Synthesis and application of a new cleavable linker for "click"-based affinity chromatography Felicetta Landi, Conny M. Johansson, Dominic J. Campopiano and Alison N. Hulme* Supporting Information 1 Chemical Synthesis S1 1.1 General methods S1 1.2 Linker synthesis S2 1.3 Solid support synthesis S4 1.4 Dithionite release S4 2 Preliminary Affinity Studies S5 2.1 Material and methods S5 2.2 Competitive capture and release of avidin S5 2.3 Capture and release of avidin from a protein mixture S5 3 Spectra S6 1 Chemical Synthesis 1.1 General methods All non-aqueous reactions were carried out under an atmosphere of nitrogen using oven-dried glassware that was cooled in a dessicator prior to use. Unless otherwise noted, starting materials and reagents were obtained from commercial suppliers and were used without further purification. Dichloromethane (DCM) and triethylamine (Et3N) were distilled from and stored over calcium hydride under a nitrogen atmosphere. Saturated aqueous solutions of inorganic salts are represented as (volume, sat. aq.). 1H and 13C NMR spectra were obtained on Bruker instuments at the stated frequency. Infra-red spectra were recorded using a Biorad FTS-7 or Perkin-Elmer Paragon 1000 FT-IR spectrometer as thin films or KBr discs. Electrospray (ESI) and fast atom bombardment (FAB) mass spectra were obtained on a Kratos MS50TC mass spectrometer. Melting points were determined on a Gallenkamp Electrothermal Melting Point apparatus and are uncorrected. Flash chromatography was carried out using Merck Kieselgel 60 (Merck 9385) under v positive pressure. Eluent compositions are quoted as /v ratios. - S1 - 1.2 Linker synthesis 4-(4’-Hydroxy-phenylazo)-benzoic acid 21 OH 4-aminobenzoic acid (1.23 g, 9.00 mmol), was dissolved in a mixture of N N hydrochloric acid (1.2 ml, conc) and water (24 ml) and the suspension obtained HO was cooled to 0 °C. Sodium nitrite (1.04 g, 15.0 mmol) dissolved in water (5 ml) O was added next and the pale yellow solution formed was stirred for 20 min. A second solution of phenol (667 mg, 7.20 mmol), sodium hydroxide (257 mg, 6.42 mmol) and potassium carbonate (1.33 g, 9.60 mmol) dissolved in water (20 ml) was prepared, cooled to 0 °C and added dropwise to the reaction mixture. The reaction was allowed to reach RT and stirred for 3 h. The brown suspension formed was acified with HCl (1 M, aq) and filtered. The filtrate was dissolved in MeOH, silica was added and the mixture dried in vacuo. Flash chromatography (dry loaded silica column, 15% MeOH in DCM) gave product 3 as an orange solid (1.57 g, 90% yield); Rf (5% MeOH in DCM = 0.25); mp 275-277 °C; νmax (neat)/cm-1 3286 (OH), 1642 (CO), 1538 (NN); 1H NMR δ (250 MHz, CD3OD) 8.19 (2H, d, J = 8.5 Hz, ArH), 7.92 (2H, d, J = 8.5 Hz, ArH), 7.90 (2H, d, J = 8.8 Hz, 13 ArH), 7.17 (2H, d, J = 8.8 Hz, ArH); C NMR δ (62.9 MHz, CD3OD) 169.23 (C), 162.88 (C), 156.84 (C), 147.58 (C), 133.00 (C), 131.81 (2 × CH), 126.46 (2 × CH), 123.21 (2 × CH), 116.87 (2 × CH); m/z (ESI−) 241 ([M−H]−, 48%), 226 (12), 112 (100). All spectroscopic data were in good agreement with the literature.1 (2-Aminoethyl)-carbamic acid tert-butyl ester O To a vigorously stirred solution of ethane-1,2-diamine (9.97 g, 167 mmol) in DCM (100 NH2 O N ml) at RT was added a solution of di-tertbutylcarbonate (6.10 g, 28.0 mmol) in DCM H (400 ml) over 5 h. The resulting solution was stirred for 24 h at RT and then concentrated in vacuo to obtain a colourless oil which was partitioned between Na2CO3 (20 ml, 2 M, aq) and DCM (20 ml). The aqueous layer was extracted with DCM (2 × 20 ml) and concentrated under reduced pressure to give the Boc-protected amine as a colourless oil (4.45 g, 99% yield); Rf (10% -1 1 MeOH in DCM) = 0.1; νmax (neat)/cm 3446 (NH), 1710 (CO); H NMR δ (360 MHz, CD3OD) 3.13 13 (2H, t, J = 6.2 Hz, CH2NHCO), 2.73 (2H, t, J = 6.2 Hz, CH2NH2), 1.44 (9H, s, 3 × CH3); C NMR δ (90.6 MHz, CD3OD), 158.68 (C), 80.18 (C), 43.26 (CH2), 42.20 (CH2), 28.79 (3 × CH3); m/z (ESI+) 183 ([M+Na]+, 8%), 161 ([M+H]+, 58). All spectroscopic data were in good agreement with the literature.2 2-[4’-(4’’-Hydroxy-phenylazo)-benzoylamino]-ethyl-carbamic acid tert-butyl ester OH To a solution of benzoic acid 2 (100 mg, 0.413 mmol), in DMF (3 ml), N O N EDC (94.0 mg, 0.500 mmol) and HOBt (27.0 mg, 0.200 mmol) were H N O N added. After stirring for 15 min N-Boc-ethylenediamine (131 mg, 0.800 H O mmol) was added and the reaction stirred for 48 h at RT. The solvent was removed in vacuo, the crude material was dissolved in DCM (2 ml) and the solution washed with HCl (5 ml, 1 M aq). The solvent was removed in vacuo. The crude product was dissolved in MeOH (2 ml), silica was added and the mixture dried in vacuo. Flash chromatography (dry loaded silica column, 2% MeOH in DCM) yielded the desired amide as an orange solid (94 mg, 60% yield). -1 Rf (5% MeOH in DCM) = 0.31; mp 169-171 °C; νmax (neat)/cm 3361 (NH), 1689 (CO), 1637 (CO), 1 1535 (NN); H NMR δ (250 Mz, (CD3)2CO) 8.06 (2H, d, J = 8.6 Hz, ArH), 7.90 (2H, d, J = 8.5 Hz, ArH), 7.89 (2H, d, J = 8.8 Hz, ArH), 7.04 (2H, d, J = 8.6 ArH), 6.47 (1H, br s NH), 3.73-3.66 (2H, m, 13 CH2), 3.55-3.48 (2H, m, CH2), 1.58 (9H, s, CH3); C NMR δ (62.9 MHz, (CD3)2CO) 167.11 (C), 162.20 (C), 161.66 (C), 155.17 (C), 147.27 (C), 137.13 (C), 129.18 (2 × CH), 126.15 (2 × CH), 123.03 (2 × CH), 116.91 (2 × CH), 79.02 (C), 41.63 (CH2), 41.06 (CH2), 28.71 (3 × CH3); m/z (ESI-) 383 ([M- - + H] , 18%), 134 (100), 113 (72); HRMS (ESI+) [M+NH4] found 402.2132, C20H28O4N5 requires 402.2136. 2-{4’’’-[4’’-(3’-Bromo-propoxy)-phenylazo]-benzoylamino}-ethyl-carbamic acid tert-butyl ester 3 O Br To a stirred solution of the phenol (800 mg, 2.08 mmol) in CH3CN N O N (30 ml) was added potassium carbonate (430 mg, 16.6 mmol) and H N O N the mixture was heated to 70 °C. 1,3-Dibromopropane (1.70 ml, H O 16.6 mmol) was added and the reaction stirred at reflux for 5 h. The solvent was removed in vacuo to give the crude bromide. Purification by flash chromatography (5% MeOH in DCM) yielded the desired bromide 3 (710 mg, 67% yield) as a yellow solid; Rf (5% -1 1 MeOH in DCM) = 0.58; mp 188-190 °C; νmax (neat)/cm 1700 (CO), 1659 (CO); H NMR δ (250 MHz, CDCl3) 7.98-7.88 (6H, m, ArH), 7.44 (1H, br s, NH), 7.26-7.21 (2H, m, ArH), 5.12 (1H, br s, NH), 4.20 (2H, t, J = 6.0 Hz, CH2), 3.64 (2H, t, J = 6.4 Hz, CH2), 3.60-3.55 (2H, m, CH2), 3.48-3.40 13 (2H, m, CH2), 2.37 (2H, qn, J = 6.0 Hz, CH2), 1.44 (9H, s, CH3); C NMR δ (62.9 MHz, CDCl3) 175.55 (C), 175.27 (C), 167.57 (C), 162.00 (C), 154.70 (C), 135.24 (C), 128.40 (2 × CH), 125.50 (2 × CH), 122.96 (2 × CH), 115.21 (2 × CH), 80.49 (C), 66.06 (CH2), 42.76 (CH2), 40.28 (CH2), 32.60 81 + 79 + (CH2), 30.14 (CH2), 28.75 (3 × CH3); m/z (ESI+) 529 ([ BrM+Na] , 38%), 527 ([ BrM+Na] , 36), - S2 - 507 ([81BrM+H]+, 2), 505 ([79BrM+H]+, 2); HRMS (FAB, 3-NOBA) [M+H]+ found 505.1461, 79 C23H30N4O4 Br requires 505.1450. 2-{4’’’-[4’’-(3’-Azido-propoxy)-phenylazo]-benzoylamino}-ethyl-carbamic acid tert-butyl ester 4 O N3 The Boc-protected bromide 3 (710 mg, 1.40 mmol) was dissolved N O N in DMF (15 ml) and sodium azide (273 mg, 4.20 mmol) was H N O N added. The solution was stirred overnight (~18 h) at RT. The H O solvent was removed in vacuo and the crude material dissolved in diethyl ether (20 ml), washed with brine (20 ml) and water (20 ml) to give the azide 4 (588 mg, 90% yield) as a pale yellow solid (90:10, E:Z); Rf (5% MeOH in DCM) = 0.58; mp 173-174 °C; νmax -1 1 (neat)/cm 3366 (NH), 3317 (NH), 2100 (N3), 1680 (CO), 1636 (CO); H NMR δ (250 MHz, CDCl3), 7.96-7.86 (5.40 H (E), m, ArH) 7.74 (0.20 H (Z), d, J = 8.5 Hz ArH), 7.35 (0.90 H (E), br s, NH), 7.07- 6.93 (1.80 H (E), m, ArH), 6.87 (0.40 H (Z), m, ArH), 6.71 (0.20 H (Z), d, J = 8.8 Hz, ArH), 5.00 (0.90 H (E), br s, NH), 4.13 (1.80 H (E), t, J = 6.0 Hz, CH2), 3.98 (0.20 H (Z), t, J = 6.0 Hz, CH2), 3.66-3.34 (6H, m, 2 × CH2NH+CH2N3), 2.14-1.94 (2H , m, CH2CH2CH2), 1.42 (8.10 H (E), s, 3 × CH3) 1.39 13 (0.90 H (Z), s, 3 × CH3); C NMR δ (62.9 MHz, CDCl3) 167.35 (C), 161.79 (C), 157.95 (C), 154.62 (C), 147.34 (C), 135.53 (C), 128.21 (2 × CH), 125.32 (2 × CH), 122.80 (2 × CH), 115.00 (2 × CH), 80.40 (C), 65.09 (CH2), 48.36 (CH2), 42.68 (CH2), 40.09 (CH2), 28.94 (CH2), 28.56 (3 × CH3); m/z (ESI+) 957 ([2M+Na]+, 27%), 490 ([M+Na]+, 100), 390 (52), 71 (43); HRMS (FAB, 3-NOBA) found + [M+H] 468.2351, C23H30N7O4 requires 468.2359.