Chemistry & Biology 16

Supplemental Data

Discovery of TNF Inhibitors

from a DNA-Encoded Chemical Library

based on Diels-Alder Cycloaddition Fabian Buller, Yixin Zhang, Jörg Scheuermann, Juliane Schäfer, Peter Bühlmann, and Dario Neri

SUPPLEMENTAL EXPERIMENTAL PROCEDURES Synthesis of Maleimide Derivatives Maleimides were synthesized starting from the corresponding amine derivatives according to the general procedure: The primary amine (50 mg) was dissolved in glacial acetic acid (8 mL) and 5-fold molar excess of maleic anhydride was added. The mixture was refluxed overnight at 120°C under argon atmosphere. The solvent was removed under vacuum and the product was HPLC purified and ESI-MS characterized. 4-(N-maleimidomethyl)benzimidamide (maleimide 6) was synthesized starting from 4-(aminomethyl)benzimidamide (Melkko et al., 2007): ([M+H]+=229.8; Mcalc=229.1). 4-(maleimido)benzoic acid (maleimide 30) was prepared from 4-(amino)benzoic acid: ([M-H]-=215.9; Mcalc=217.0). 2-maleimido-5-chloro-2'-fluorobenzophenone (maleimide 69) was prepared from 2-Amino-5-chloro-2'-fluorobenzophenone: (([M+H]+=329.7; Mcalc=329.0). N-(2,3-dihydro-1H-inden-5-yl)-maleimide (maleimide 70) was prepared from 2,3-dihydro-1H-inden-5-amine: ([M+H]+=213.8; Mcalc=213.1). N-(2-dimethylamino-2-phenylethyl)-maleimide (maleimide 74) was prepared from N1, N1-dimethyl-1-phenylethane-1,2-diamine: ([M+H]+=244.9; Mcalc=244.1). (R)-2-(maleimido)-3-(4-hydroxyphenyl)propanoic acid (maleimide 79) was prepared from D-tyrosine: ([M-H]-=260.0; Mcalc=261.1). N-(1-methylpiperidin-4-yl)-maleimide (maleimide 86) was prepared from 1-methylpiperidin-4-amine: ([M+H]+=194.8; Mcalc=194.1). N-(3,4-dichlorophenyl)maleimide (maleimide 110) was prepared from 3,4-dichloroaniline. 1,2-Diphenylethylmaleimide (maleimide 171) was prepared from 1,2-Diphenylethylamine: ([M+Na]+=299.8; Mcalc=277.1). 2-maleimido-6-(4-(4-iodophenyl)butanamido)hexanoic acid (maleimide 199) was prepared from 2-amino-6-(4-(4-iodophenyl)butanamido)hexanoic acid: ([M-H]-=496.9; Mcalc=498.1) (Dumelin et al., 2008). 2-maleimido-2',5-dichlorobenzophenone was prepared from 2-Amino-2',5-dichlorobenzophenone ([M+H]+=345.8; Mcalc=345.0). N-(2-(maleimido)ethyl)-6-(5-methyl-2-oxoimidazolidin-4-yl)hexyl amide (maleimide 200) was synthesized from D-desthiobiotin and N-(2-Aminoethyl)maleimide trifluoroacetate salt as described previously ([M+H]+=336.9; Mcalc=336.2) (Buller et al., 2008). Synthesis of Triethyleneglycol-fluorescein-conjugates carrying a 2,4-hexadiene Moiety Triethyleneglycol-fluorescein-conjugates carrying a 2,4-hexadiene moiety were prepared from amino-triethyleneglycol-fluorescein and 2,4-hexadiene derivatives of carboxylic acids: Amino-triethyleneglycol-fluorescein was synthesized from 5-carboxy-fluorescein: 5-carboxy-fluorescein (200 mg, Carbogen AMCIS AG, Bubendorf, Switzerland) was dissolved in dry DMF (10 mL). N-Hydroxysuccinimide (NHS, 67.2 mg, 1.1eq) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl, 111.9 mg, 1.1eq) were added and the mixture was stirred in the dark for 4h at 30°C. Next, 4,7,10-Trioxa-1,13-tridecanediamine (585 mg, 5eq), TEA (2.2 mL) and water (2 mL) were added and the mixture was stirred overnight at 30°C. The solvent was removed under vacuum and the product was HPLC purified ([M+H]+=579.1; Mcalc=578.2). Amino-triethyleneglycol-fluorescein was then coupled to the carboxylic acid moiety of the diene-derivatives used for library construction (Buller et al., 2008). In general, the corresponding diene-derivatives (28.5 µmol, dissolved in 285 µL DMSO) were activated with EDC-HCl (6 mg, 31 µmol) and N-hydroxysulfosuccinimide (7.6 mg, 35 µmol) for 3h at 30°C. Next, Amino-triethyleneglycol-fluorescein (23 µmol, in DMSO/H2O) and TEA (39 µL) were added and the mixture was stirred overnight at 30°C. Subsequently the reaction was quenched with Tris and the product was HPLC purified and ESI-MS characterized: Fluorescein-diene 1 ([M+H]+=919.5; Mcalc=918.4); Fluorescein-diene 3 ([M+H]+=842.4; Mcalc=841.4); Fluorescein-diene 5 ([M+H]+=840.4; Mcalc=839.4); Fluorescein-diene 6 ([M+H]+=924.5; Mcalc=923.4); Fluorescein-diene 7 ([M+H]+=874.4; Mcalc=873.4); Fluorescein-diene 9 ([M+H]+=876.4; Mcalc=875.4); Fluorescein-diene 10 ([M+H]+=910.4; Mcalc=909.4); Fluorescein-diene 12 ([M+H]+=855.4; Mcalc=854.4); Fluorescein-diene 19 ([M+H]+=966.3; Mcalc=965.4); Fluorescein-diene 20 was prepared from N-(Boc)-O-(hexa-2(E),4(E)-dienyl)-L-4-hydroxyproline (31 mg, 100 µmol) (Buller et al., 2008). The carboxylic acid

moiety of the proline-diene derivative was activated in DMF using EDC-HCl (21 mg, 110 µmol) and NHS (13 mg, 110 µmol) for 3h at room temperature, and amino-triethyleneglycol-fluorescein (58 mg, 100 µmol) and triethylamine (69 µL) were added. The mixture was stirred overnight at room temperature. After removal of the volatile components under vacuum, a solution of 1 M ZnCl2 in Et2O (10eq) was added, and the mixture was stirred under an argon atmosphere for 16h at RT. The solvent was removed under vacuum, the product was dissolved in acetonitrile and the precipitate was removed by filtration. The product was subsequently HPLC purified and ESI-MS characterized ([M+H]+=772.3; Mcalc=771.3). Synthesis of Triethyleneglycol-fluorescein-conjugates by Diels-Alder Cycloadditions In general, fluorescein-conjugates for polarization measurements were prepared by the Diels-Alder cycloaddition of maleimides to fluorescein-dienes. In the Diels-Alder reaction, fluorescein-diene derivatives (see above, 2 µmol) were mixed with maleimide derivatives (see above, 3 µmol) in 1:1 DMSO:H2O containing 0.05 % trifluoroacetic acid. The mixture was stirred overnight at 37°C before HPLC purification using a linear gradient from 0% to 100% MeCN with 0.1% TFA. The purity of the final compounds was confirmed by the UV trace (260 nm/450 nm) on an analytical HPLC using a XTerra RP18 column (5 µm, 10x150 mm) with a gradient from 0% to 80% MeCN with 100 mM TEAA/H2O, pH 7. ESI-MS analysis of the fluorescein-conjugates yielded: 1-199: [M+H]+=1417.6; Mcalc=1416.5; 1-200: [M+H]+=1255.7 Mcalc=1254.6; 3-69: [M+H]+=1171.5; Mcalc=1170.4; 3-74: [M+H]+=1086.6; Mcalc=1085.5; 3-200: [M+H]+=1178.7; Mcalc=1177.6; 5-69: [M+H]+=1169.5; Mcalc=1168.4; 5-110: [M+H]+=1080.4; Mcalc=1081.3; 5-171: [M+H]+=1116.5; Mcalc=1117.5; 5-200: [M+H]+=1176.7; Mcalc=1175.6; 6-79: [M+H]+=1185.5; Mcalc=1184.5; 6-86: [M+H]+=1118.6; Mcalc=1117.5; 6-199: [M+H]+=1422.6; Mcalc=1421.5; 7-70: [M+H]+=1087.8; Mcalc=1086.5; 9-200: [M+H]+=1212.6; Mcalc=1211.6; 10-171: [M+H]+=1087.8; Mcalc=1186.5; 12-69: [M+H]+=1184.5; Mcalc=1183.4; 19-30: [M+H]+=1183.5; Mcalc=1182.4; 20-74: [M+H]+=1016.5; Mcalc=1015.4. 1a: [M+H]+=1185.4; Mcalc=1184.4; 1b: [M+H]+=1269.4; Mcalc=1268.4. All fluorescein-conjugates were dissolved in dry DMSO. The concentration was determined by measuring absorbance at 495 nm (ε495nm, PBS, pH 7.4= 67760). Synthesis of Trypsin Inhibitors The trypsin inhbitors were derived from 4-(N-maleimidomethyl)benzimidamide by Diels-Alder cycloaddition reactions with the corresponding diene derivatives used for library construction (Buller et al., 2008). 4-(N-maleimidomethyl)benzimidamide (5 mg, 22 µmol) was incubated with equimolar amounts of the diene derivative in 400 µL DMSO/H2O (1:1) containing 0.05 % trifluoroacetic acid overnight at 37°C. The cycloadducts were HPLC purified and ESI-MS analyzed: 1-6: [M+H]+=588.2; Mcalc=587.3; 3-6: [M+H]+=511.0; Mcalc=510.2; 6-6: [M+H]+=593.2; Mcalc=592.3; 12-6: [M+H]+ =524.1; Mcalc=523.2; 19-6: [M+H]+=635.2; Mcalc=634.3. The compound concentrations were determined by NMR, using nitromethane as an internal standard. Synthesis of TNF ligands (2a-c) 2-Amino-2',5-dichlorobenzophenone (69.3 mg, 1eq) was dissolved in glacial acetic acid. After addition of trimellitic anhydride (50.0 mg, 1eq) the mixture was refluxed overnight yielding 2-(4-chloro-2-(2-chlorobenzoyl)phenyl)-1,3-dioxoisoindoline-5-carboxylic acid (2a) which was HPLC purified ([M-H]-=437.8; Mcalc=439.0). Compound 2a (4.8 mg, 1eq) was activated in DMF using EDC-HCl (2.1 mg, 1eq) and NHS (1.5 mg, 1.2 eq) for 3h at room temperature. Next, L-tryrosine (6.0 mg, 3 eq) and triethylamine (14 µL) were added and the mixture was stirred overnight at room temperature yielding compound 2b, which was HPLC purified ([M+H]+=602.7; Mcalc=602.0). Compound 2c was prepared by coupling of L-methyl-tryptophan (21.3mg, 1 eq) to 2a (37.0 mg, 1 eq) using HATU (32 mg, 1eq) and DIPEA (25 µL) in dry DMF. The mixture was stirred at room temperature for 3 h. After the addition of 5 M HCl (final 2.5 M), the mixture was heated to 90°C overnight. The final product was then HPLC purified ([M+H]+=625.8; Mcalc=625.1). The corresponding fluorescein-conjugates of compound 2a-c were prepared by activating the carboxylic acid moiety of compound 2a-c (3.5 µmol) in DMSO using EDC (3.5 µmol) and S-NHS (4.2 µmol) for 2h at room temperature. Amino-triethyleneglycol-fluorescein (3.5 µmol) and triethylamine (2 µL) were added and the mixture was stirred overnight at room temperature. Fluorescein-conjugates were then HPLC purified and ESI-MS characterized. Fluorescein-conjugate of 2a: [M+H]+=1001.8; Mcalc=1000.8; Fluorescein-conjugate of 2b: [M+H]+=1163.0; Mcalc=1162.3; Fluorescein-conjugate of 2c: [M+H]+=1186.2; Mcalc=1185.3.

Figure S1. Selection against Tris-quenched Resin and Matrix Metalloprotease 3 (A) Sequence enrichment profile after high-throughput sequencing of a selection experiment against Tris-quenched resin. DNA-conjugates with Code 2 = 120, 123, 136 and 175 were enriched. (B) Precursors of building block 2. Compounds carrying these building blocks were excluded for hit identification in protein selection experiments. (C) Sequence enrichment profile after selection against matrix metalloprotease 3, background binders with code 2 equal to 120, 123, 136, 175 were omitted for clarity.

Figure S2. Binding Analysis by Fluorescence Polarization with additional TNF Ligands The affinity of TNF ligands (Table 2) was determined by fluorescence polarization against the trimeric EDB-TNF protein (A). Ligands selected for TNF showed no binding against the EDB-tag alone (B).

Figure S3. Binding Analysis of BH3I-1 to Bcl-xL Isothermal titration calorimetry measurements (A) were performed using a VP-ITC instrument (Microcal). Bcl-xL (34 µM) in PBS containing 2% DMSO was titrated with a 318 µM solution of BH3I-1 (B) in PBS containing 2% DMSO at 26°C. After an initial dummy injection of 2 µL, 29 injections of 10 µL each were performed. The resulting titration curves were then processed and fitted with the Origin 7 software (Microcal) yielding a Kd of 4.4 µM.

SUPPLEMENTAL REFERENCES Buller, F., Mannocci, L., Zhang, Y., Dumelin, C.E., Scheuermann, J., and Neri, D. (2008). Design and synthesis of a novel DNA-encoded chemical library using Diels-Alder cycloadditions. Bioorg Med Chem Lett 18, 5926-5931. Dumelin, C.E., Trussel, S., Buller, F., Trachsel, E., Bootz, F., Zhang, Y., Mannocci, L., Beck, S.C., Drumea-Mirancea, M., Seeliger, M.W. et al. (2008). A portable albumin binder from a DNA-encoded chemical library. Angew Chem Int Ed Engl 47, 3196-3201. Melkko, S., Zhang, Y., Dumelin, C.E., Scheuermann, J., and Neri, D. (2007). Isolation of high-affinity trypsin inhibitors from a DNA-encoded chemical library. Angew Chem Int Ed Engl 46, 4671-4674.