Supporting Information. Phosphorylation of capsaicinoid derivatives provides highly potent and selective inhibitors of the transcription factor STAT5b

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1 Supporting Information Phosphorylation of capsaicinoid derivatives provides highly potent and selective inhibitors of the transcription factor STAT5b Nagarajan Elumalai #, Angela Berg #, Stefan Rubner, and Thorsten Berg* Table of Contents Table S Figure S Figure S Figure S Figure S Figure S Plasmid construction and protein expression... 5 Fluorescence polarization assays... 5 Cell culture... 5 Western Blots... 6 Cell viability assay... 6 Apoptosis assay... 6 Synthesis and characterization of compounds... 7 Supporting References NMR spectra

2 Table S1 Protein IC 50 (µm) or maximum inhibition K i (µm) STAT5b 0.75 ± ± 0.01 STAT5a 26 ± ± 0.7 STAT1 6 ± 5 % inhibition at 40 µm N/A STAT3 4 ± 9 % inhibition at 40 µm N/A STAT4 35 ± 3 17 ± 1 STAT ± ± 0.8 Lck SH2 18 ± 2 inhibition at 40 µm N/A Table S1: Activity of compound 6a against the SH2 domains of STAT proteins and the tyrosine kinase Lck as analyzed in fluorescence polarization assays. The conversion of IC50 values to Ki values was carried out according to the published equation. 1 N/A = not applicable. Figure S1 Figure S1: Docking of compound 6a into the STAT5b SH2 domain. Docking suggests a potential hydrogen bond between STAT5b Asn642 and the amide function of 6a. 2

3 Figure S2 Figure S2: Time course of the inhibition of STAT5b-GFP Tyr699 phosphorylation in K562 cells. K562 cells were transfected with STAT5b-GFP for 24 h and subsequently treated with 10 µm of compound 7 for the indicated periods of time. Figure S3 Figure S3: Relative amounts of tyrosine-phosphorylated STAT5a/b and total STAT5a/b in K562 cells and MDA-MB-231 cells analyzed by Western Blot. 3

4 Figure S4 Figure S4: Representative results of the flow cytometry analysis carried out on K562 cells using the indicated concentrations of 7. Apoptotic cells, as characterized by Annexin V staining and the absence of 7-AAD staining, are depicted in the lower right-hand section of the flow cytometry plot. Figure S5 Figure S5: Representative results of the flow cytometry analysis carried out on MDA-MB-2331 cells using the indicated concentrations of 7. Apoptotic cells, as characterized by Annexin V staining and the absence of 7-AAD staining, are depicted in the lower right-hand section of the flow cytometry plot. 4

5 Plasmid construction and protein expression Cloning, expression, and purification protocols for STAT1, STAT3, STAT4, STAT5a, STAT5b, STAT6 and the Lck SH2 domain have been described previously. 2, 3 Cloning of the GFP-STAT5a and GFP-STAT5b plasmids has also been described. 3 Fluorescence polarization assays For competition-based fluorescence polarization assays, the ability of the test compounds to displace fluorophore-labeled peptides from their respective binding proteins was analyzed as previously described. 3 Peptide sequences were: STAT1: 5-carboxyfluorescein- GY(PO 3H 2)DKPHVL; STAT3: 5-carboxyfluorescein-GY(PO 3H 2)LPQTV-NH 2; STAT4: 5- carboxyfluorescein-gy(po 3H 2)LPQNID-OH; STAT5a and STAT5b: 5-carboxyfluorescein- GY(PO 3H 2)LVLDKW; STAT6: 5-carboxyfluorescein-GY(PO 3H 2)VPWQDLI-OH; Lck SH2: 5- carboxyfluorescein-gy(po 3H 2)EEIP. Due to protein instability, STAT2 was not analyzed. Final concentration of 5-carboxyfluorescein-labeled peptides: 10 nm. Proteins were used at the following final concentrations, which correspond to the K d-values of the interactions with the respective fluorophore-labeled peptide: STAT1: 420 nm; STAT3: 270 nm; STAT4: 130 nm; STAT5a: 130 nm; STAT5b: 100 nm; STAT6: 310 nm; Lck SH2: 30 nm. Pipetting was carried out partly using a Biomek FX workstation (Beckman-Coulter). Proteins were incubated with the test compounds for 60 min, prior to addition of the fluorophore-labeled peptides. After another 60 min, fluorescence polarization was read using an Infinite F500 plate reader (Tecan) in black 384-well plates (Corning # 3573). Final concentrations of the buffer components: 10 mm Tris (ph 8.0), 50 mm NaCl, 1 mm EDTA, 1 mm DTT, 0.1 % Nonidet P-40 substitute, and 2 % DMSO. Percent inhibition was calculated based on curve fits using SigmaPlot (SPSS Science Software). IC 50 data were converted to K i-values using the published equation. 1 Cell culture K562 and MDA-MB-231 cells were obtained from the DSMZ (Braunschweig, Germany). K562 cells were cultured in RPMI 1640 medium (Invitrogen), containing 10 % (v/v) FBS (Gibco Life Technologies), 2 mm L-glutamine (PAA Laboratories) and 1 % (v/v) penicillin/streptomycin (PAA Laboratories) at 37 C, 5% CO 2 and 95% humidity. MDA-MB-231 cells were cultured in Leibovitz s L-15 medium (Invitrogen) supplemented with 10 % (v/v) FBS (Gibco Life Technologies) and 1 % (v/v) penicillin/streptomycin (PAA Laboratories) at 37 C and 95 % humidity with no additional CO 2. 5

6 Western Blots Transfection of cultured K562 cells and Western blotting was performed as previously described. 3 In brief, K562 cells were transfected with plasmid encoding either STAT5a-GFP or STAT5b-GFP, using Fugene HD Transfection Reagent (Promega; 1 x 10 6 cells per well in 1 ml medium with a 4:1 ratio of Fugene:DNA). After 24 h, the cells were treated with compound 7 or DMSO for 4 h (final DMSO concentration: 0.2 %). After harvesting, cells were lysed (lysis buffer composition: 50 mm Tris-HCl ph 7.5, 150 mm NaCl, 10 mm Na 4P 2O 7, 10 % glycerol, 1 % Triton X-100, 1 mm EDTA, 100 ng/ml aprotinin, 1 mm Na 3PO 4, 10 mm NaF, 1 mm PMSF). After separation of the cell lysate components (SDS-PAGE, 10% polyacrylamide gel), they were transferred to a nitrocellulose membrane. Two gels and two membranes were prepared for each experiment. One membrane was probed with Stat5 antibodies (first with anti-pstat5 antibody; then anti-stat5, then anti- -actin. The other membrane was first probed with anti-gfp antibody, then with anti- actin antibody) to confirm even transfection. All primary antibodies are rabbit antibodies from Cell Signaling. Primary antibodies were detected by -rabbit-hrp secondary antibody (Dako) and ECL (Western Lightning Plus chemiluminescence reagent, Perkin-Elmer), and visualized using an ImageQuant digital imaging system (GE Healthcare). Quantitation was carried out using ImageJ software (NIH). 4 Cell viability assay K562 cells or MDA-MB-231 cells were seeded at a density of 1 x 10 4 cells per well in a 96-well tissue culture plate (Corning #3596) and were treated with compound 7 at the indicated concentrations (final DMSO concentration: 0.2 %) for 48 h. Subsequently, 10 µl WST-1 solution (Roche, 1:10 dilution with cell culture medium) was added to each well, and incubated for 1 h. The absorbance at 450 nm was analyzed, using the absorbance at 650 nm as a reference. Apoptosis assay K562 cells (2.5 x 10 5 cells per well) or MDA-MB-231 cells (1 x 10 5 cells per well) were seeded in 24-well tissue culture plates (Corning #3526), and were treated with compound 7 at the indicated concentrations (final DMSO concentration: 0.2 %) for 48 h. After 48 h, K562 suspension cells were harvested. MDA-MB-231 were washed once with warm phosphate buffered saline (PBS), and were incubated with Accutase (BD Bioscience #561527) at 37 C for 10 min. The cell culture supernatant from each well was used for neutralization of Accutase and for cell resuspension to obtain single-cell suspensions. After harvesting of either cell type, cells were centrifuged at 3000 rpm at 4 C for 5 min. Subsequently, cells were washed twice with cold PBS and centrifuged again. 6

7 Cell staining was carried out using the PE Annexin V Apoptosis Detection Kit I (BD Bioscience, #559763). Cells were resuspended in 1 x Binding Buffer, and were incubated with PE Annexin V and 7-AAD at 4 C for 30 min. Subsequently, apoptosis was measured using a LSR II flow cytometer (BD Bioscience). Synthesis and characterization of compounds Dibenzyl (2-methoxy-4-((8-methylnonanamido)methyl)phenyl) phosphate (2a) To the solution of dihydrocapsaicin (1a) (40 mg, 0.13 mmol) in anhydrous acetonitrile (4 ml) was added CCl 4 (0.063 ml, 0.65 mmol), DIEA (0.045 ml, 0.26 mmol) and a catalytic amount of DMAP at 0 C. Dibenzyl phosphite (0.043 ml, mmol) was added dropwise, and the mixture was stirred for 1 h at 0 C. Upon completion of the reaction (TLC control), 1 ml of 0.5 M KH 2PO 4 was added. The reaction mixture was repeatedly extracted with ethyl acetate, and the combined organic phases were subsequently washed with 5 % aqueous NaCl solution, H 2O, and dried over Na 2SO 4. The volatiles were removed under reduced pressure and purified by flash column chromatography (8.5:1.5 DCM/acetone) to obtain 2a as an oily liquid (60 mg, 81 %); R f = 0.35 (DCM/acetone 9:1); 1 H NMR (400 MHz, CDCl 3) δ = 0.88 (d, J=6.6, 6H), (m, 2H), (m, 6H), 1.52 (dq, J=13.2, 6.6, 1H), (m, 2H), 2.22 (t, J=7.6, 2H), 3.78 (s, 3H), 4.37 (d, J=5.7, 2H), 5.18 (d, J=7.8, 4H), 6.09 (t, 1H), 6.76 (dd, J=8.3, 1.9, 1H), (m, 1H), 7.13 (dd, J=8.2, 1.4, 1H), 7.35 (s, 10H); 13 C NMR (101 MHz, CDCl 3) δ = 22.7, 25.8, 27.3, 28.0, 29.4, 29.7, 36.8, 39.0, 43.2, 55.9, (d, J CP=5.8), 112.3, 119.9, 119.9, 121.4, 121.4, 127.9, 128.5, 128.5, (d, J CP=7.3), 136.7, (d, J CP=7.1), (d, J CP=5.0), 173.1; 31 P NMR (162 MHz, CDCl 3) δ = (m, J HP=7.9); UV/Vis (nm) : 280, 274, 221; IR (Film): ν = 3306, 3089, 3066, 3034, 2952, 2926, 2854, 1650, 1601, 1545, 1512, 1464, 1456, 1420, 1383, 1365, 1355, 1280, 1241, 1212, 1156, 1126, 1081, 1036, 1020, 1000, 957, 883, 815, 740, 697, 640, 600, 515, 491, 459; HRMS (ESI) C 32H 42NNaO 6P calcd: [M+Na + ], found:

8 Dibenzyl (2-methoxy-4-(nonanamidomethyl) phenyl) phosphate (2b) To the solution of N-vanillyl nonanamide (1b) (150 mg, 0.51 mmol) in 6 ml anhydrous acetonitrile was added CCl 4 (0.25 ml, 2.55 mmol), DIEA (0.178 ml, 1 mmol) and a catalytic amount of DMAP at 0 C. Dibenzyl phosphite (0.17 ml, 0.75 mmol) was added dropwise, and the solution was stirred for 1 h at 0 C. Upon completion of the reaction (TLC control), 3 ml of 0.5 M KH 2PO 4 were added. The reaction mixture was repeatedly extracted with ethyl acetate, and the combined organic phase was subsequently washed with 5 % aqueous NaCl solution, H 2O, and dried over Na 2SO 4. The volatiles were removed under reduced pressure and purified by flash column chromatography (ethyl acetate/hexane 1:1 2:1) to obtain 2b as an oily liquid (260 mg, 92%); R f = 0.17 (ethyl acetate/hexane 2:1); 1 H NMR (400 MHz, CDCl 3) δ = (m, 3H), (m, 10H), (m, 2H), (m, 2H), 3.76 (s, 3H), 4.36 (d, J=5.7, 2H), 5.16 (d, J=7.9, 4H), 5.88 (t, J=5.9, 1H), 6.74 (dd, J=8.2, 2.0, 1H), 6.84 (s, 1H), 7.12 (dd, J=8.1, 1.4, 1H), 7.33 (s, 10H); 13 C NMR (101 MHz, CDCl 3) δ = 14.20, 22.75, 25.91, 29.27, 29.44, 29.47, 31.93, 36.90, 43.35, 55.99, 69.92, 69.98, , , , , , δ = (d, J CP=7.4), (d, J CP=1.6), (d, J CP=7.2), (d, J CP=5.1), ; 31 P NMR (162 MHz, CDCl 3) δ = (s, 1P); UV/Vis (nm) : 274, 208; IR (Film): ν = 3306, 3089, 3066, 3034, 2954, 2926, 2854, 1651, 1602, 1545, 1512, 1463, 1456, 1420, 1380, 1354, 1281, 1212, 1156, 1126, 1081, 1036, 1020, 1001, 957, 882, 814, 741, 697, 602, 515, 509, 495, 458; MS (ESI) C 31H 40NNaO 6P calcd: [M+Na + ], found: Methoxy-4-((8-methylnonanamido)methyl)phenyl dihydrogen phosphate (3a) To a solution of 2a (50 mg, mmol) in absolute ethanol (6 ml) was added 10 % Pd on charcoal (20 mg) under argon. The argon atmosphere was exchanged for a hydrogen atmosphere. After completion of the reaction (0.5 h, RP-TLC control), the mixture was filtered through celite, and washed with ethanol. After removal of the solvent under reduced pressure, 8

9 the product was dissolved in water and lyophilized to obtain 3a as a white solid (32 mg, 94 %); Melting point: C; 1 H NMR (400 MHz, DMSO-d 6) δ = 0.85 (d, J=6.6, 6H), (m, 2H), (m, 7H), (m, 3H), 2.12 (t, J=7.4, 2H), 3.72 (s, 3H), 4.18 (d, J=5.7, 2H), (m, 1H), (m, 1H), 7.31 (d, J=8.2, 1H), 8.23 (t, J=5.8, 1H); 13 C NMR (101 MHz, DMSO) δ = 23.0, 25.8, 27.2, 27.9, 29.2, 29.5, 35.8, 38.9, 40.4, 40.6, 42.3, 56.0, 112.3, 119.3, 120.5, (m), (m), 172.5; 31 P{H} NMR (162 MHz, DMSO) δ = -5.0 (s, 1P); HRMS (ESI) C 18H 29NO 6P calcd: [M-H + ], found: Methoxy-4-(nonanamidomethyl) phenyl dihydrogen phosphate (3b) To a solution of 2b (150 mg, 0.26 mmol) in absolute ethanol (20 ml) was added 10 % Pd on charcoal (30 mg) under argon. The argon atmosphere was exchanged for a hydrogen atmosphere. After completion of the reaction (0.5 h, RP-TLC control), the mixture was filtered through celite, and washed with ethanol. After removal of the solvent under reduced pressure, the product was dissolved in water, and washed with dichloromethane (DCM) (2 x 5 ml). The aqueous layer was lyophilized to obtain 3b as a fluffy white solid (90 mg, 93%); Melting point: C; 1 H NMR (400 MHz, DMSO-d 6) δ = 0.84 (t, J=6.8, 3H), 1.23 (s, 10H), (m, 2H), 2.10 (t, J=7.4, 2H), 3.72 (s, 3H), 4.18 (d, J=5.9, 2H), 6.70 (dd, J=8.2, 2.0, 1H), 6.88 (s, 1H), 7.20 (d, J=8.2, 1H), 8.22 (t, J=5.8, 1H); 13 C NMR (101 MHz, DMSO) δ = 14.4, 22.6, 25.8, 29.1, 29.2, 29.2, 31.7, 35.8, 40.4, 40.6, 42.2, 56.0, 112.3, 119.3, 120.6, 136.0, (d, J CP=5.0), 150.6, 172.5; 31 P{H} NMR (162 MHz, DMSO-d 6) δ = (s, 1P); UV/Vis (nm): 275, 221, 205; IR (Film): ν = 3646, 3557,3434, 3288, 3073, 2954, 2925, 2870, 2851, 2360, 2338, 2248, 1636, 1604, 1548, 1518, 1467, 1419, 1303, 1281, 1269, 1234, 1218, 1203, 1154, 1126, 1026, 974, 927, 850, 819, 739, 722, 638, 517, 501; HRMS (ESI) C 17H 27NO 6P calcd: [M-H + ], found: N-(3, 4-dihydroxybenzyl)-8-methylnonanamide (4a) 9

10 To the solution of dihydrocapsaicin (1a) (95 mg, 0.31 mmol) in dry DCM was slowly added BBr 3 (2 ml of a 1 M solution in DCM) at 0 ºC. The mixture was stirred overnight at room temperature. After completion of the reaction (TLC control), excess BBr 3 was quenched by addition of 5 ml of methanol were added, and the volatiles were removed under reduced pressure. The quenching procedure was repeated two times, and the dark crude product was purified by column chromatography (2% methanol in DCM) to obtain 4a as an off-white solid (43 mg, 47%); R f = 0.25 (5 % methanol in DCM); 1 H NMR (300 MHz, CDCl 3) δ = 0.84 (d, J=6.6, 6H), 1.11 (d, J=6.3, 2H), 1.23 (s, 6H), 1.48 (dt, J=13.1, 6.6, 1H), 1.60 (s, 2H), 2.20 (s, 3H), 4.26 (s, 2H), 6.16 (s, 1H), 6.59 (d, J=7.5, 1H), (m, 2H); 13 C NMR (75 MHz, CDCl 3) δ = 22.75, 25.94, 27.33, 28.05, 29.40, 29.66, 36.93, 39.04, 43.69, , , , , , , , ; MS (ESI) C 34H 53N 2O 6 calcd: [M-H + ], found: N-(3,4-dihydroxybenzyl)nonanamide (4b) To a solution of N-vanillyl nonanamide (1b) (100 mg, 0.34 mmol) in dry DCM was slowly added BBr 3 (2 ml of a 1 M solution in DCM) at 0 C. The mixture was stirred overnight at room temperature. After completion of the reaction (TLC control), the reaction was quenched by addition of 5 ml of methanol, and the volatiles were removed under reduced pressure. The quenching procedure was repeated two times, and the dark crude product was purified by column chromatography (2% 5% methanol in DCM) to obtain 4b as an off-white solid (47 mg, 50 %); R f = 0.45 (10 % methanol in DCM); 1 H NMR (400 MHz, CDCl 3 + CD 3OD) δ = 0.82 (t, J=6.8, 3H), (m, 10H), 1.56 (p, J=7.4, 2H), 2.13 (t, 2H), 2.84 (s, 3H), 4.19 (s, 2H), 6.57 (dd, J=8.1, 2.1, 1H), 6.70 (d, J=2.1, 1H), 6.72 (d, J=8.1, 1H); MS (ESI) C 16H 25NNaO 3 calcd: [M+Na + ], found: Tetrabenzyl (4-((8-methylnonanamido) methyl)-1,2-phenylene) bis(phosphate) (5a) 10

11 To the solution of 4a (48 mg, 0.16 mmol) in anhydrous acetonitrile was added CCl 4 (0.13 ml, 1.6 mmol), DIEA (0.10 ml, 0.64 mmol) and a catalytic amount of DMAP at 0 C. Dibenzyl phosphite (0.09 ml, 0.48 mmol) was added dropwise, and the mixture was stirred for 1 h at 0 C. Upon completion of the reaction (TLC control), 3 ml of 0.5M KH 2PO 4 were added. The reaction mixture was repeatedly extracted with ethyl acetate, and the combined organic phases were subsequently washed with 5% NaCl solution and H 2O and dried over Na 2SO 4. The volatiles were removed under reduced pressure and purified by flash column chromatography (1% 2% methanol in DCM) to yield 5a as an oily liquid (129 mg, 99%); R f = 0.37 (5 % methanol in DCM); 1 H NMR (400 MHz, CDCl 3) δ = 0.86 (d, J=6.6, 6H), (m, 2H), (m, 7H), 1.51 (hept, J=13.2, 6.6, 1H), (m, 2H), 2.17 (dd, J=8.6, 6.8, 2H), 4.31 (d, J=5.6, 2H), (m, 8H), 5.70 (t, J=5.7, 1H), 7.00 (dd, J=8.4, 1.3, 1H), (m, 1H), (m, 20H); 13 C NMR (101 MHz, CDCl 3) δ = 22.77, 25.84, 27.36, 28.07, 29.53, 29.75, 36.84, 39.09, 42.70, 70.23, 70.27, 70.29, 70.33, 77.47, (d, J CP=2.3), (d, J CP=2.3), , , , , , , , , , (t, J CP=6.6), (t, J CP=6.5), ; 31 P{H} NMR (162 MHz, CDCl 3) δ = (s, 1P), (s, 1P); MS (ESI) C 44H 51NNaO 9P 2: [M+Na + ], found: Tetrabenzyl (4-(nonanamidomethyl)-1,2-phenylene) bis(phosphate) (5b) To the solution of 4b (50 mg, 0.18 mmol) in anhydrous acetonitrile (5 ml) was added CCl 4 (0.175 ml, 1.8 mmol), DIEA (0.13 ml, 0.72 mmol) and a catalytic of DMAP at 0 C. Dibenzyl phosphite (0.12 ml, 0.54 mmol) was added dropwise, and the mixture was stirred for 1 h. Upon completion of reaction (TLC control) 3 ml of 0.5 M KH 2PO 4 were added. The reaction mixture was repeatedly extracted with ethyl acetate and the combined organic phase was subsequently washed with 5% NaCl solution and H 2O and dried over Na 2SO 4. The volatiles were removed under reduced pressure and purified by flash column chromatography (2 % 5 % methanol in DCM) to obtain 5b as an oily liquid (130 mg, yield 90%); R f = 0.52 (10 % methanol in DCM); 1 H NMR (400 MHz, CD 3OD) δ = 0.87 (t, J=6.9, 3H), (m, 10H), 1.61 (p, J=7.1, 2H), 2.21 (t, J=7.5, 2H), 4.28 (s, 2H), (m, 8H), 7.09 (dd, J=8.5, 2.1, 1H), (m, 22H); 13 C NMR (101 MHz, CD 3OD) δ = 14.44, 23.69, 23.71, 27.04, 30.29, 30.31, 30.36, 30.40, 32.98, 37.09, 43.07, 11

12 49.63, 71.72, (dd, J=5.7, 2.0), 71.80, , , , , , , , , (d, J=7.1), , , (d, J CP=6.2), (d, J CP=6.3), ; 31 P{H} NMR (162 MHz, CD 3OD) δ = (s, 1P), (s, 1P); MS (ESI) C 44H 51NNaO 9P 2 calcd [M+Na + ], found: ((8-Methylnonanamido) methyl)-1,2-phenylene bis(dihydrogen phosphate) (6a) To a solution of 5a (125 mg) in absolute ethanol (20 ml) was added 10 % Pd on charcoal (30 mg) under argon. The argon atmosphere was exchanged for a hydrogen atmosphere. After completion of the reaction (2 h, RP-TLC control), the mixture was filtered through celite and washed with ethanol. After removal of the solvent under reduced pressure, the product was dissolved in water and washed with dichloromethane (DCM) (2 x 5 ml). The aqueous layer was lyophilized to obtain 6a as a light brown hygroscopic solid (70 mg, quant); 1 H NMR (300 MHz, DMSO-d 6) δ = 0.83 (d, J=6.5, 6H), (m, 8H), (m, 3H), 2.09 (t, J=7.5, 2H), 4.16 (d, J=5.7, 2H), 6.94 (d, J=8.1, 1H), (m, 2H), 8.28 (d, J=6.0, 1H); 13 C NMR (75 MHz, DMSO-d 6) δ = 22.55, 25.28, 26.70, 27.39, 28.79, 29.06, 35.38, 38.46, 41.36, , , , , , , , ; 31 P{H} NMR (162 MHz, DMSO-d 6) δ = (s, 1P), (s, 1P); UV/Vis: λ (nm) = 274, 268, 205; IR (Film): ν = 3433, 2920, 2851, 1691, 1660, 1642, 1465, 1430, 1384, 1070, 725, 644, 593, 455, 423, 406; HRMS (ESI) C 17H 28NO 9P 2: calcd [M-H + ], found: (Nonanamidomethyl)-1,2-phenylene bis(dihydrogen phosphate) (6b) To a solution of 5b (80 mg) in absolute ethanol (20 ml) was added 10 % Pd on charcoal (25 mg) under argon. The argon atmosphere was exchanged for a hydrogen atmosphere. After completion of the reaction (2 h, RP-TLC control), the mixture was filtered through celite, and washed with 12

13 ethanol. After removal of the solvent under reduced pressure, the product was dissolved in water and washed with dichloromethane (DCM) (2 x 5 ml). The aqueous layer was lyophilized to obtain 6b as an off-white hygroscopic solid (41 mg, 93%). 1 H NMR (400 MHz, DMSO-d 6) δ = 0.84 (t, J=6.7, 3H), 1.23 (s, 10H), 1.49 (t, J=7.2, 2H), 2.09 (t, J=7.5, 2H), 4.16 (d, J=5.8, 2H), 6.50 (bs, 4H), 6.93 (d, J=8.1, 1H), (m, 2H), 8.26 (t, J=6.1, 1H); 13 C NMR (101 MHz, DMSO-d 6) δ = 13.97, 22.09, 25.27, 28.60, 28.75, 31.27, 35.36, 38.89, 40.14, 41.37, , , , , , , ; 31 P{H} NMR (162 MHz, DMSO-d 6) δ = -4.6 (s, 1P), -4.5 (s, 1P); UV/Vis: λ (nm) = 274, 268, 206; IR (KBr): ν = 3421, 2956, 2928, 2856, 2321, 1644, 1636, 1595, 1511, 1467, 1427, 1285, 1212, 1155, 1121, 1022, 981, 822, 724, 705, 510, 500, 458; HRMS (ESI) C 16H 26NO 9P 2 calcd: [M-H + ], found: ((((4-((8-Methylnonanamido)methyl)-1,2-phenylene)bis(oxy))bis(oxo-l5-phosphanetriyl)) tetrakis (oxy))tetrakis(methylene) tetrakis(2,2-dimethylpropanoate) (7) 6a (30 mg, 0.07 mmol) was suspended in dry acetonitrile. Diisopropylethylamine (0.092 ml, 0.53 mmol) and iodomethyl pivalate (130 mg, 0.53 mmol) were added. After stirring at room temperature for 24 h, the suspension had turned into a clear solution. Volatiles were removed in vacuo. The product was purified by column chromatography (hexane / acetone 4:1) to yield 7 as a colorless oil (25 mg, 39%); R f = 0.5 (hexane / acetone 4:1); 1 H NMR (400 MHz, CDCl 3) δ = 0.85 (d, J=6.6, 6H), 1.19 (dd, J=3.6, 0.7, 39H), (m, 7H), 1.50 (hept, J=6.7, 1H), 1.65 (p, J=7.6, 2H), (m, 2H), 4.39 (d, J=5.7, 2H), (m, 8H), 5.96 (t, J=5.4, 1H), (m, 1H), (m, 2H); 13 C NMR (101 MHz, CDCl 3) δ = 22.76, 25.83, 26.89, 27.35, 28.06, 29.53, 29.74, 36.80, 38.84, 39.09, 42.69, 83.29, 83.34, (d, J CP=2.4), (d, J CP=2.4), , , (t, J CP=6.7), (t, J CP=6.6), , , ; 31 P{H} NMR (162 MHz, CDCl 3) δ = (s, 1P), -9.6 (s, 1P); HRMS (ESI) C 41H 69NNaO 17P 2 calcd: [M+Na + ], found:

14 Supporting References (1) Nikolovska-Coleska, Z., Wang, R., Fang, X., Pan, H., Tomita, Y., Li, P., Roller, P. P., Krajewski, K., Saito, N. G., Stuckey, J. A., and Wang, S. (2004) Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization, Anal. Biochem. 332, (2) Gräber, M., Hell, M., Gröst, C., Friberg, A., Sperl, B., Sattler, M., and Berg, T. (2013) Oral Disinfectants Inhibit Protein-Protein Interactions Mediated by the Anti-Apoptotic Protein Bcl-xL and Induce Apoptosis in Human Oral Tumor Cells, Angew. Chem. Int. Ed. 52, (3) Elumalai, N., Berg, A., Natarajan, K., Scharow, A., and Berg, T. (2015) Nanomolar Inhibitors of the Transcription Factor STAT5b with High Selectivity over STAT5a, Angew. Chem. Int. Ed. 54, (4) Schneider, C. A., Rasband, W. S., and Eliceiri, K. W. (2012) NIH Image to ImageJ: 25 years of image analysis, Nat. Methods 9,

15 NMR spectra 1 H NMR of compound 2a 13 C NMR of compound 2a 15

16 31 P NMR of compound 2a 1 H NMR of compound 2b 16

17 13 C NMR of compound 2b 31 P NMR of compound 2b ( 1 H-decoupled) 17

18 1 H NMR of compound 3a 13 C NMR of compound 3a 18

19 31 P NMR of compound 3a ( 1 H-decoupled) 1 H NMR of compound 3b 19

20 13 C NMR of compound 3b 31 P NMR of compound 3b ( 1 H-decoupled) 20

21 1 H NMR of compound 5a 13 C NMR of compound 5a 21

22 31 P NMR of compound 5a ( 1 H-decoupled) 1 H NMR of compound 5b 22

23 13 C NMR of compound 5b 31 P NMR of compound 5b ( 1 H-decoupled) 23

24 1 H NMR of compound 6a 13 C NMR of compound 6a 24

25 31 P NMR of compound 6a ( 1 H-decoupled) 1 H NMR of compound 6b 25

26 13 C NMR of compound 6b 31 P NMR of compound 6b ( 1 H-decoupled) 26

27 1 H NMR of compound 7 13 C NMR of compound 7 27

28 31 P NMR of compound 7 ( 1 H-decoupled) 28