyl peptide 6.

6: MALDI-TOF
Ligation with 9: 500 µL of a degassed 0.2 M pH 7.1 sodium phosphate buffer containing 25 mM MPAA, 50 mM TCEP and 6 M guanidine hydrochloride were added to 1.3 mg of the peptide 9 (final concentration 2 mM) and 0.7 mg peptide 6 (final concentration 1 mM) under argon. The ligation was carried out at 37°C and monitored by RP-HPLC (Chromolith, gradient: 20-70 % B/A over 6 min). For this, aliquots of 2 µL were diluted in 108 µL of 1.5 % TFA in water and 100 µL were injected in HPLC. 9c-Study of the hydrolysis of the thioester precursor 9 and epimerization at the ligation site during a typical NCL with 9 In order to quantify the hydrolysis of the thioester and the epimerization at the ligation site during a classical NCL with 6 and 9 (page S60), HPLC standards S14 and 15 were synthesized.
Supplementary scheme S18: Hydrolysis of peptide 7 to give HPLC standard S14. 4 Reaction conditions inspired by Kent et al. 4 Peptide 7 (1 mM) was incubated under argon in 100 µL of a deoxygenated 200 mM pH 9 sodium phosphate buffer containing 25 mM MPAA, 50 mM TCEP, 200 mM βmercaptoethanol and 6 M guanidine hydrochloride. The reaction was monitored by RP-HPLC (Chromolith, gradient: 20-70 % B/A over 6 min). After 16h at room temperature, the starting material was consumed and the product S14 was characterized and purified to serve as a HPLC standard. An HPLC co-injection of the NCL mixture after 24h using 9 with HPLC standards S14 and 15 allowed determining the amount of hydrolysis of the thioester moiety and epimerization at the ligation site after a day of reaction.
9d-Influence of the buffer Supplementary scheme S20: NCL in HEPES buffer with 9.
The ligation was carried out at 37°C and monitored by RP-HPLC (Chromolith, gradient: 20-70 % B/A over 6 min). For this, aliquots of 2 µL were diluted in 108 µL of 1.5 % TFA in water and 100 µL were injected in HPLC.
Supplementary figure S60: Comparison of reaction kinetics using phosphate or HEPES buffer using 9.
9e-Influence of masking phenol or thiol groups Supplementary scheme S21: Synthesis of O-methylated peptide 12.
Tentagel R resin (476 mg, 0.21 mmol/g, 0.1 mmol) was introduced into a polypropylene syringe fitted with a polypropylene frit and a PTFE stopcock and swollen with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Then, Fmoc-Rink-OH (270 mg, 0.5 mmol, 5 equiv.) and HATU (190 mg, 0.5 mmol, 5 equiv.) were dissolved in NMP (2 mL) prior to addition of iPr 2 NEt (175 µL, 1 mmol, 10 equiv. then the syringe was drained and the resin was washed with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Fmoc-Gly-OH (297 mg, 1 mmol, 10 equiv.) and HCTU (372 mg,0.90 mmol,9 equiv.) were dissolved in NMP (2 mL) prior to addition of iPr 2 NEt (348 µL, 2 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Then, Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Fmoc-Cys(StBu)-OH (432 mg, 1 mmol, 10 equiv.) and HCTU (372 mg,0.90 mmol,9 equiv.) were dissolved in NMP (10 mL) prior to addition of iPr 2 NEt (348 µL, 2 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1.5 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. The 2-hydroxy-5-nitrobenzyl linker was then introduced following the general procedure (p S5). The Ac-LYRAA sequence was installed by standard SPPS procedure (protocol p S3), an extended double coupling (2 × 2h) was performed for the first alanine and a normal double coupling (2 × 30 min) was performed for the arginine. Peptide resin was then treated with 20% piperidine in NMP (10 mL, 3 × 3 min), washed with NMP then CH 2 Cl 2 . Methylation of the phenol group was performed using a solution of MeI (50 equiv.), iPr 2 NEt (50 equiv.) in DMF for 4 h and the resin was washed with NMP and CH 2 Cl 2 . Finally, peptide-resin was cleaved following the general procedure (p S3) to give compound 12, which was purified by semi-preparative HPLC (Nucleosil, gradient: 40-45 % B/A over 10 min). Tentagel R resin (476 mg, 0.21 mmol/g, 0.1 mmol) was introduced into a polypropylene syringe fitted with a polypropylene frit and a PTFE stopcock and swollen with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Then, Fmoc-Rink-OH (270 mg, 0.5 mmol, 5 equiv.) and HATU (190 mg, 0.5 mmol, 5 equiv.) were dissolved in NMP (2 mL) prior to addition of iPr 2 NEt (175 µL, 1 mmol, 10 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1.5 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Fmoc-Gly-OH (297 mg, 1 mmol, 10 equiv.) and HCTU (372 mg, 0.90 mmol, 9 equiv.) were dissolved in NMP (2 mL) prior to addition of iPr 2 NEt (348 µL, 2 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Then, Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Fmoc-Cys(Acm)-OH (414.5 mg, 1 mmol, 10 equiv.) and HCTU (372 mg, 0.90 mmol, 9 equiv.) were dissolved in NMP (10 mL) prior to addition of iPr 2 NEt (348 µL, 2 mmol, 20 equiv.). The resulting S69 solution was immediately added to the resin. The syringe was left for 1.5 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. The 2-hydroxy-5-nitrobenzyl linker was then introduced following the general procedure (p S5). The Ac-LYRAA sequence was installed by standard SPPS procedure (protocol p S3), an extended double coupling (2 × 2h) was performed for the first alanine and a normal double coupling (2 × 30 min) was performed for the arginine. Peptide resin was then treated with 20% piperidine in NMP (10 mL, 3 × 3 min), washed with NMP then CH 2 Cl 2 . Finally, the peptide was cleaved following the general procedure p S3 to give compound 13. 13 has been purified by semi-preparative HPLC (Nucleosil, gradient: 20-35 % B/A over 5 min). 500 µL of different degassed 0.2 M sodium phosphate buffers (pH 5.6, 6.1, 6.6, 7.1, 7.6) containing 25 mM MPAA, 50 mM TCEP and 6 M guanidine hydrochloride were added to 0.8 mg of the peptide 9 (final concentration 1.5 mM) and 0.69 mg peptide 6 (final concentration 1 mM) under argon. The ligation was carried out at 37°C and monitored by RP-HPLC (Chromolith, gradient: 20-70 % B/A over 6 min). For this, aliquots of 2 µL were diluted in 108 µL of 1.5 % TFA in water and 100 µL were injected in HPLC. Peptide 10 was obtained through automated SPPS (protocol p S3) and using the automated reductive amination (protocol p S34) starting from Fmoc-Rink tentagel resin (120 mg, 0.21 mmol/g, 25 µmol). Fmoc-Ser(OtBu)-OH was coupled for 3 × 30 min on the (Hnb)C(StBu)G-Rink Tentagel resin. The complete sequence was installed through standard Fmoc-SPPS (protocol p S3), Fmoc-Arg(Pbf)-OHwas coupled twice (2 × 30 min) and a final piperidine treatment (20 % in NMP, 3 mL, 3 min, ×3) was performed. Peptide-resin was cleaved (protocol p S3) and peptide 10 was purified by semi-preparative RP-HPLC (Nucleosil, gradient: 40-55 % B/A over 12 min).
Peptide 11 was obtained through automated SPPS (protocol p S3) and using the automated reductive amination (protocol p S34) starting from Fmoc-Rink tentagel resin (120 mg, 0.21 mmol/g, 25 µmol). Fmoc-Val-OH has been coupled for 5 × 30 min on the (Hnb)C(StBu)G-Rink Tentagel resin. The complete sequence was installed through standard Fmoc-SPPS (protocol p S3), Fmoc-Arg(Pbf)-OH was coupled for 2 × 30 min and a final piperidine (20 % in NMP, 3mL, 3 min, ×3) was performed. Peptide-resin was cleaved (protocol p S3) and peptide 11 was purified by semi-preparative RP-HPLC (Nucleosil, gradient: 40-55 % B/A over 12 min). Tentagel R resin (476 mg, 0.21 mmol/g, 0.1 mmol) was introduced into a polypropylene syringe fitted with a polypropylene frit and a PTFE stopcock and swollen with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Then, Fmoc-Rink-OH (270 mg, 0.5 mmol, 5 equiv.) and HATU (190 mg, 0.5 mmol, 5 equiv.) were dissolved in NMP (2 mL) prior to addition of iPr 2 NEt (175 µL, 1 mmol, 10 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1.5 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Fmoc-Gly-OH (297 mg, 1 mmol, 10 equiv.) and HCTU (372 mg, 0.90 mmol, 9 equiv.) were dissolved in NMP (2 mL) prior to addition of iPr 2 NEt (348 µL, 2 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Then, Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. Fmoc-Cys(StBu)-OH (432 mg, 1 mmol, 10 equiv.) and HCTU (372 mg, 0.90 mmol, 9 equiv.) were dissolved in NMP (10 mL) prior to addition of iPr 2 NEt (348 µL, 2 mmol, 20 equiv.). The resulting solution was immediately added to the resin. The syringe was left for 1.5 h under gentle stirring, then the syringe was drained and the resin was washed with NMP. Fmoc group was removed by three successive treatments with 20% piperidine in NMP (10 mL) for 3 min. The N-2-hydroxy-5-nitrobenzyl group was then introduced following the general procedure (p S5). Then, the complete MT7  sequence was installed on a quarter of the resin trough standard Fmoc-SPPS (25 µmol, protocol S3). After cleavage of the resin (protocol p S3), the crude product 18 was pre-purified using a . For this, aliquots of 2 µL were diluted in 108 µL of 1.5% TFA in water and 100 µL were injected to the HPLC.After 24h, the reaction mixture was acidified with TFA (30 µL) and 2 mL of 1.5% TFA in water; this solution was extracted with diethylether (4 × 10 mL) to remove the MPAA. The ligation product precipitated; after centrifugation and removal of the supernatant, the solid was diluted in 400 µL of solvent B and 1.2 mL of solvent A (V total = 1.6 mL); 45 mg of TCEP were added (final 100 mM) and the pH was adjusted to 4.5, after 20 min the pH was adjusted to 1 and the ligation product SXX was purified by semi-preparative RP-HPLC (Nucleosil, gradient: 50-60 % B/A over 10 min). 130 nmol were obtained (yield = 18.4 %, UV titration at 280 nm, molar extinction coefficient = 21320 L.mol -1 .cm −1 ).