Traceless β-mercaptan-assisted activation of valinyl benzimidazolinones in peptide ligations† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc04148a

An internal activation strategy-enabled traceless ligation at sterically hindered Val-Xaa site is accomplished under thiol additive-free conditions assisted by a β-mercaptan on the C-terminal valine residue.

Analytical thin layer chromatography was performed using Merck TLC silica gel 60-F254 glass plates. Flash chromatography was performed using 200-300 mesh silica gel (Qingdao Haiyang Chemical Co., Ltd.). Filtration for crude peptide was performed using a Bulk GHP Acrodisc ® 13 mm syringe filter with 0.22 μm GHP membrane. Ultrapure argon (≥99.999%) was used in all ligation and desulfurization reactions. Yields refer to chromatographically and spectroscopically pure materials unless otherwise stated.
1 H NMR spectra were recorded on Bruker Avance III 400 MHz at ambient temperature using CDCl3 as solvent unless otherwise stated, referenced to TMS or residual solvent. 13 C NMR spectra were recorded at 100.0 MHz at ambient temperature using CDCl3 as solvent unless otherwise stated. Chemical shifts are reported in parts per million relative to CDCl3( 1 H, δ 7.26; 13 C, δ 77.0). Data for 1 H NMR are reported as follows: chemical shift, integration, multiplicity (ovrlp = overlapping, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) and coupling constants (J Hz). All 13 C NMR spectra were recorded as chemical shift (δ). Infrared spectra were recorded on a Nicolet Nexus 670 FT-IR spectrophotometer. High-resolution mass spectra were obtained in the Chemical Instrumentation Center, Peking University Health Science Center using a Waters Q-TOF mass spectrometer (Xevo G2 Q-TOF). Low-resolution mass spectra analyses were performed with a Waters SQD mass spectrometer (Alliance e2695-SQD). Melting

Synthesis of the N-9-fluorenylmethoxycarbonyl-methylthio-L-penicillamine (2)
To a solution of penicillamine (300 mg, 2.0 mmol) in 15 mL of degassed EtOH was added methyl methanethiosulfonate (MMTS, 0.2 mL, 2.0 mmol) dropwise. The mixture was stirred at room temperature for 4 h. Upon the full consumption of penicillamine, the reaction was concentrated in vacuo to afford compound S1 without further purification.
A solution of compound S3 S2 (160 mg, 1.0 equiv) in methanol (3.7 mL) was treated with 1N NaOH solution (5.5 ml, 5.0 equiv) at 0˚C for 10 min. The reaction mixture was carefully neutralized using 1N HCl and water at 0˚C. The resulting mixture was extracted with ethyl acetate (×3). The combined extracts were washed with water and brine, dried with MgSO4 and concentrated in vacuo. The obtained crude intermediate was used directly in next step without purification, which was mixed with S2 (270 mg, S1 H. T. Pham, N.-L. T. Nguyen, F. Duus, T. X. T. Luu, Phosphorus, Sulfur, and Silicon and the Related Elements 2015, 190, 1934-1941. S2 K. K. Pasunooti, R. Yang, B. Banerjee, T. Yap, C. F. Liu, Org. Lett. 2016, 18, 2696-2699 3.5 equiv) in anhydrous CH2Cl2 (3 mL), followed by the addition of TEA (80 µL, 1.2 equiv) at 0 ˚C. The reaction mixture was stirred at room temperature overnight, then quenched with 1N HCl and water at 0˚C. The resulting mixture was extracted with ethyl acetate (×3). The combined extracts were washed with water and brine, dried with MgSO4 and concentrated in vacuo to afford crude compound S4, which was treated with TFA/DCM = 9:1 (v/v) solution at room temperature for 2 hours. The solvent was removed under a nitrogen atmosphere. The residue was co-evaporated with toluene, and dissolved in 3 mL dioxane/H2O (1:1, v/v), followed by the addition of Boc2O (216 µL, 2.0 equiv) and TEA (80 µL, 1.3 equiv) at 0 ˚C. After stirring at room temperature for 24 hours, the reaction was quenched with 1N HCl and water at 0 ˚C. The resulting mixture was extracted with ethyl acetate (×3). The combined extracts were washed with water and brine, dried with MgSO4 and concentrated in vacuo. The crude residue was purified using silica gel column chromatography eluting with petroleum ether/EtOAc

Pre-load an amino acid to 2-chlorotritylchloride resin
The first Fmoc-amino acid residue was loaded to 2-chlorotritylchloride resin before Fmoc-SPPS following the general procedure below.

Determination of resin loading S4
Dry Fmoc amino-acid resin (approx. 5 μmol with respect to Fmoc) was weighted into a clean test tube, followed by the addition of 2 mL of 2% DBU in DMF. The mixture was agitated gently for 30 min, and then diluted to 10 mL with CH3CN. 2 mL of the resulting solution was taken out and diluted to 25 mL in a 50 mL centrifuge tube as the test solution. A reference solution was prepared in the same manner without the addition of resin.
Two matched silica UV cells were filled with reference solution to blank the U.V.
spectrophotometer. The solution in one of the silica UV cells was changed to the test S3 J. B. Blanco-Canosa, P. E. Dawson, Angew. Chem. Int. Ed. 2008, 47, 6851-6855. S4 Peptide Synthesis, 2010/2011 solution after washing with the test solution for three times. The optical density at 304 nm was recorded for three times and the average value was calculated as Abssample. The Fmoc loading of resin could be calculated using the equation below: Fmoc loading: mmol/g = Abssample ×16.4/(mg of resin).

Automated solid-phase peptide synthesis
Automated peptide synthesis was performed on a Pioneer peptide synthesis system (GEN600611) or a CS Bio peptide synthesizer (CX136XT).
The employed 2-chlorotritylchloride resin (1.147 mmol/g) employed in SPPS was purchased from GL Biochem, and Rink MBHA resin (0.42 mmol/g) was purchased from CS Bio. S12

Preparation of peptidyl acids and peptidyl amides
Upon completion of the automated synthesis on a 0.05 mmol scale, the peptide resin was washed into a peptide synthesis vessel using DCM. Resin cleavage and global deprotection was performed under the treatment of TFA/H2O/TIPS (95:2.5:2.5, v/v/v) solution for 2 hours. The resin was then removed by filtration, and the filtrate was concentrated under a nitrogen atmosphere. The residue was washed with cold diethyl ether to give a white solid, which was then dissolved in a mixture of acetonitrile and water containing 5% of acetic acid. The resulting solution was ready for HPLC purification after filtration. Utilization of 2-chlorotrityl resin and Rink-MBHA resin afforded the peptidyl acids and peptidyl amides respectively.
For side-chain protected peptide, the resin after SPPS was treated with DCM/TFE/AcOH (3:1:1, v/v/v) solution 40 min (×3), the combined cleavage solution was concentrated under reduced pressure, and lyophilized to remove the residual acid.

Preparation of C-terminal Pen-Nbz containing peptides
Fmoc-Pen(SMe)-OH (2) (5.0 equiv) was coupled to pre-loaded Dbz-Rink-MBHA resin (1.0 equiv, loading = 0.307 g/mol) manually using DIC (5.0 equiv) and oxyma pure ( solution for 2 hours. The resin was then removed by filtration, and the filtrate was concentrated under a nitrogen atmosphere. The residue was washed with cold diethyl ether to give a white solid, which was then dissolved in a mixture of acetonitrile and water containing 5% of acetic acid. The resulting solution was ready for HPLC S13 purification after filtration.

Native Chemical Ligation
To a mixture of the west-side peptide (1.0 equiv), and east-side peptide containing thiolamino acid at the N-terminus (1.2 equiv), was added appropriate volume of ligation buffer (6 M Gn·HCl, 200 mM Na2HPO4, 20 mM TCEP·HCl, pH 7.0) under an argon atmosphere, the concentration of the west-side peptides is approximately 3 mM, and the resulting solution was stirred at room temperature (25 °C) and monitored using LC-MS. The reaction was quenched with H2O/MeCN/AcOH (90:5:5, v/v/v) and purified using HPLC.

Metal-free Desulfurization
To a solution of the thiol-containing peptide (3 mM

One-pot Ligation-Desulfurization
Upon completion of the ligation as indicated by LC-MS analysis, to the reaction vessel was added proper volume of 0.5 M Bond-breaker ® TCEP solution (Pierce), 20 μL of 2methyl-2-propanethiol and 100 μL of radical initiator VA-044 (0.1 M in degassed water).
The reaction mixture was stirred at 37 °C and monitored by LC-MS. Upon completion, the reaction was quenched by adding H2O/MeCN/ AcOH (90:5:5, v/v/v) and further purified using HPLC. S14

Peptide 8c
Peptide 8c was prepared according to General Procedure 3.3 using Pioneer peptide S20 synthesizer on a 0.05 mmol scale. Purification of the crude peptide using preparative HPLC (10 to 30% solvent B over 30min, Agilent Eclipse XDB-C18 column) afforded peptide 8c as a white solid after lyophilization (11.2 mg, 41%

Peptide 8d
Peptide 8d was prepared according to General Procedure 3.3 using Pioneer peptide synthesizer on a 0.05 mmol scale. Purification of the crude peptide using preparative HPLC (10 to 30% solvent B over 30min, Agilent Eclipse XDB-C18 column) afforded peptide 8d as a white solid after lyophilization (6.1 mg, 11%). Analytical HPLC: tR =

Peptidyl Pen-Nbz 7h
Peptide 7h was prepared according to General Procedure 3.4 using CS Bio peptide synthesizer on a 0.03 mmol scale. Purification of the crude peptide using preparative HPLC (25 to 35% solvent B over 30min, Proto-300 C4 column) afforded peptide 7h as
The filtrate was concentrated under an argon atmosphere, and lyophilized to remove the residual acid. To the obtained powder (1.0 equiv) was added leucine thioester (2.0 equiv) and HATU (2.0 equiv), and the mixture was dissolved in anhydrous DCM, followed by the addition of DIEA (4.0 equiv) at 0 °C. The reaction mixture was stirred at room temperature for 30 min. After removal of the solvent under a nitrogen atmosphere, the resulting residue was treated with a solution of TFA/H2O/TIPS (95:2.5:2.5, v/v/v) for 2 hours, and then concentrated under a nitrogen atmosphere. The residue was washed with cold diethyl ether to afford a white solid, which was then dissolved in a mixture of acetonitrile and water containing 5% of acetic acid. The resulting solution was ready for HPLC purification after filtration. Purification of the crude peptide using preparative HPLC (15 to 30% solvent B over 30min, Proto-300 C4 column) afforded peptide 13 as a white solid after lyophilization (23.6 mg, 10%).

Peptide 15b
Peptide 15b was prepared according to General Procedure 3.3 using CS Bio peptide synthesizer on a 0.05 mmol scale, and the N-terminal thio-valine derivative was coupled manually using Boc-Val( γ SSMe)-OH S2 (1.5 equiv) and HATU (1.5 equiv) for 30 min
The filtrate was concentrated under a nitrogen atmosphere, and lyophilized to remove the residual acid. To a solution of the obtained powder (1.0 equiv) and 6-Chlorobenzotriazole-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyClock, 3.0 equiv) in anhydrous DMF, ethyl 3-mercaptopropionate (30 equiv) was added followed by the addition of DIEA (5.0 equiv) at -20 °C. The reaction mixture was stirred at the same temperature for 2 hours. S6 After removal of the solvent under lyophilization, the resulting residue was treated with a solution of TFA/ H2O/TIPS (95:2.5:2.5,v/v/v) for 2 hours, and then concentrated under a nitrogen atmosphere. The residue was washed with cold diethyl ether to afford a white solid, which was then dissolved in a mixture of acetonitrile and water containing 5% of acetic acid. The resulting solution was ready for HPLC purification after filtration. Purification of the crude peptide using preparative HPLC (20 to 35% solvent B over 30min, Proto-300 C4 column) afforded peptide 19 as a white solid after lyophilization (61.5 mg, 22%).

Ligation reactions between S6 and 8a
Peptidyl Val-Nbz S6 (1.0 equiv) and cysteinyl peptide 8a (1.2 equiv) were subjected to the ligation conditions following General Procedure 3.5 as described previously. The reaction was stirred at room temperature for 8 hours, and the progress was monitored with LC-MS. As shown in Figure S24, a complex mixture was generated with low conversion to the ligation product after 8 h.

Ligation reactions between S7 and 8a
Peptidyl Cys-Nbz S7 (1.0 equiv) and cysteinyl peptide 8a (1.2 equiv) were subjected to the ligation conditions following General Procedure 3.5 as described previously. The reaction was stirred at room temperature for 2 hours, and the progress was monitored with LC-MS. As shown in Figure S25, large amount of S7 decomposed after 2 hours, and only trace amount of ligation product was generated. Figure S25. UV trace of the ligation reaction between peptides S7 and 8a.

Ligation reactions between 7a and S8
Peptidyl Pen-Nbz 7a (1.0 equiv) and alanyl peptide S8 (1.2 equiv) were subjected to the ligation conditions following General Procedure 3.5 as described previously. The reaction was stirred at room temperature for 8 hours, and the progress was monitored with LC-MS. As shown in Figure S26, large amount of byproduct was generated and trace amount of ligation product was observed.

One-pot ligation and desulfurization reactions between 7a and 8a
0.70 mg of peptidyl Nbz 7a and 0.81 mg of cysteinyl peptide 8a were subjected to the ligation conditions following General Procedure 3.5 as described previously. The reaction was stirred for 2 h at room temperature, followed by the addition of Bond-

One-pot ligation and desulfurization reactions between 7b and 8b
0.70 mg of peptidyl Nbz 7b and 0.60 mg of cysteinyl peptide 8b were subjected to the ligation conditions following General Procedure 3.5 as described previously. The reaction was stirred for 2 h at room temperature, followed by the addition of Bond-

One-pot ligation and desulfurization reactions between 7c and 8c
0.95 mg of peptidyl Nbz 7c and 0.73 mg of cysteinyl peptide 8c were subjected to the ligation conditions following General Procedure 3.5 as described previously. The reaction was stirred for 2 h at room temperature, followed by the addition of Bond-

One-pot ligation and desulfurization reactions between 7d and 8d
0.42 mg of peptidyl Nbz 7d and 1.37 mg of cysteinyl peptide 8d were subjected to the ligation conditions following General Procedure 3.5 as described previously. The reaction was stirred for 2 h at room temperature, followed by the addition of Bond-

One-pot ligation and desulfurization reactions between 7a and 8e
0.60 mg of peptidyl Nbz 7a and 0.64 mg of cysteinyl peptide 8e were subjected to the ligation conditions following General Procedure 3.5 as described previously. The reaction was stirred for 2 h at room temperature, followed by the addition of Bond-

One-pot ligation and desulfurization reactions between 7a and 8f
0.68 mg of peptidyl Nbz 7a and 0.9 mg of cysteinyl peptide 8f were subjected to the ligation conditions following General Procedure 3.5 as described previously. The reaction was stirred for 2 h at room temperature, followed by the addition of Bondbreaker ® TCEP solution, t BuSH and VA -044 following General Procedure 3.7. The reaction was stirred for another 3 h at 37 °C under an argon atmosphere, and quenched with 1.0 mL of CH3CN/H2O/AcOH (5/90/5) solution. The resulting mixture was subjected to LC-MS analysis, and purified using preparative HPLC (10-

One-pot ligation and desulfurization reactions between 7a and 8g
0.50 mg of peptidyl Nbz 7a and 0.48 mg of cysteinyl peptide 8g were subjected to the ligation conditions following General Procedure 3.5 as described previously. The reaction was stirred for 2 h at room temperature, followed by the addition of Bondbreaker ® TCEP solution, t BuSH and VA-044 following General Procedure 3.7. The reaction was stirred for another 3 h at 37 °C under an argon atmosphere, and quenched with 1.0 mL of CH3CN/H2O/AcOH (5/90/5) solution. The resulting mixture was subjected to LC-MS analysis, and purified using preparative HPLC (10-30% solvent B over 30 min, Agilent Eclipse XDB-C18 column). The fractions containing pure peptide was concentrated affording 12f (0.14 mg, 18%) as a white solid.

One-pot ligation and desulfurization reactions between 7d and S9
0.83 mg of peptidyl Nbz 7d and 0.70 mg of cysteinyl peptide S9 were subjected to the ligation conditions following General Procedure 3.5 as described previously. The reaction was stirred for 8 h at room temperature, followed by the addition of Bondbreaker ® TCEP solution, t BuSH and VA-044 following General Procedure 3.7. The S40 reaction was stirred for another 3 h at 37 °C under an argon atmosphere, and quenched with 1.0 mL of CH3CN/H2O/AcOH (5/90/5) solution. As shown in Figure S35, only trace amount of ligation and desulfurized products were observed, which failed to afford isolable amount of product after preparative HPLC purification.

One-pot ligation and desulfurization reactions between 7h and 8h
3.9 mg of peptidyl Nbz 7h (1.0 equiv)and 4.7 mg of cysteinyl peptide 8h (1.5 equiv) were subjected to the ligation buffer (6 M Gn·HCl, 200 mM Na2HPO4, 50 mM TCEP·HCl, pH 6.9) under an argon atmosphere, the concentration of the 7h is approximately 5 mM, and the resulting solution was stirred at room temperature for 8 hours, followed by the addition of Bond-breaker ® TCEP solution, t BuSH and VA-044 following General Procedure 3.7. The reaction was stirred for another 8 h at 37 °C under an argon atmosphere, and quenched with 1.0 mL of CH3CN/H2O/AcOH (5/90/5) solution. The resulting mixture was subjected to LC-MS analysis, and purified using preparative HPLC (15-30% solvent B over 30 min, Beim Brueckle C4 column). The fractions containing pure peptide was concentrated affording 12h (3.5 mg, 55%) as a white solid. Analytical HPLC for 12h: tR = 24.7 min (15 to 35% solvent B over 30min,

Desulfurization of 16
Purified peptide 16 was subjected to desulfurization conditions following General Procedure 3.6 as described previously. The reaction mixture was allowed to stirred at 37 °C for 8 hours, and quenched with 1.0 mL of CH3CN/H2O/AcOH (5/90/5) solution.

One-pot three segment ligation for the preparation of peptide 22
Peptidyl Pen-Nbz 18 (6.8 mg, 1.0 equiv) and peptidyl thioester 19 (5.0 mg, 1.2 equiv) were subjected to the ligation buffer (6 M Gn·HCl, 100 mM Na2HPO4, 100 mM sodium ascorbate, 50 mM TCEP·HCl, pH 7.5) under an argon atmosphere, and the resulting solution was stirred overnight at room temperature. To the reaction mixture was then added a solution of 21 (5.9 mg, 1.4 equiv) in buffer containing MPAA. The reaction was stirred for another 8 hours at room temperature, and quenched with 1.0 mL of CH3CN/H2O/AcOH (5/90/5) solution. The resulting mixture was subjected to LC-MS analysis, and purified using preparative HPLC (27-

Desulfurization of 22
Peptide 22 was subjected to desulfurization conditions following General Procedure 3.6 as described previously. The reaction mixture was allowed to stirred at 37 °C for 8 hours, and quenched with 1.0 mL of CH3CN/H2O/AcOH (5/90/5) solution. The resulting mixture was subjected to LC-MS analysis, and purified using preparative HPLC (27-38% solvent B over 30 min, Proto 300-C4 column). The fractions containing pure peptide was concentrated affording 23 (5.0 mg, 35%, 3 steps) as a white solid.