аЯрЁБс>ўџ VXўџџџUџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџьЅС[ јПї>bjbjтт "\€j€j?%џџџџџџlўўўўўўў8H,tmЖ”^ђ"ЬЮЮЮЮЮЮ$# CЈђ5ўђв ўў'в в в њўўЬв Ьв њв ЬўўЬˆ  ‡. Сў ЬЬ=0mЬы ОыЬв ўўўўйSupplementary information for: “A library approach to the generation of bisubstrate analog sulfotransferase inhibitors” Joshua I. Armstrong, Xue Ge, Dawn E. Verdugo, Katharine A. Winans, Julie A. Leary, and Carolyn R. Bertozzi General. All chemical reagents were used as purchased from Sigma and Aldrich. Thin-layer chromatography was performed using Analtech Uniplate silica gel plates. Flash chromatography was performed using Merck 60 Х 230-300 mesh silica gel. All reaction solvents were distilled under a N2 atmosphere with the exception of DMF which was purchased anhydrous and stored over roasted Х molecular sieves. CH2Cl2 and CH3CN were dried over CaH2. All 1H NMR and 13C NMR spectra were recorded using a Bruker DRX-500 MHz spectrometer as noted. Chemical shifts are reported in d№ relative to tetramethylsilane.31P NMR spectra were recorded using a Bruker AMX-400 MHz spectrometer. Chemical shifts are reported in d№ relative to H3PO4. Coupling constants are reported in Hz. Electrospray (ES) mass spectra were obtained on a Hewlett Packard 1100 MSD. Fast atom bombardment (FAB+) spectra and elemental analyses were obtained at the U.C. Berkeley Mass Spectral and Microanalytical Laboratories, respectively. 5’-[2-[(N-Fluorenemethyloxycarbonyl)-aminooxy]]-acetylamino-N-benzoyl-2’,5’-dideoxyadenosine (5). To a solution of 3 ADDIN ENRfu 1 (2.7 g, 7.1 mmol) in MeOH (60 mL) was added AcOH (410 mL, 7.1 mmol) and 10% Pd/C (805 mg). H2 was bubbled through the suspension with vigorous stirring for 3 h. The suspension was then filtered, concentrated, redissolved in 25 mL of MeOH and passed through a column (1.5 cm X 5 cm) of Amberlyst A21 resin (OH- form). The solution was concentrated to provide 1.6 g (62%) crude aminonucleoside as an off-white glass and was used without further purification. To 3 mL of anhydrous DMF was added 2-ethoxy-1-ethyoxycarbonyl-1,2-dihydroquinoline (382 mg, 1.55 mmol) and FmocNHOCH2COOH ADDIN ENRfu 2 (386 mg, 1.23 mmol). The solution was stirred at rt under Ar for 30 min before adding a solution of the crude aminonucleoside (364 mg, 1.00 mmol) in 3 mL of anhydrous DMF via syringe. After 2.5 h, the solution was concentrated and the resulting gel was purified by silica gel chromatography (2% MeOH in CH2Cl2) to provide 5 as a white amorphous solid (410 mg, 61%). 1H NMR (CD3OD, 500 MHz): d№ №ppm = 2.40-2.45 (m, 1H), 2.82-2.87 (m, 1H), 3.57 (dd, 1H, J=4 Hz, J=14 Hz), 3.66 (dd, 1H, J=6 Hz, J=14 Hz), 4.07-4.10 (m, 2H), 4.27-4.33 (m, 4H), 4.51 (app dt, 1H, J=4 Hz, J=6 Hz), 6.37 (app t, 1H, J=7 Hz), 7.14-7.19 (m, 2H), 7.24-7.28 (m, 2H), 7.45-7.51 (m, 4H), 7.59 (app t, 1H, J=7 Hz), 7.65 (dd, 2H, J=3 Hz, J=7.5 Hz), 7.99 (d, 2H, J=7 Hz), 8.45 (s, 1H), 8.63 (s, 1H). 13C NMR (CD3OD, 125 MHz): d№ №ppm = 38.9, 40.4, 46.5, 67.0, 71.6, 75.0, 84.8, 85.5, 119.4, 123.7, 124.5, 126.6, 127.3, 127.9, 128.2, 132.2, 133.4, 140.1, 142.9, 143.2, 149.4, 151.3, 151.6, 158.4, 166.3, 169.9. MS (ES (+)): m/e = 650.3 [M+H]+. Anal. Calcd: C, 62.86; H, 4.81; N, 15.09. Found: C, 62.56; H, 5.16; N, 14.87. 3’-[[bis[2-(4-nitrophenyl)ethoxy]phosphinyl]oxy]- 5’-[2-[(N-Fluorenemethyloxycarbonyl)-aminooxy]]-acetylamino-N-benzoyl-2’,5’-dideoxyadenosine (6). To a solution of 5 (390 mg, 0.60 mmol) in CH2Cl2 (12 mL) was added tetrazole (210 mg, 3.0 mmol) followed by di(p-nitrophenethyl) diisopropylphosphoramidite ADDIN ENRfu 3 (340 mg, 0.73 mmol) in 3 portions over 5 min. After 6 h at rt the suspension was chilled to –35 АC and 260 mg mCPBA was added. After 1 h the solution was warmed to rt, diluted with 100 mL of CHCl3 and washed with sat. NaHCO3 (20 mL) and brine (20 mL). The organic layer was concentrated and the resulting residue purified by silica gel chromatography (2% MeOH in CH2Cl2) to provide 6 as a clear glass (454 mg, 73%). 1H NMR (CDCl3, 500 MHz): d№ №ppm = 2.52-2.55 (m, 1H), 2.93-3.04 (m, 5H), 3.56 (dt, 1H, J=5.5 Hz, J=14 Hz), 3.67 (app dt, 1H, J=6 Hz, J=14 Hz), 4.10 (app t, 1H, J=7 Hz), 4.17-4.27 (m, 5H), 4.32-4.35 (m, 4H), 5.02-5.03 (m, 1H), 6.26 (t, 1H, J=6 Hz), 7.16-7.19 (m, 2H), 7.28-7.33 (m, 6H), 7.41-7.48 (m, 4H), 7.53-7.56 (m, 1H), 7.67 (d, 2H, J=7.5), 8.06-8.13 (m, 6H), 9.19 (s, 1H), 9.29 (s, 1H). 13C NMR (CDCl3, 125 MHz): d№ №ppm = 36.2, 37.6, 40.2, 46.7, 67.5, 67.8, 76.0, 78.4, 84.2, 84.7, 120.0, 123.7, 123.9, 124.9, 127.0, 127.8, 127.9, 128.8, 129.8, 132.9, 133.3, 141.2, 142.0, 143.1, 144.5, 146.9, 149.6, 151.3, 152.4, 158.2, 164.9, 169.4. 31P NMR (CDCl3, 164 MHz): d№ №ppm = -5.01. MS (ES (+)): m/e = 1028.5 [M+H]+. 3 -Phosphonooxy-5 -(2-aminooxy)-acetylamino-2 ,5 -dideoxyadenosine, ammonium salt (2). To a stirred solution of 6 (280 mg, 0.27 mmol) in anhydrous CH3CN (2.5 mL) was added 450 m№L of n-PrNH2. After 1 h at rt the solution was concentrated in vacuo, resuspended in 2.5 mL of anhydrous CH3CN and bis(trimethylsilyl) acetamide (BSA, 1.4 mL, 5.5 mmol) was added via syringe. After 5 min 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 410 m№L, 2.7 mmol) was added slowly via syringe. After 6 h the reaction was quenched with the addition of 5 drops of H2O. The brown solution was concentrated and partitioned between 5 mL of H2O and 5 mL of CHCl3. The organic layer was extracted with 2 X 1 mL of H2O and the combined aqueous layer was washed with 1 mL of CHCl3. Lyophilization followed by purification via C18 reversed-phase HPLC (0 to 20% CH3CN in 25 mM NH4OAc) provided 2 as a clear glass (65 mg, 57%). 1H NMR (D2O, 500 MHz): d№ №ppm: 2.68-2.76 (m, 1H), 2.82-2.91 (m, 1H), 3.55 (d, 2H, J=8 Hz), 4.06 (app s, 2H), 4.30 (app dt, 1H, J=8 Hz, J=14 Hz), 4.74-4.81 (m, 1H), 6.33 (app t, 1H, J=11 Hz), 8.08 (s, 1H), 8.24 (s, 1H). 13C NMR (D2O, 125 MHz): d№ ppm = 37.5, 41.1, 74.5, 74.9, 83.9, 84.4, 119.4, 139.9, 149.2, 152.7, 156.1, 170.6. 31P NMR (D2O, 164 MHz): d№ ppm: 2.60. MS (ES (+)): m/e = 404.4 [M+H]+. Library synthesis (1). To 10 m№L of each of 447 aldehydes (20 mM DMSO) distributed in individual wells of 96-well plates was added 10 m№L of 2 in DMSO (25 mM). The plates were incubated at rt in the dark for 48 h then frozen. Immobilized enzyme mass spectrometry (IEMS) assay. Enzyme immobilization was accomplished by reductive amination of lysine residues on the protein and aldehyde groups on the Aminolink (agarose) coupling gel (Pierce). Approximately 4.0-4.5 mg (500 m№L from the purification) of EST (expressed and purified as previously described ADDIN ENRfu 4) was added to 1 mL of Aminolink coupling gel slurry (0.5 mL coupling gel) in a 5-mL polypropylene affinity column (Pierce) that was equilibrated with 0.01 M NaHxPO4, pH 7.0 (coupling buffer). Additional coupling buffer (300 m№L) was then added, followed by 200 m№L of 1 M NaCNBH3 prepared in H2O. The column was capped and gently mixed using a rocking platform shaker for 2 h and stored at 4 АC overnight. The column was then drained and washed with 4 mL of coupling buffer. To block excess reactive sites on the Aminolink gel, 1 mL of 0.01 M Tris-HCl buffer, pH 7.4, was added to the column and mixed for 1 h. The column was then washed successively with 15 mL of coupling buffer, followed by 20 mL of a 0.05 M NaCl solution and finally with 30 mL of a 0.01 M NH4OAc buffer, pH 7.0. Library components were combined (0.5 m№L/compound) in batches of 30 to 60 compounds. Three non-inhibiting compounds (S-ethyl glutathione, S-butyl glutathione, and S-hexyl glutathione) were added as internal standards and the library was diluted to a final volume of 400 m№L in 0.01 M NH4OAc buffer, pH 7.5. A 300-m№L aliquot of the library solution was incubated with the immobilized EST for 1 h at rt with gentle rocking. Aliquots of pre- and post-incubation solutions were mixed with MeOH for electrospray ionization. Spectra were acquired on an Apex II FT-ICR mass spectrometer (Bruker, Billerica, MA) equipped with a 7-T shielded superconducting magnet. Ions (in positive polarity mode) were formed in a pneumatically assisted electrospray source (Analytica, Branford, CT) employing an off-axis electrospray probe at 1 m№L/min. Ions were externally accumulated in an rf-only hexapole for 2 s before transfer into the ICR cell for mass analysis. Library compound intensity was calculated by dividing its relative ion intensity by the intensity sum of three internal standards. Radiolabel-transfer TLC assay. EST inhibitory activity was measure in 50 m№L of 20 mM Tris-HCl (pH 7.4) containing 1 mM EDTA using 20 m№g EST, 50 nM estrone (Km(estrone) is 50 nM), and 200 m№M (DMSO stocks) of each inhibitor. The reactions were initiated by the addition of 2.5 nM enzymatically synthesized 35S-PAPS (Km(PAPS) is 2.5 nM) and quenched with 50 m№L MeOH after 10 min incubation at rt. Half of the quenched reaction was applied to the loading lane of Whatman 10 X 20 cm LKTLC silica plates and eluted with 8:2:1 nBuOH:MeOH:H2O at 4 АC (3 h). The plates were then air-dried and the amount of radiolabel incorporated into estrone sulfate was quantified by phosphorimaging. Inhibition was calculated as the percent incorporation of radiolabel into estrone sulfate relative to control experiments with DMSO (triplicate).  ADDIN ENBbu  (1) Mag, M.; Engels, J. W. Nucleosides Nucleotides 1988, 7, 725. (2) Trevisiol, E.; Defrancq, E.; Lhomme, J.; Laayoun, A.; Cros, P. Tetrahedron 2000, V56, 6501. (3) Uhlmann, E.; Engels, J. W. Tetrahedron Lett. 1986, 27, 1023. (4) Verdugo, D. E.; Cancilla, M. T.; Ge, X.; Gray, N. S.; Chang, Y.-T.; Schultz, P. G.; Negishi, M.; Leary, J. A.; Bertozzi, C. R. J. Med. Chem. 2001, in press. 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