Supporting Information Photolytic Cleavage of Leader Peptides


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Supporting Information
Photolytic Cleavage of Leader Peptides
Noah Bindman, Remco Merkx, Robert Koehler, Nicholas Herrman, and Wilfred A. van der Donk*
Howard Hughes Medical Institute, and Roger Adams Laboratory, Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue,
Urbana, IL, 61801

Table of Contents 1. Experimental details for synthesis and
characterization of compounds 14-17 2. Differential Scanning Calorimetry for compounds 14-17 3. Experimental details for peptides 18-23 4. Mass spectra of 21a and 23a 5. HPLC traces of 21a-i and 23a-i 6. Experimental details for lacticin analogs

S2 - S10 S11 – S13 S13 – S16 S17 S17 – S24 S24 – S29

Supplementary Material (ESI) for Chemical Communications S1
This journal is (c) The Royal Society of Chemistry 2010

Materials. Fmoc amino acids and resins were purchased from Advanced ChemTech, Novabiochem, or Chem-Impex. HOBt (1-hydroxybenzotriazole) was purchased from Chem-Impex, and DIC (N,N’ diisopropylcarbodiimide) and HCTU (O-(1H-6 chlorobenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate) were purchased from Advanced ChemTech. Solvents commonly used in peptide synthesis and purification, including dimethylformamide (DMF), dichloromethane (DCM) trifluoroacetic acid (TFA), and acetonitrile (MeCN) were obtained in HPLC grade or better and used directly without further purification. Tri-n-butylphosphine, piperidine, and N-methyl morpholine were purchased from Acros whereas pyridine, diisopropylethylamine (DIPEA), and β-mercaptoethanol were purchased from Sigma Aldrich. Tris-(2-carboxyethyl)phosphine (TCEP) was obtained from Molecular Probes as the TCEP-HCl salt. The ligand tris-(benzyltriazolylmethyl)amine (TBTA), tetrakis(MeCN)copper(I) hexafluorophosphate and hydrazine were purchased from Sigma Aldrich and 3-butyn-1-amine hydrochloride was purchased from AB Chem Inc. For the synthesis of the photocleavable linker, p-toluenesulfonyl chloride, trimethylsilyl chloride, sodium azide, and N-hydroxysuccinimidyl carbonate were purchased from Sigma Aldrich. The irradiation used for photochemical reactions was generated using a UVP Blak-Ray lamp (Ultraviolet Products, San Gabriel, CA) with a 365 nm filter.
Synthetic design of photolabile linker 17.
Experimental:
2-Hydroxy-2-(2-nitrophenyl)ethyl 4-methylbenzenesulfonate (14): Into a 50 mL round-bottomed flask was placed p-toluenesulfonyl chloride (1.46 g, 7.64 mmol, 1.4 equiv) and 1-(2-nitrophenyl)-1,2-ethanediol 13 (1.0 g, 5.46 mmol, 1 equiv). The flask was then sparged with N2 for five min. Distilled pyridine (25 mL) was added and the stirred reaction was placed in a 0 oC icebath. The mixture was stirred for 18 h at 0 oC, was allowed to warm to room temperature, and was then quenched with H2O (80 mL). Crude product was extracted with Et2O (2 × 50 mL) and combined extracts were washed successively with 1 M KHSO4 (3 × 30 mL), sat. aqueous NaHCO3 (3 × 30 mL) and sat. aqueous NaCl (2 × 50 mL). The organic layer was dried over MgSO4, concentrated by rotary evaporation, and then purified by SiO2 gel column
Supplementary Material (ESI) for Chemical Communications S2
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chromatography (eluted with hexanes/EtOAc (4:1); Rf = 0.065) yielding compound 14 (1.65 g, 90%) as an orange oil. 1H NMR: (399.947 MHz, CDCl3) δ=2.43 (s, 3H, CH3), 3.95 (bs, 1H, OH) 4.14 (dd, J = 7.3, 3.2 Hz, 1H, CH2), 4.36 (dd, J = 2.7, 7.8 Hz, 1H, CH2), 5.51 (dd, J = 2.7, 4.7 Hz, 1H, CH), 7.31 (d, J = 8.1 Hz, 2H, CHtosyl), 7.44 (t, J = 7.8 Hz, 1H, CHphenyl), 7.65 (t, J = 7.3 Hz, 1H, CHphenyl), 7.75 (d, J = 8.3 Hz, 2H, CHtosyl), 7.85 (d, J = 7.8 Hz, 1H, CHphenyl), 7.95 (d, J = 8.1 Hz, 1H, CHphenyl). 13C NMR: (100.527 MHz, CDCl3) δ=21.9, 67.9, 73.5, 124.9, 128.2, 129.4, 130.3, 132.5, 134.1, 134.4, 145.4, 147.7. HRMS [M+H]+ C15H16NO6S calc’d = 338.0698, found = 338.0704.
1H NMR of 14. Supplementary Material (ESI) for Chemical Communications S3
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13C NMR of 14.
2-(2-Nitrophenyl)-2-((trimethylsilyl)oxy)ethyl 4-methylbenzenesulfonate (15) Into a 50 mL round-bottomed flask was placed a solution of p-toluenesulfonyl derivative 14 (1.69 g, 5.0 mmol, 1 equiv). The flask was purged with a nitrogen stream for five min followed by the addition of distilled pyridine (25 mL). To this flask was added TMSCl (2.17 g, 20 mmol, 4 equiv) dropwise and the solution was stirred for 1 h at room temperature. At this time the reaction was quenched with H2O (80 mL), crude product was extracted with Et2O (2 × 50 mL), and the combined extracts were washed successively with 1 M KHSO4 (3 × 30 mL), sat. aqueous NaHCO3 (3 × 30 mL), and sat. aqueous NaCl (2 × 50 mL). The organic layer was dried over MgSO4 and concentrated by rotary evaporation, yielding compound 15 (1.84 g, 90%) as a tan solid.
Supplementary Material (ESI) for Chemical Communications S4
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1H NMR: (399.746 MHz, CDCl3) δ=0.01 (s, 9H, CH3), 2.41 (s, 3H, CH3), 4.05 (dd, J = 6.6, 4.4 Hz, 1H, CH2), 4.16 (dd, J = 2.9, 6.8 Hz, 1H, CH2), 5.47 (dd, J = 6.6, 6.6 Hz, 1H, CH), 7.27 (d, J = 8.1 Hz, 2H, CHtosyl), 7.43 (t, J = 7.8 Hz, 1H, CHphenyl), 7.61 (t, J = 7.7 Hz, 1H, CHphenyl), 7.70 (d, J = 8.3 Hz, 2H, CHtosyl), 7.79 (d, J = 7.9 Hz, 1H, CHphenyl), 7.89 (d, J = 8.3 Hz, 1H, CHphenyl). 13C NMR: (100.527 MHz, CDCl3) δ=-0.02, 21.8, 68.4, 73.7, 124.6, 128.1, 129.1, 129.8, 130.0, 133.1, 133.7, 135.6, 144.9, 147.8. HRMS [M+Na]+ C18H23N6NaSSi calc’d = 432.0913, found = 432.0917. M.P. = 83-87 oC.
1H NMR of 15. Supplementary Material (ESI) for Chemical Communications S5
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13C NMR of 15.
2-Azido-1-(2-nitrophenyl) ethanol (16): Into a 25 mL round-bottomed flask was placed sodium azide (0.52 g, 2.52 mmol, 2 equiv). The flask was purged with a nitrogen stream for five min. To this flask was added trimethylsilyl ether derivative 15 (0.52 g, 1.26 mmol, 1 equiv) in DMF (9 mL) and the reaction was stirred at 80 °C for 10 h. At this time the reaction was quenched with H2O (80 mL), crude product was extracted with Et2O (2 × 50 mL), and the combined extracts were washed with sat. aqueous NaCl (2 × 50 mL). The organic layer was dried with MgSO4, concentrated by rotary evaporation, and purified by SiO2 gel column chromatography (eluted with hexanes/EtOAc (4:1); Rf = 0.29) yielding 16 (0.23 g, 85%) as a red oil.
Supplementary Material (ESI) for Chemical Communications S6
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1H NMR: (399.947 MHz, CDCl3) δ=2.64 (d, J = 3.7 Hz, 1H, OH), 3.44 (dd, J = 7.6, 4.9 Hz, 1H, CH2), 3.74 (dd, J = 3.0, 9.5 Hz, 1H, CH2), 5.49 (m, 1H, CH), 7.47 (t, J = 7.8 Hz, 1H, CHphenyl), 7.69 (t, J = 8.0 Hz, 1H, CHphenyl), 7.90 (d, J = 7.8 Hz, 1H, CHphenyl), 8.00 (d, J = 8.3 Hz, 1H, CHphenyl). 13C NMR: (100.527 MHz, CDCl3) δ=57.4, 69.2, 125.0, 128.8, 129.2, 134.1, 136.3, 147.9. HRMS [M + Na]+ C8H8N4O3Na calc’d = 231.0494, found = 231.0483.
1H NMR of 16. Supplementary Material (ESI) for Chemical Communications S7
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13C NMR of 16.
2-Azido-1-(2-nitrophenyl)ethyl (2,5-dioxopyrrolidin-1-yl) carbonate (17): Into a 10 mL round-bottomed flask was placed a solution of azide derivative 16 (41.6 mg, 0.2 mmol, 1 equiv) in DMF (1.0 mL) and N-hydroxysuccinimidyl carbonate (85.0 mg, 0.33 mmol, 1.65 equiv) was added followed by NEt3 (83.6 µL, 0.60 mmol, 3 equiv). The reaction was stirred at room temperature for 16 h. At this time, solvent was removed by rotary evaporation and crude product was purified by SiO2 gel column chromatography (eluted with hexanes/EtOAc (1:1); Rf = 0.45). Fractions were collected and concentrated by rotary evaporation yielding 17 (55.9 mg, 80% yield) as a white solid.
1H NMR: (499.947 MHz, CDCl3) δ=2.80 (s, 4H, CH2), 3.76 (dd, J = 6.4, 7.1 Hz, 1H, CH2), 3.93 (dd, J = 3.0, 10.7 Hz, 1H, CH2), 6.46 (dd, J = 3.0, 3.7 Hz, 1H, CH), 7.57 (m, J = 4.3 Hz, 1H, CHphenyl), 7.77 (d, J = 4.1 Hz, 2H, CHphenyl) 8.11 (d, J = 8.3 Hz, 1H, CHphenyl).
Supplementary Material (ESI) for Chemical Communications S8
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13C NMR: (100.578 MHz, CDCl3) δ=25.6, 54.5, 77.8, 125.5, 128.3, 130.4, 131.2, 134.7, 147.5, 150.9, 168.4. HRMS [M+H]+ C13H12N5O7 calc’d = 350.0737, found = 350.0727. M.P. = 110-115 oC.
1H NMR of 17. Supplementary Material (ESI) for Chemical Communications S9
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13C NMR of 17.
Differential Scanning Calorimetry (DSC) of compounds 14-17. Because of the large ratio of oxygen and nitrogen atoms to carbon atoms, compounds 1417 were analyzed by DSC to determine whether they qualify as Class 5 explosives. Each compound was tested in comparison to benzoyl peroxide (BPO) as standard reference material.1 Briefly, if the heat quantities of the decomposition reactions of compounds 1417 were found to be lower than the heat quantity of decomposition of BPO then the compound was judged not to be a Class 5 explosive.

Supplementary Material (ESI) for Chemical Communications This journal is (c) The Royal Society of Chemistry 2010

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Supporting Information Photolytic Cleavage of Leader Peptides