Sharpless asymmetric dihydroxylation of styrene derivative 6 afforded chiral triols R)-7( and S)-7-4( and S)-4-configured enantiomers represent eutomers with eudismic ratios of up to 29 for the ester R)-18(. The methyl ether R)-15-21( was used as precursor for the radiosynthesis of [18F]-R)-20(-20 was able to label those regions of the brain, which were reported to have high density of σ1 receptors. It was named after the ligand SKF-10,047 and initially regarded as opioid receptor subtype  or [18F], have already been developed -labeled PET tracer based on this scaffold.The fluoroalkyl substituted 2-benzofurans tagonist , enantiopyrans 3  and 2-be2Cl2: distilled from calcium hydride. Flash chromatography: silica gel 60 (40–63 µm); parentheses include: diameter of the column (ø), height of the stationary phase (h), eluent and fraction size (V). Thin layer chromatography: TLC silica gel 60 F254 on aluminum sheets. Melting points (mp): uncorrected. Polarimetry: sodium D line (589 nm); length (l): 1 dm; temperature +20 °C; unit of specific rotation [deg mL dm−1 g−1] is omitted; parentheses include: concentration of the sample [mg/mL] and solvent: 1H-NMR: 400.3 MHz; 13C-NMR: 100.3 MHz; chemical shifts in [ppm] against TMS; in some cases, 1H and 13C-NMR spectroscopy were supported by 2D NMR techniques. IR spectroscopy: ATR technique.Solvents: THF: distilled from sodium/benzophenone; CHTM, 2.6 μm, C18, 100 Å; 50 mm/2.1 mm, guard column: Security Guard Standard C18 Cartridge, 4 mm/2mm, temperature: 30 °C, solvents: A: acetonitrile-NH4HCOO (10 mM) = 10:90 + 0.1% (v/v) HCO2H, B: acetonitrile-NH4HCO2 (10 mM) = 90:10 + 0.1% (v/v) HCO2H, gradient elution: (A%): 0–5 min: gradient from 100% to 0%, flow rate: 0.4 mL/min, 5–6.5 min: 0%, flow rate: 0.4 mL/min, 6.5–7 min: gradient from 0% to 100%, flow rate: 0.4 mL/min, 10 min: 100%, flow rate: 0.6 mL/min, injection volume: 0.5–1 μL, sample temperature: 5 °C, UV detection wavelength: 200–350 nm.Mass spectrometry: Exact masses (APCI and LC-MS): Deviations of the found exact masses from the calculated exact masses: 5 mDa or less. LC-HRMS: column: Kinetex® 60 RP-select B (5 µm), LiChroCART® 250-4 mm cartridge; guard Column: LiChrospher® 60 RP-select B (5 µm), LiCroCART® 4-4 mm cartridge (No.: 1.50963.0001) using manu-CART® NT cartridge holder; solvents: A: water with 0.05% (v/v) trifluoroacetic acid; B: acetonitrile with 0.05% (v/v) trifluoroacetic acid; gradient elution: (A %): 0–4 min: 90%, 4–29 min: gradient from 90% to 0%, 29–31 min: 0%, 31–31.5 min: gradient from 0% to 90%, 31.5–40 min: 90%; flow rate: 1.0 mL/min; injection volume: 5.0 µL; UV detection wavelength: 210 nm; stop time: 30.0 min. Chiral HPLC for determination of enantiomeric purity (method 2): UV detection wavelength: 210 nm; stop time: 30.0 min; parentheses include: column, solvent, flow rate, injection volume.HPLC for determination of compound purity (method 1): column: LiChrosphertert-Butyl-4-hydroxy-4-(2-vinylphenyl)piperidine-1-carboxylate (6)5, 3.1 g, 16.9 mmol) was dissolved in THF (125 mL). The solution was cooled to −78 °C under N2 atmosphere. A solution of n-butyllithium in hexanes  was added dropwise and the mixture was stirred for 15 min. Then tert-butyl 4-oxopiperidine-1-carboxylate , dissolved in THF (50 mL), was added and the mixture was stirred at −78 °C for 2.5 h. Then the solution was warmed to ambient temperature. A solution of LiBH4 in THF  was added dropwise and the mixture was stirred for 1 h at ambient temperature. The reaction was stopped by the addition of water and a 1 M aqueous solution of HCl. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give 6 as a colorless solid , mp 104 °C, yield 3.83 g (75%). C18H25NO3 (303.4 g/mol). Purity (HPLC method 1): 99.4%, tR = 20.4 min. Exact mass (APCI): m/z = 304.1882 . 1H-NMR (CDCl3): δ (ppm) = 1.47 3), 1.64 , 1.93–2.10 2), 3.32–3.36 2), 3.89–4.13 2), 5.28 , 5.51 , 7.24–7.31 , 7.34–7.39 , 7.45–7.50 , 7.65 . 13C NMR (CDCl3): δ (ppm) = 28.6 3), 37.4 2), 39.5 2), 40.3 2), 72.5 , 79.6 3), 115.7 , 124.9 , 127.7 , 127.8 , 129.1 , 137.9 , 138.0 , 143.8 , 155.0 3). FT-IR (neat): (cm−1) = 3387 (O-H), 2967, 2932, (C-H), 1655 (C=O), 756 .2-Bromostyrene (tert-Butyl (R)-4-[2-phenyl]-4-hydroxypiperidine-1-carboxylate (R)-7tert-butyl alcohol (600 mL) and water (600 mL). The mixture was cooled to 0 °C, 6  was added and the reaction mixture was stirred at 0 °C for 3 d. Then sodium sulfite (29 g) was added and the mixture was allowed to warm to room temperature and stirred for 20 min. Ethyl acetate was added to the reaction mixture, and after separation of the layers, the aqueous layer was extracted with ethyl acetate (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give R)-7, mp 93 °C, yield 5.4 g (82%). C18H27NO5 (337.4 g/mol). Specific rotation: : = ‒24.5 . Purity (HPLC method 1): 99.5%, tR = 15.3 min.AD-mix-β (27.1 g) was added to a mixture of tert-Butyl (S)-4-phenyl]-4-hydroxypiperidine-1-carboxylate (S)-7tert-butyl alcohol (325 mL) and water (325 mL). The mixture was cooled to 0 °C, 6  was added and the reaction mixture was stirred overnight at 0 °C. Then methanesulfonamide  was added and the mixture was stirred overnight at ambient temperature. Then sodium sulfite (16.2 g) was added and the mixture stirred for 30 min. Ethyl acetate was added to the reaction mixture, and after separation of the layers, the aqueous layer was extracted with ethyl acetate (3×). The combined organic layers were washed with a 2 M aqueous solution of NaOH, dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give S)-7, mp 87 °C, yield 2.7 (74%) C18H27NO5 (337.4 g/mol). Specific rotation: : = +25.8 . Purity (HPLC method 1): 95.5%, tR = 15.5 min.AD-mix-α (15.2 g) was added to a mixture of R)-7( and S)-7. 1H-NMR (CDCl3): δ (ppm) = 1.47 3), 1.82–2.11 2), 3.24 2), 3.78 , 3.86 , 3.95–4.05 2), 5.64 , 7.23–7.32 , 7.48–7.52 . Signals for the OH protons are not visible in the spectrum. 13C-NMR (CDCl3): δ (ppm) = 28.6 3), 38.3 2), 38.6 2), 39.4 2), 40.0 2), 68.0 , 72.1 , 72.7 , 79.8 3), 125.6 , 127.8 , 127.9 , 129.1 , 139.5 , 145.0 , 155.1 3). IR (neat):  (cm−1) = 3387 (O-H), 2974, 2925, (C-H), 1663 (C=O), 1246, 1161 (C-O-C ester), 756 .LC-HRMS: tert-Butyl (S)-3-[(tosyloxy)methyl]-3H-spiro[[2]benzofuran-1,4′-piperidine]-1′-carboxylate (S)-8R)-7 was dissolved in CH2Cl2 (10 mL). 4-Dimethylaminopyridine , triethylamine  and 4-toulenesulfonyl chloride  were added and the mixture was stirred for 3 h at ambient temperature. Then water was added and after separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give S)-8, mp 124 °C, yield 69 mg (50%). C25H31NO6S (473.6 g/mol). Specific rotation: : = +19.2 . Purity (HPLC method 1): 98.8%, tR = 22.8 min. Exact mass (APCI): m/z = 474.1969 . 1H-NMR (CDCl3): δ (ppm) = 1.48 3), 1.51–1.70 2), 1.70 2), 1.82 2), 2.44 , 3.02 2), 3.12 2), 3.95–4.07 2), 4.15 , 4.24 , 5.37 , 7.04–7.08 , 7.12–7.17 , 7.24–7.35 ), 7.71–7.75 . 13C-NMR (CDCl3): δ (ppm) = 21.8 , 28.6 3), 37.2 2), 37.9 2), 40.4 2), 40.6 2), 72.2 , 79.4 , 79.6 3), 85.4 , 121.1 , 122.0 , 128.1 , 128.3 , 128.8 , 130.0 , 133.0 , 136.9 , 145.1 , 145.8 , 155.1 3). IR (neat):  (cm−1) = 2978, 2870 (C-H), 1686 (C=O), 1362 (O=S=O), 1234, 1173 , 1069 , 768 .tert-Butyl (R)-4-hydroxy-3,4-dihydrospiro[[2]benzopyran-1,4′-piperidine]-1′-carboxylate (R)-11R)-7 was dissolved in THF (25 mL). Dibutyltin oxide , triethylamine  and toluene-4-sulfonyl chloride  were added and the mixture was stirred for 3 h at ambient temperature. Then water and CH2Cl2 were added. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give R)-11, mp 161 °C, yield 538 mg (62%). C18H25NO4 (319.4 g/mol). Specific rotation: : = −4.9 . Purity (HPLC method 1): 96.0%, tR = 18.6 min. Enantiomeric ratio : (R):(S) = 92.5:7.5, tR = 11.4 min.tert-Butyl (S)-4-hydroxy-3,4-dihydrospiro[[2]benzopyran-1,4′-piperidine]-1′-carboxylate (S)-11S)-7 was dissolved in THF (15 mL). Dibutyltin oxide , triethylamine  and toluene-4-sulfonyl chloride  were added and the mixture was stirred for 3 days at ambient temperature. Then water and CH2Cl2 were added. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give S)-11, mp 158 °C, yield 447 mg (47%). C18H25NO4 (319.4 g/mol). Specific rotation:: = +5.2 . Purity (HPLC method 1): 98.3%, tR = 18.3 min. Enantiomeric ratio : (R):(S) = 11.4:88.6, tR = 20.7 min.R)-11( and S)-11. 1H-NMR (CDCl3): δ (ppm) = 1.49 3), 1.69–1.83 2), 1.90–2.05 2), 3.11 2), 3.22 2), 3.92 , 3.98 , 3.99–4.07 2), 4.54 , 7.10 , 7.25–7.34 , 7.42 . A signal for the OH proton is not visible in the spectrum. 13C-NMR (CDCl3): δ (ppm) = 28.6 3), 34.3 2), 37.6 2), 39.5 2), 40.1 2), 64.8 , 66.0 , 73.8 , 79.7 3), 125.2 , 127.3 , 128.6 , 129.1 , 135.3 , 141.2 , 155.1 3). IR (neat):  [cm−1] = 3314 (O-H), 2974, 2928 (C-H), 1686 (C=O), 1169 (C-O-C ether), 768 .Exact mass (APCI): R)-1′-Benzyl-3,4-dihydrospiro[[2]benzopyran-1,4′-piperidin]-4-ol (R)-12(R)-11 was dissolved in CH2Cl2 (4 mL). The solution was cooled to 0 °C. Then trifluoroacetic acid (200 μL) was added and the mixture was stirred for 3.5 h at 0 °C. Then a 2 m aqueous solution of sodium hydroxide (4 mL) was added, and after separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The residue was dissolved in CH2Cl2 (5 mL), benzaldehyde  and sodium triacetoxyborohydride  were added and the mixture was stirred overnight at ambient temperature. The reaction was stopped by the addition of a 2 M aqueous solution of sodium hydroxide, and after separation of the layers, the aqueous layer was extracted with CH2Cl2 (4×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give R)-12, mp 55 °C), yield 57 mg (56%). C20H23NO2 (309.4 g/mol). Specific rotation: : = −8.4 . Purity (HPLC method 1): 95.3%, tR = 13.5 min. Enantiomeric ratio : (R):(S) = 96.1:3.9, tR = 9.7 min).S)-1′-Benzyl-3,4-dihydrospiro-4-ol (S)-12(R)-11 was dissolved in CH2Cl2 (10 mL). Trifluoroacetic acid (200 μL) was added and the mixture was stirred overnight at ambient temperature. Then water was added, and after separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The residue was dissolved in CH2Cl2 (10 mL), benzaldehyde  and sodium triacetoxyborohydride  were added and the mixture was stirred for 5.5 h at ambient temperature. Then benzaldehyde  and sodium triacetoxyborohydride  were added and the mixture was stirred overnight at ambient temperature). The reaction was stopped by the addition of a 2 M aqueous solution of sodium hydroxide, and after separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give S)-12 mp 53 °C, yield 12 mg (22%). C20H23NO2 (309.4 g/mol). Specific rotation: : = +8.8 . Purity (HPLC method 1): 98.3%, tR = 13.4 min. Enantiomeric ratio : (R):(S) = 11.9:88.1, tR = 12.9 min.R)-12( and S)-12. 1H-NMR (CDCl3): δ (ppm) = 1.78–1.98 2), 2.18 2), 2.39 2), 2.46–2.54 2), 2.75 2), 3.58 , 3.89 , 3.97 , 4.51 , 7.20–7.41 . A signal for the OH proton is not visible in the spectrum. 13C-NMR (CDCl3): δ (ppm) = 34.7 2), 38.0 2), 49.3 2), 49.3 2), 63.5 , 64.5 , 66.1 , 73.9 , 125.3 , 127.0 , 127.1 , 128.3 , 128.5 , 128.9 , 129.4 , 135.5 , 138.6 , 141.8 . IR (neat): (cm−1) = 3329 (O-H), 2924, 2817 (C-H), 1072 (C-O-C), 733 , 698 (monosubst. arom.).Exact mass (APCI): tert-Butyl (R)-4-methoxy-3,4-dihydrospiro[[2]benzopyran-1,4′-piperidine]-1′-carboxylate (R)-13R)-10 was dissolved in THF (12 mL). NaH  was added and the mixture was stirred for 1 h at ambient temperature. Then iodomethane  was added dropwise and the mixture was stirred for 1 h at ambient temperature. The solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give R)-13, yield 465 mg (99%). C19H27NO4 (333.4 g/mol). Specific rotation:= −8.0 . Purity (HPLC method 1): 96.6%, tR = 20.4 min.tert-Butyl (S)-4-methoxy-3,4-dihydrospiro-1′-carboxylate (S)(-13)S)-10 was dissolved in THF (2.5 mL). NaH  was added and the mixture was stirred for 1 h at ambient temperature. Then iodomethane  was added dropwise and the mixture was stirred overnight at ambient temperature. The solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give S)-13, yield 128 mg (69%). C19H27NO4 (333.4 g/mol). Specific rotation: : = +7.4 . Purity (HPLC method 1): 97.7%, tR = 20.4 min.R)-13( and S)-13. 1H-NMR (CDCl3): δ (ppm) = 1.49 3), 1.75 2), 1.84–1.99 2), 3.03–3.28 2), 3.50 , 3.93–4.06 2 (2), CHCH2O (2)), 4.19 , 7.11 , 7.25 , 7.30 , 7.37 . 13C-NMR (CDCl3): δ (ppm) = 28.6 3), 34.9 2), 37.0 2), 39.4 2), 40.2 2), 56.9 , 61.6 , 73.5 , 74.0 , 79.5 3), 125.1 , 126.7 , 128.4 , 129.1 , 132.8 , 141.8 , 155.0 3). IR (neat): (cm−1) = 2970, 2928 (C-H), 1686 (C=O), 1084 (C-O-C ether), 756 .Exact mass (APCI): tert-Butyl (R)-4-ethoxy-3,4-dihyrospiro[[2]benzopyran-1,4′-piperidine]-1′-carboxylate (R)-14R)-10 was dissolved in THF (15 mL). NaH  was added and the mixture was stirred for 1 h at ambient temperature. Then iodoethane  was added dropwise and the mixture was stirred for 2.5 h at ambient temperature. A 1 m solution of lithium bis(trimethylsilyl)amide (4.5 mL) was added and the mixture was heated to reflux overnight. The mixture was allowed to cool to ambient temperature and stirred overnight. Then water and CH2Cl2 were added. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent removed in vacuo. The crude product was purified by flash column chromatography  to give R)-14, yield 66 mg (29%). C20H29NO4 (347.4 g/mol). Specific rotation: : = −2.2 . Purity (HPLC method 1): 96.3%, tR = 21.3 min.tert-Butyl (S)-4-ethoxy-3,4-dihydrospiro[[2]benzopyran-1,4′-piperidine]-1′-carboxylate (S)-14S)-10 was dissolved in THF (5 mL). A 1 m solution of lithium bis(trimethylsilyl)amide (6.3 mL) was added and the mixture was stirred for 1 h at ambient temperature. Then iodoethane  was added dropwise and the mixture was stirred for 16 h at ambient temperature. NaH  and iodoethane  were added and the mixture was heated to reflux overnight. The mixture was allowed to cool to ambient temperature and stirred for 3 days. Then water was added. After separation of the layers, the aqueous layer was extracted with ethyl acetate (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography three times  to give S)-14, yield 110 mg (50%). C20H29NO4 (347.4 g/mol). Specific rotation:: = +2.4 . Purity (HPLC method 1): 98.4%, tR = 21.5 min.R)-14( and S)-14. 1H-NMR (CDCl3): δ (ppm) = 1.28 , 1.49 3), 1.76–1.86 2), 1.86–1.96 2), 3.04–3.26 2), 3.64–3.78 , 3.86–4.08 2), 3.89 , 4.00 , 4.35 , 7.08 , 7.20–7.32 , 7.42 . 13C-NMR (CDCl3): δ (ppm) = 15.9 , 28.7 3), 35.8 2), 36.4 2), 39.5 2), 40.3 2), 62.1 , 64.9 , 72.4 , 73.7 3), 79.6 , 125.1 , 126.8 , 128.1 , 128.5 , 134.0 , 141.8 , 155.1 3). IR (neat): (cm−1) = 2970, 2928 (C-H), 1690 (C=O), 1092 (C-O-C ether), 756 .Exact mass (APCI): R)-1′-Benzyl-4-methoxy-3,4-dihydrospiro[[2]benzopyran-1,4′-piperidine] (R)-15(R)-13  was dissolved in CH2Cl2 (5 mL). The solution was cooled to 0 °C. Trifluoroacetic acid (0.7 mL) was added and the mixture was stirred for 2 h at 0 °C. Then a 2 M aqueous solution of NaOH was added. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The residue was dissolved in CH2Cl2 (5 mL). Benzaldehyde  and sodium triacetoxyborohydride  were added and the mixture was stirred for 26 h at ambient temperature. Then a 2 M aqueous solution of NaOH (3 mL) and water (3 mL) were added. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give R)-15, yield 31 mg (9%). C21H25NO2 (323.4 G/mol). Specific rotation: : = −8.6 . Purity (HPLC method 1): 98.2%, tR = 15.8 min. Enantiomeric ratio : (R):(S) = 94.8:5.2, tR = 7.2 min.(S)-1′-Benzyl-4-methoxy-3,4-dihydrospiro[[2]benzopyran-1,4′-piperidine] (S)-15(S)-13 was dissolved in CH2Cl2 (5 mL). Trifluoroacetic acid (200 μL) was added and the mixture was stirred for 4.5 h at ambient temperature. Then a 2 M aqueous solution of NaOH was added. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The residue was dissolved in CH2Cl2 (10 mL). Benzaldehyde  and sodium triacetoxyborohydride  were added and the mixture was stirred overnight at ambient temperature. The reaction was stopped by the addition of a 2 M aqueous solution of NaOH. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography twice  to give S)-15, yield 39 mg (80%). C21H25NO2 (323.4 g/mol). Specific rotation: : = +7.7 . Purity (HPLC method 1): 97.3%, tR = 15.6 min. Enantiomeric ratio : (R):(S) = 9.0:91.0, tR = 8.6 min.R)-15( and S)-15. 1H-NMR (CDCl3): δ (ppm) = 1.87–1.95 2), 2.13 2), 2.36–2.44 2), 2.51 2), 2.70–2.79 2), 3.50 , 3.56 , 3.60 , 3.93–4.06 , 4.19 , 7.21–7.39 . 13C-NMR (CDCl3): δ (ppm) = 35.5 2), 37.3 2), 49.4 2), 49.4 2), 56.9 , 61.3 , 63.5 , 73.5 , 74.1 , 125.3 , 126.5 , 127.1 , 128.3 , 128.3 , 129.0 , 129.4 , 133.1 , 138.7 , 142.5 . IR (neat): (cm−1) = 2924, 2816 (C-H), 1088 (C-O-C), 737 , 737 (monosubst. arom.).Exact mass (APCI): R)-1′-Benzyl-4-ethoxy-3,4-dihydrospiro[[2]benzopyran-1,4′-piperidine] (R)-16(R)-14 was dissolved in CH2Cl2 (10 mL). Trifluoroacetic acid (200 μL) was added and the mixture was stirred for 3 h at ambient temperature. Then a 2 M aqueous solution of NaOH (10 mL) was added. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The residue was dissolved in CH2Cl2 (10 mL). Benzaldehyde  and (after 45 min) sodium triacetoxyborohydride  were added and the mixture was stirred overnight at ambient temperature. The reaction was stopped by the addition of a 2 M aqueous solution of NaOH. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (2×) and ethyl acetate (1×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography twice  to give R)-16, yield 33 mg (70%). C22H27NO2 (337.5 g/mol). Specific rotation: : = −3.3 . Purity (HPLC method 1): 99.1%, tR = 17.0 min.S)-1′-Benzyl-4-ethoxy-3,4-dihydrospiro[[2]benzopyran-1,4′-piperidine] (S)-16(S)-14 was dissolved in CH2Cl2 (5 mL). Trifluoroacetic acid (300 μL) was added and the mixture was stirred for 2 h at ambient temperature. Then a 2 M aqueous solution of NaOH was added. After separation of the layers, the aqueous layer was extracted with ethyl acetate (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The residue was dissolved in CH2Cl2 (5 mL). Benzaldehyde  and after 15 min, sodium triacetoxyborohydride  were added and the mixture was stirred at ambient temperature for 8 h. The reaction was stopped by the addition of a 2 M aqueous solution of NaOH. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give (S)-16 as a pale yellow oil , yield 28 mg (46%). C22H27NO2 (337.5 g/mol). Specific rotation: : = +2.6 . Purity (HPLC method 1): 96.6%, tR = 17.0 min.R)-16( and S)-16 1H-NMR (CDCl3): δ (ppm) = 1.20 , 1.74–2.04 2), 2.28–2.24 2), 2.62–2.70 2), 3.50 , 3.58–3.68 , 3.79 , 3.93 , 4.28 , 7.11–7.35 . 13C-NMR (CDCl3): δ (ppm) = 15.9 , 36.3 2), 36.7 2), 49.3 2), 49.5 2), 61.8 , 63.6 , 64.8 , 72.5 , 73.7 , 125.2 , 126.6 , 127.1 , 128.0 , 128.2 , 128.3 , 129.4 , 134.2 , 138.7 , 142.4 . IR (neat): (cm−1) = 2928, 2812 (C-H), 1092 (C-O-C ether), 737 , 698 (monosubst. arom.).Exact mass (APCI): tert-Butyl (R)-4-(2-ethoxy-2-oxoethoxy)-3,4-dihydrospiro[[2]benzopyran-1,4′-piperidine]-1′-carboxylate ((R)-17)R)-11 was dissolved in THF (60 mL). A 1 M solution of lithium bis(trimethyl-silyl)amide  was added and the mixture was stirred for 1 h at ambient temperature. Then ethyl 2-bromoacetate  and tetrabutylammonium iodide  were added and the mixture was heated to reflux overnight. The solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give R)-17, yield 1.2 g (59%). C22H31NO6 (405.5 g/mol). Specific rotation:: = −14.7 . Purity (HPLC method 1): 97.3%, tR = 21.3 min.tert-Butyl (S)-4-(2-ethoxy-2-oxoethoxy)-3,4-dihydrospiro-1′-carboxylate (S)-17S)-11 was dissolved in THF (50 mL). A 1 M solution of lithium bis(trimethyl-silyl)amide  was added and the mixture was stirred for 45 min at ambient temperature. Then ethyl 2-bromoacetate  and tetrabutylammonium iodide  were added and the mixture was heated to reflux overnight. The solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give S)-17, yield 1.4 g (55%). C22H31NO6 (405.5 g/mol). Specific rotation: : = +14.3 . Purity (HPLC method 1): 96.0%, tR = 20.9 min.R)-17( and S)-17. 1H-NMR (CDCl3): δ (ppm) = 1.29 , 1.49 3), 1.68–1.79 2), 1.83–2.02 2), 3.01–3.29 2), 3.93–4.11 2 (2H), CHCH2O (2H)), 4.17-4.28 , OCH2CO2 (2)), 4.52 , 7.11 , 7.25–7.35 , 7.55 . 13C-NMR (CDCl3): δ (ppm) = 14.4 , 28.7 3), 34.7 2), 37.1 2), 39.5 2), 40.1 2), 61.0 , 62.0 , 65.7 , 72.6 , 73.6 3), 79.6 , 125.0 , 127.0 , 128.8 , 129.5 , 131.8 , 142.1 , 155.0 3), 170.9 . IR (neat): (cm−1) = 2974, 2928 (C-H), 1751, 1690 (C=O), 1165, 1099 (C-O-C ether), 759 .Exact mass (APCI): R)-2-[oxy]acetate (R)-18Ethyl (R)-17 was dissolved in CH2Cl2 (4 mL). Trifluoroacetic acid (200 μL) was added and the mixture was stirred overnight at ambient temperature. Then water was added. After separation of the layers, the aqueous layer was extracted with ethyl acetate (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The residue was dissolved in CH2Cl2 (2 mL). Benzaldehyde  and sodium triacetoxyborohydride  were added and the mixture was stirred at ambient temperature for 4 days. The reaction was stopped by the addition of a 2 M aqueous solution of NaOH. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give R)-18, yield 36 mg (43%). C24H29NO4 (395.5 g/mol). Specific rotation: : = −16.0 . Purity (HPLC method 1): 95.4%, tR = 17.7 min.S)-2-[oxy]acetate (S)-18Ethyl (S)-17 was dissolved in CH2Cl2 (60 mL). Trifluoroacetic acid (3.5 mL) was added and the mixture was stirred for 7 h at ambient temperature. Then a 2 M aqueous solution of NaOH was added. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The residue was dissolved in CH2Cl2 (50 mL). Benzaldehyde  and, after 15 min, sodium triacetoxyborohydride  were added and the mixture was stirred overnight at ambient temperature. The reaction was stopped by the addition of a 2 M aqueous solution of NaOH and worked up as described for R)-18 to give S)-18, yield 36 mg (43%). C24H29NO4 (395.5 g/mol). Specific rotation:: = +15.3 . Purity (HPLC method 1): 93.1%, tR = 17.3 min.R)-18( and S)-18. 1H-NMR (CDCl3): δ (ppm) = 1.30 , 1.87–1.95 2), 2.16 2), 2.34–2.45 2), 2.47–2.55 2), 2.70–2.81 2), 3.57 , 3.61 , 3.98 , 4.05 , 4.16–4.29 , OCH2CO2 (2H)), 4.53 , 7.22–7.41 , 7.53–7.57 . 13C-NMR (CDCl3): δ (ppm) = 14.4 , 35.1 2), 37.4 2), 49.3 2), 49.4 2), 61.0 , 61.7 , 63.5 , 65.6 , 72.7 , 73.6 , 125.2 , 126.8 , 127.1 , 128.3 , 128.6 , 129.4 , 129.4 , 132.0 , 138.6 , 142.8 , 170.9 . IR (neat): (cm−1) = 2920, 2866 (C-H), 1748 (C=O), 1099, 1053 (C-O-C ether), 737 , 698 (monosubst. arom.).Exact mass (APCI): R)-2-[oxy]ethanol (R)-19(R)-18 was dissolved in THF (5 mL). A 1 m solution of LiAlH4 in THF  was added and the mixture was stirred overnight at ambient temperature. Then water was added. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were washed with water (2×) and brine (1×), dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give R)-19, yield 254 mg (59%). C22H27NO3 (353.5 g/mol). Specific rotation: : = −2.2 . Purity (HPLC method 1): 95.1%, tR = 13.8 min.S)-2-[oxy]ethanol (S)-19(S)-18 was dissolved in THF (10 mL). A 1 M solution of LiAlH4 in THF  was added and the mixture was stirred overnight at ambient temperature. Then water and CH2Cl2 were added. The reaction was worked up as described for R)-19 to give S)-19, yield 302 mg (75%). C22H27NO3 (353.5 g/mol). Specific rotation:: = +2.1 . Purity (HPLC method 1): 94.3%, tR = 13.9 min.R)-19( and S)-19. 1H-NMR (CDCl3): δ (ppm) = 1.85–1.99 2), 2.13 2), 2.30–2.44 , 2.40 2), 2.45–2.53 2), 2.70–2.79 2), 3.58 , 3.72–3.81 , 3.94 , 4.01 , 4.36 , 7.22–7.39 . 13C-NMR (CDCl3): δ (ppm) = 35.4 2), 37.4 2), 49.3 2), 49.4 2), 61.6 , 62.2 , 63.5 , 70.1 , 73.1 , 73.7 ,125.4 , 126.7 , 127.1 , 128.3 , 128.5 , 128.9 , 129.4 , 133.1 , 138.7 , 142.5 . IR (neat): (cm−1) = 3399 (O-H), 2924, 2816 (C-H), 1092, 1076 , 741 , 698 (monosubst. arom.).Exact mass (APCI): R)-1′-Benzyl-4-(2-fluoroethoxy)-3,4-dihydrospiro[[2]benzopyran-1,4′-piperidine] (R)-20 and triethylamine trihydrofluoride  were dissolved in CH2Cl2 (1 mL). The solution was cooled to −78 °C. R)-19 was added and the mixture was stirred at ‒78 °C for 1 h, then at 0 °C for 1 h and at ambient temperature for 1 h. A 5% aqueous solution of NaHCO3 (3 mL) was added and the mixture was stirred for 15 min. The aqueous layer was extracted with CH2Cl2 (2×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give R)-20, yield 27 mg (54%). C22H26FNO2 (355.4 g/mol). Specific rotation: : = −6.4 . Purity (HPLC method 1): 97.0%, tR = 16.4 min.(Diethylamino)difluorosulfonium tetrafluoroborate -1′-Benzyl-4-(2-fluoroethoxy)-3,4-dohydrospiro[[2]benzopyran-1,4′-piperidine] (S)-20 and triethylamine trihydrofluoride  were dissolved in CH2Cl2 (2 mL). The solution was cooled to −78 °C. S)-19 dissolved in CH2Cl2 (2 mL), was added and the mixture was stirred at -78 °C for 1 h, then at 0 °C for 1 h and at ambient temperature overnight. A 20% aqueous solution of NaHCO3 (4 mL) was added and the mixture was stirred for 15 min. The aqueous layer was extracted with CH2Cl2 (2×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give S)-20, yield 86 mg (71%). C22H26FNO2 (355.4 g/mol). Specific rotation: : = +5.9 . Purity (HPLC method 1): 95.1%, tR = 16.1 min.(Diethylamino)difluorosulfonium tetrafluoroborate -20( and S)-20. 1H-NMR (CDCl3): δ (ppm) = 1.85–2.00 2), 2.10 2), 2.39 2), 2.48 2), 2.69–2.78 2), 3.57 , 3.78–3.87 , 3.87–3.94 , 3.94 , 4.01 , 4.45 , 4.59 , 7.21–7.38 , 7.41–7.44 . 13C-NMR (CDCl3): δ (ppm) = 35.7 2), 37.0 2), 49.3 2), 49.4 2), 61.8 , 63.5 , 68.0 , 73.1 , 73.6 , 83.5 , 125.3 , 126.7 , 127.2 , 128.4 , 128.7 , 129.5 , 129.6 , 133.0 , 138.4 , 142.5 . IR (neat): (cm−1) = 2934, 2812 (C-H), 1096 , 737 , 698 (monosubst. arom.).Exact mass (APCI): R)-2-[oxy]ethyl} 4-methylbenzene-sulfonate (R)-21{(R)-19 was dissolved in CH2Cl2 (13 mL). 4-Dimethylaminopyridine , triethylamine  and 4-toulenesulfonyl chloride  were added and the mixture was stirred overnight at ambient temperature. Then a 2 M aqueous solution of NaOH was added. After separation of the layers, the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash column chromatography  to give R)-21, yield 59 mg (46%). C29H33NO5S (507.6 g/mol). Specific rotation: : = −5.9 . Purity (HPLC method 1): 94.5%, tR = 20.3 min.S)-2-[oxy]ethyl} 4-methylbenzene-sulfonate (S)-21{(S)-19 was dissolved in CH2Cl2 (20 mL). Triethylamine , 4-dimethylaminopyridine  and 4-toulenesulfonyl chloride  were added and the mixture was stirred overnight at ambient temperature. Then reaction was worked up as described for R)-21 to give S)-21, yield 66 mg (32%). C29H33NO5S (507.6 g/mol). Specific rotation:  : = +7.5 . Purity (HPLC method 1): 95.1%, tR = 19.6 min.R)-21( and S)-21. 1H-NMR (CDCl3): δ (ppm) = 1.78–1.90 2), 1.91–2.03 2), 2.14 2), 2.41 , 2.45–2.62 2), 2.77–2.82 2), 3.62 , 3.70–3.87 , CHCH2O (1H)), 3.91 , 4.09–4.25 , 4.33 , 7.08–7.57 , 7.71–7.83 . 13C-NMR (CDCl3): δ (ppm) = 21.8 , 35.2 2), 36.8 2), 49.2 2), 49.3 2), 61.7 , 63.3 , 66.1 , 69.7 , 73.0 , 73.4 , 125.2 , 126.1 , 126.7 , 128.1 , 128.4 , 128.7 , 128.8 , 129.6 , 129,9 , 132.2 , 133.0 , 133.9 , 144.9 , 146.0 . IR (neat): [cm−1] = 2924, 2812 (C-H), 1358(m), 1177 (O=S=O), 1096 (C-O-C), 741 , 698 (monosubst. arom.).Exact mass (APCI): 1 and σ2 receptor binding assays were commercially available . Homogenizer: Elvehjem Potter . Cooling centrifuge model Rotina 35R  and High-speed cooling centrifuge model Sorvall RC-5C plus . Multiplates: standard 96-well multiplates . Shaker: self-made device with adjustable temperature and tumbling speed (scientific workshop of the institute). Vortexer: Vortex Genie 2 . Harvester: MicroBeta FilterMate-96 Harvester. Filter: Printed Filtermat Typ A and B. Scintillator: Meltilex (Typ A or B) solid state scintillator. Scintillation analyzer: MicroBeta Trilux . Chemicals and reagents were purchased from different commercial sources and of analytical grade.The guinea pig brain and rat liver for the σg for 10 min at 4 °C. The supernatant was separated and centrifuged at 23,500 × g for 20 min at 4 °C. The pellet was resuspended in 5‑6 volumes of buffer  and centrifuged again at 23,500 × g . This procedure was repeated twice. The final pellet was resuspended in 5‑6 volumes of buffer and frozen (−80 °C) in 1.5 mL portions containing about 1.5 mg protein/mL.According to ,36,37: fg for 10 min at 4 °C. The supernatant was separated and centrifuged at 31,000 × g for 20 min at 4 °C. The pellet was resuspended in 5–6 volumes of buffer  and incubated at room temperature for 30 min. After the incubation, the suspension was centrifuged again at 31,000 × g for 20 min at 4 °C. The final pellet was resuspended in 5–6 volumes of buffer and stored at −80 °C in 1.5 mL portions containing about 2 mg protein/mL.According to ,36,37: t2O and 5 mL phosphoric acid  were added to this solution, the mixture was stirred and filled to a total volume of 50.0 mL with deionized water. The calibration was carried out using bovine serum albumin as a standard in 9 concentrations . In a 96‑well standard multiplate, 10 µL of the calibration solution or 10 µL of the membrane receptor preparation were mixed with 190 µL of the Bradford solution, respectively. After 5 min, the UV absorption of the protein-dye complex at λ = 595 nm was measured with a platereader .The protein concentration was determined by the method of Bradford , modifie−5, 10−6, 10−7, 10−8, 10−9 and 10−10 mol/L), 50 µL of corresponding radioligand solution and 50 µL of the respective receptor preparation into each well of the multiplate . The receptor preparation was always added last. During the incubation, the multiplates were shaken at a speed of 500–600 rpm at the specified temperature. Unless otherwise noted, the assays were terminated after 120 min by rapid filtration using the harvester. During the filtration each well was washed five times with 300 µL of water. Subsequently, the filtermats were dried at 95 °C. The solid scintillator was melted on the dried filtermats at a temperature of 95 °C for 5 min. After solidifying of the scintillator at room temperature, the trapped radioactivity in the filtermats was measured with the scintillation analyzer. Each position on the filtermat corresponding to one well of the multiplate was measured for 5 min with the [3H]-counting protocol. The overall counting efficiency was 20%. The IC50-values were calculated with the program GraphPad Prism® 3.0  by non-linear regression analysis. Subsequently, the IC50 values were transformed into Ki-values using the equation of Cheng and Prusoff -(+)-pentazocine . The thawed membrane preparation of guinea pig brain cortex (about 100 μg of protein) was incubated with various concentrations of test compounds, 2 nM [3H]-(+)-pentazocine, and TRIS buffer  at 37 °C. The non-specific binding was determined with 10 μM unlabeled (+)‑pentazocine. The Kd-value of (+)-pentazocine is 2.9 nM DTG . The thawed membrane preparation of rat liver (about 100 µg of protein) was incubated with various concentrations of the test compound, 3 nM [3H]DTG and buffer containing (+)-pentazocine (500 nM (+)-pentazocine in 50 mM TRIS, pH 8.0) at room temperature. The non-specific binding was determined with 10 μM non-labeled DTG. The Kd value of [3H]DTG is 17.9 nM F-K222-carbonate complex. To this mixture a solution of tosylate precursor R)-21( (2.0–2.5 mg) in acetonitrile (0.5 mL) was added and the reaction mixture was heated to 82 °C for 20 min . These optimized reaction conditions led to reproducible labelling yields of 25%–32% (n = 8) as determined by radio-TLC (petroleum ether-ethyl acetate 7: 3 (v/v). TLC retention values: [18F]R)-20--21: Rf = 0.27.Aqueous [18F]-R), phosphate-saline-solution  and 0.01 M TRIS-HCl (pH 7.4). In-vitro stability in native mouse plasma was investigated by incubation of 200 µL plasma plus 5 µL [18F]-R)-20( (~50 MBq) in isotonic NaCl (containing 10% ethanol) for 30 min at 37 °C.For pharmacological characterization of the radiotracer [18F]-(R)-20, the distribution coefficient logD was determined by conventional shake-flask experiments using n-octanol/phosphate buffer pH 7.2, n-octanol/phosphate-saline-solution pH 7.2 and n-octanol/TRIS-HCl pH 7.4 extraction systems. The amount of the radiotracer [18F]-(R)-20 in the respective layer was determined using a calibrated γ-counter . The determination of the logD7,4 value by HPLC  was achieved after calibration using reference compounds following the EU guideline 67/548/EWG based on OECD guidelines 2004 -(S)-20 was recorded six times. Furthermore, the logD value was calculated via ACD ChemSketch2012 software.To get information about the blood-brain barrier permeability of -R)-20 into the left or right vena caudalis lateralis. Metabolites were investigated in plasma, urine, brain and liver samples. Urine samples were analyzed directly. Plasma samples were obtained by centrifugation of EDTA blood  obtained by heart puncture. Brain and liver samples were acquired by homogenization of the organs in ice-cold 50 mM TRIS-HCl buffer (pH 7.4) using a PotterS® Homogeizer (B. Braun). The tissues were treated in a borosilicate glass cylinder by 10 strokes of a PTFE plunge at a speed of 800–1000 min−1.The metabolism of -(R)-20 was administered via right vena caudalis lateralis without (31.0 MBq) or with (31.7 MBq) co-application 1 mg/kg haloperidol   and a longer reaction time did not lead to reproducible high yields of diol (R)-7. It was assumed that the reason for the low yields of (R)-7 was the low solubility of alkene 6 in a 1:1 tert-butanol-water solvent mixture. Despite the addition of different cosolvents  to the solvent mixture, the yields were not improved. However, the yield was considerably raised when increasing the amount of solvent mixture from 10 mL to 60 mL/mmol alkene. The (R)-configured triol (R)-7 was obtained in a reproducible yield of 82%. Analogously the (S)-configured enantiomer (S)-7 was accessible in 74% yield, when using AD-mix-α for the dihydroxylation step.The Sharpless Asymmetric Dihydroxylation of terminal alkene 6 with AD-mix-β employing the standard protocol ,46,47, lR)-7 . IncreasR)-7 with tosyl chloride, NEt3 and DMAP provided the 2-benzofuran (S)-8 in 50% yield. It is assumed that tosyl chloride reacts predominantly with the primary OH-moiety of triol (R)-7. In the presence of base the resulting primary tosylate forms an oxirane, which is opened by the tertiary alcohol giving the five-membered 2-benzofuran. Finally the primary alcohol reacts with a second equivalent tosyl chloride to form the tosylate (S)-8. Reaction of 2-(2-bromophenyl)oxirane (R)-9 with n-BuLi and subsequently with 1-Boc-piperidin-4-one led to an analogous 2-benzofuran ((S)-10) upon regioselective opening of the oxirane ring by the intermediate lithium alcoholate fluorine as well as the radioactive isotope [18F]fluorine into the side chain. Reaction of (R)-19 with XtalFluor-E® provided the fluoroethoxy derivative (R)-20 in 54% yield. For the radiosynthesis the alcohol (R)-11 had to be activated for nucleophilic substitution (see S)-configured enantiomers (S)-17–20 were prepared in the same manner.In order to introduce a fluorine atom into the substituent in 4-position of the 2-benzopyran ring the alcohol (1 and σ2 receptor affinities were measured in competition experiments with radioligands. The σ1 receptor binding assay was carried out using a receptor preparation from guinea pig brain and [3H]-(+)-pentazocine as a high-affinity and selective radioligand. The σ2 receptor affinity was conducted with a receptor preparation from rat liver and [3H]di-o-toylguanidine ([3H]DTG) was used as radioligand. Since DTG is not selective for the σ2 subtype over the σ1 subtype, σ1 receptors binding sites were masked by addition of non-labeled (+)-pentazocine fluoride using the K[18F]F-K222-carbonate complex, prepared from a 1:1 mixture of K2CO3 and Kryptofix K222. Using this complex, the precursor (R)-21 was readily transformed into the 18F-labeled radiotracer by heating in acetonitrile at 82 °C for 20 min. According to radio-TLC and radio-HPLC analyses, only a few radioactive by-products were formed. Thus, the crude reaction mixture was diluted with water to 4 mL and directly applied to semi-preparative HPLC. The radiotracer eluted at ca. 32 min and was completely free from radioactive and non-radioactive impurities. -20. Combined isolated fractions were diluted with water (50 mL), adsorbed on a Sep-Pak C18 Plus cartridge and desorbed with pure MeOH in small portions. Adsorption of the total activity (≥95%) and elution with MeOH (≥95%) were achieved resulting in a total volume of 1.25 to 1.5 mL. The solvent was carefully evaporated at 60 °C, and the final product was dissolved in 0.9% NaCl solution containing 5% EtOH.The one-step introduction of urities.  Interest18F]-R)-20. The logD7.4 value obtained by HPLC method, however, differed by one order of magnitude (2.97±0.32) from these values. The range of the experimentally determined logD7.4 values is very similar to the corresponding logD7.4 values of fluspidine , which has been shown to have excellent brain uptake properties.Determination of the distribution coefficient by the shake flask method provided a logD18F]-R)-20 was similar to but not identical with the retention time of [18F]fluoride. In brain samples (n = 4) the fraction of the non-metabolized radiotracer accounted for 91%–95% (radio-TLC) and 89%–92% (radio-HPLC) with good reproducibility. Only one highly polar (hydrophilic) radiometabolite (M1) was detected. Its retention time (18F]-R)(-20 remained unchanged as determined by acetonitrile and methanol extraction). Results from radio-HPLC and radio-TLC agreed well. The recovery of total radioactivity was 75% for acetonitrile extraction and up to 90% for methanol extraction. The main radiometabolite in plasma samples was the very polar metabolite M1 . Additionally, two small peaks for metabolites M2 (tR ~24.7 min) and M3 tR ~26.0 min) were detected with an intensity of lower than 3%.Analysis of the plasma samples (n = 3) at 30 min p.i. revealed fast biotransformation (15% and 12% of [18F]-R)-20 was observed.In urine samples (n = 3), a large amount of radiometabolites and a very low amount of the parent radiotracer [18F]-R)-20 remained unchanged after 30 min. The recovery of radioactivity was 45% for acetonitrile and 89% for methanol extraction. The same radiometabolite profile as in plasma and urine samples was found.The results obtained by analysis of liver homogenates are based on a single experiment and should be treated with caution. About 50% to 56% of the parent radiotracer [18F]-R)-20(-20 in liver samples was lower (50%) compared with those of [18F]2b (69%) and [18F]2c (65%) in liver samples. These results indicate that the 2-benzopyran based radiotracer [18F]-R)-20. Although this result indicates target specific binding of [18F]-R)(-20 of 1.5 at 30 min p.i. is lower than previously reported ratios of approx. 3 at 30 min after injection of [18F]fluspidine (2b) [18F-labeled fluorobutyl-radiotracer 2d [1 affinity of [18F]-R)-20( (Ki = 4.7 nM) compared to fluspidine (2b) (Ki = 0.59 nM) and the fluorobutyl derivative 2d (Ki = 1.2 nM).In order to investigate the spatial distribution of [ine (2b)  or the cracer 2d . This ma18F]-R)guanidine [18F]-R) and the pons (pontine reticular nucleus). High to moderate uptake was noted in midbrain, cortex, hippocampus, cerebellum and layers of the olfactory bulb. Low uptake was found in parts of the olfactory bulb, striatum and basal forebrain. 2D densitometric evaluation of whole brain sections confirmed the general blocking effect of haloperidol with a mean decrease of radiotracer uptake in the slices of the whole brain hemisphere of about 40%.  showed residual binding B after c1 receptor ligands. Some of the compounds show an eudismic ratio up to 29 (ester 18), indicating an enantioselective interaction of the σ1 receptor with these ligands. The very potent fluorinated fluoroethoxy derivative R)-20( was developed as fluorinated PET-tracer. The radiosynthesis based on a one-step nucleophilic substitution of tosylate R)-21( provided the PET tracer [18F]-R)-20-20-20-20( represents a very good alternative fluorinated PET tracer for imaging of σ1 receptors in the brain.In this manuscript the asymmetric synthesis of spirocyclic 2-benzopyrans in enantiomerically pure form is described for the first time. The key step of the synthesis is the asymmetric dihydroxylation according to Sharpless, which allows the preparation of 4-substituted spirocyclic 2-benzopyrans. These compounds represent a new type of potent and subtype selective σ


Inhibitors of soluble epoxide hydrolase (sEH) represent one of the novel pharmaceutical approaches for treating hypertension, vascular inflammation, pain and other cardiovascular related diseases. Most of the potent sEH inhibitors reported in literature often suffer from poor solubility and bioavailability. Toward improving pharmacokinetic profile beside favorable potency, two series of 4-benzamidobenzoic acid hydrazide derivatives with hydrazide group as a novel secondary pharmacophore against sEH enzyme were developed. The designed compounds were synthesized in acceptable yield and their in vitro assay was determined. Most of the synthesized compounds have appropriate physical properties and exhibited considerable  EETs vessels , 4. In a vessels . Therefo vessels -6. in-vivo availability ..in-vivo According to the pharmacophore model suggested for sEH inhibitors , 13, 14,ChemistryAll laboratory grade reagents were obtained commercially from Aldrich or Merck Company. The reactions were monitored by thin layer chromatography (TLC) performed on commercially available Merck precoated plates . The structures of the synthesized compounds were confirmed by IR, LC/MS and 1HNMR. Perkin Elmer 843 IR and Agilent 6410 (QQQ) LC/MS were used to obtain IR and Mass spectra respectively.1HNMR spectra were recorded on a Bruker advance II (500 MHz) spectrophotometer using [D6] DMSO as a solvent. Water solubility was determined experimentally in 1.0 mL of sodium phosphate buffer  at 25 ± 1 °C , 15. TheGeneral procedure for the preparation of 4-(4-substitutedbenzamido)benzoic acid (2a-2e) A solution of 4-aminobenzoic acid (1.68 mmol) and para substituted benzoylchlorides (1.68 mmol) in dry THF, in presence of anhydrous Na2CO3 (1.68 mmol) was stirred at room temperature for 6-12 h. The solvent was evaporated and the precipitate was washed with water and recrystallized from ethanol 96% to give final products.4-benzamidobenzoic acid (2a)Yield: 85%; white crystalline powder; mp: 150-151 °C, IR (KBr): υ (cm-1) 2887-3045 (OH), 3340 (NH), 1675, 1650 (C=O); LC-MS (ESI) m/z = 242 , 264 .4-(4-fluorobenzamido)benzoic acid (2b)Yield: 80%; white crystalline powder; mp: 155-156 °C, IR (KBr): υ (cm-1) 2857-3125 (OH), 3379 (NH), 1696, 1685 (C=O); LC-MS (ESI) m/z = 260 , 282 .4-(4-chlorobenzamido)benzoic acid (2c)Yield: 75%; white crystalline powder; mp: 138-140 °C, IR (KBr): υ (cm-1) 2730-3045 (OH), 3320 (NH), 1695, 1670 (C=O); LC-MS (ESI) m/z = 276 , 298 .4-(4-methylbenzamido)benzoic acid (2d)Yield: 80%; white crystalline powder; mp: 145-147 °C, IR (KBr): υ (cm-1) 2630-3145 (OH), 3340 (NH), 1670, 1690 (C=O); LC-MS (ESI) m/z = 256 , 278 .4-(4-methoxybenzamido)benzoic acid (2e)Yield: 60%; cream crystalline powder; mp: 150-152 °C, IR (KBr): υ (cm-1) 2807-3015 (OH), 3320 (NH), 1695, 1660 (C=O); LC-MS (ESI) m/z = 272 , 294 .General procedure for the preparation of Ethyl 4-(4-substitutedbenzamido)benzoate (3a-3e)8.06 mmol of 2 was dissolved in ethanol (15 mL) and concentrated sulfuric acid (0.5 mL) was added. The solution was refluxed for 24 h. Then, ethanol was evaporated and the remnant was alkalized after being cooled in the ice bath with NaOH 20% and extracted with diethyl ether. The diethyl ether phase was washed first with aqueous NaOH 20% and water and dried with anhydrous sodium sulfate and evaporated.Ethyl 4-benzamidobenzoate (3a)Yield: 60%; white crystalline powder; mp: 95-97, IR (KBr): υ (cm-1) 3340 (NH), 1720, 1670 (C=O); LC-MS (ESI) m/z = 270 , 292 .Ethyl 4-(4-fluorobenzamido)benzoate (3b)Yield: 65%; white crystalline powder; mp: 90-92 °C, IR (KBr): υ (cm-1) 3330 (NH), 1715, 1670 (C=O); LC-MS (ESI) m/z = 288 , 310 .Ethyl 4-(4-chlorobenzamido)benzoate (3c)Yield: 63%; white crystalline powder; mp: 96-97 °C, IR (KBr): υ (cm-1) 3315 (NH), 1690, 1730 (C=O); LC-MS (ESI) m/z = 304 .Ethyl 4-(4-methylbenzamido) benzoate (3d)Ethyl 4-(4-methoxybenzamido)benzoate (3e)Yield: 65%; white crystalline powder; mp: 99-100 °C, IR (KBr): υ (cm-1) 3340 (NH), 1730, 1680 (C=O); LC-MS (ESI) m/z = 284 , 306 . Yield: 58%; cream crystalline powder; mp: 105-106 °C, IR (KBr): υ (cm-1) 3330 (NH), 1715, 1690 (C=O); LC-MS (ESI) m/z = 300 .General procedure for the preparation of 4-substituted-N-(4-hydrazinecarbonyl) phenyl)benzamide (4a-4e)7.24 mmol of 3 and 10 mL hydrazine hydrate (200 mmol) were added to 10 mL ethanol. The mixture was stirred for 12 h at room temperature. Afterward, the solvent was evaporated and white precipitates washed with diethyl ether, and recrystallized from a mixture of ethanol and a few drops of water.N-(4-(hydrazinecarbonyl)phenyl)benzamide (4a)Yield: 80%; white crystalline powder; mp: 120-122 °C, IR (KBr): υ (cm-1) 3397, 3307 (NH), 1647 (C=O); LC-MS (ESI) m/z = 256 .4-Fluoro-N-(4-hydrazinecarbonyl)phenyl)benzamide (4b)Yield: 70%; white crystalline powder; mp: 125-126 °C, IR (KBr): υ (cm-1) 3336, 3317 (NH), 1659 (C=O); LC-MS (ESI) m/z = 274 .4-Chloro-N-(4-hydrazinecarbonyl)phenyl)benzamide (4c)Yield: 75%; white crystalline powder; mp: 127-129 °C, IR (KBr): υ (cm-1) 3340, 3327 (NH), 1670 (C=O); LC-MS (ESI) m/z = 290 .4-Methyl-N-(4-hydrazinecarbonyl)phenyl) benzamide (4d)Yield: 80%; white crystalline powder; mp: 120-121 °C, IR (KBr): υ (cm-1) 3336, 3307 (NH), 1659 (C=O); LC-MS (ESI) m/z = 270 .4-Methoxy-N-(4-hydrazinecarbonyl) phenyl)benzamide (4e)Yield: 70%; white crystalline powder; mp: 130-131 °C, IR (KBr): υ (cm-1) 3320, 3307 (NH), 1680 (C=O); LC-MS (ESI) m/z = 286 .General procedure for the preparation of 2-(2-(4-(4-substitutedbenzamido)benzoyl)hydrazinyl)carbonylbenzoic acid (5a-5e ) A solution of 4 (1.68 mmol) and phtalic anhydrides (1.68 mmol) in dry toluene was stirred at room temperature for overnight. The solvent was evaporated and the precipitate was washed with water and recrystallized from ethanol 96%.2-(2-(4-benzamido)benzoyl) hydrazinylcarbonylbenzoic acid (5a)J = 8.0 Hz), 7.90 , 7.95 , 8.06 , 10.35 , 12.00 .Yield: 70%; white crystalline powder; mp: 170 °C, IR (KBr): υ (cm-1) 2665-3315 (OH), 3268, 3231 (NH), 1694, 1666 (C=O); LC-MS (ESI) m/z = 404 ; 1HNMR (DMSO/500 MHz): 7.57 , 7.70 benzoyl)hydrazinyl)carbonylbenzoic acid (5b)J = 8.7 Hz), 7.58 , 7.74 , 7.90 , 7.95 , 8.06 , 10.35 , 10.45 , 12.01 .Yield: 65%; white crystalline powder; mp: 172 °C, IR (KBr): υ (cm-1) 2657-3330 (OH), 3235, 3224 (NH), 1710, 1667 (C=O); LC-MS (ESI) m/z = 422 , 444 ; 1HNMR (DMSO/500 MHz): 7.34 benzoyl)hydrazinyl)carbonylbenzoic acid (5c)J = 8.7 Hz), 7.55 , 7.74 , 7.80 , 7.90 , 8.00 , 10.05 , 10.35 , 12.20 .Yield: 70%; white crystalline powder; mp: 173 °C, IR (KBr): υ (cm-1) 2673-3270 (OH), 3250, 2865 (NH), 1677, 1657 (C=O); LC-MS (ESI) m/z = 438 ; 1HNMR (DMSO/500 MHz): 7.14 benzoyl)hydrazinyl)carbonylbenzoic acid (5d)J = 8.1 Hz), 7.25 , 7.44 , 7.70 , 7.80 , 7.90 , 10.25 , 12.20 .Yield: 65%; white crystalline powder; mp: 174 °C, IR (KBr): υ (cm-1) 2859-3400 (OH), 3308, 3227 (NH), 1700, 1680 (C=O); LC-MS (ESI) m/z = 418 , 440 ; 1HNMR (DMSO/500 MHz): 2.39 , 7.04 benzoyl)hydrazinyl)carbonylbenzoic acid (5e)J = Rezaee zavareh E et al. / IJPR (2014), 13 (supplement): 51-59 56 Yield: 68%; white crystalline powder; mp: 170 °C, IR (KBr): υ (cm-1) 2940-3492 (OH), 3318, 3228 (NH), 1712, 1643 (C=O); LC-MS (ESI) m/z = 434 , 456 ; 1HNMR (DMSO/500 MHz): 3.84 , 6.95 , 7.30 , 7.60 , 7.80 , 10.00 , 12.00 .8.1 Hz), 7.05 , 7.20 benzoyl)hydrazinyl)-4-oxobutanoic acid (6a-6e) A solution of 4 (1.68 mmol) and succinic anhydride (1.68 mmol) in dry toluene was stirred at room temperature for 18 h. The solvent was evaporated and the precipitate was washed with water and recrystallized from methanol.4-(2-(4-benzamidobenzoyl) hydrazinyl)-4-oxobutanoic acid (6a)J = 8.7, 3.5 Hz), 10.18 , 12.10 .Yield: 80%; white crystalline powder; mp: 172 °C, IR (KBr): υ (cm-1) 2890-3402 (OH), 3235, 3021 (NH), 1684, 1667 (C=O); LC-MS (ESI) m/z = 356 , 378 ; 1HNMR (DMSO/500 MHz): 2.40 , 7.57 , 7.95 , 8.27 benzoyl)hydrazinyl)-4-oxobutanoic acid (6b)J = 8.7 Hz), 7.90 , 8.07 , 10.18 , 10.44 , 10.57 , 12.10 .Yield: 72%; white crystalline powder; mp: 170 °C, IR (KBr): υ (cm-1) 2936-3373 (OH), 3306, 3262 (NH), 1685, 1649 (C=O); LC-MS (ESI) m/z = 374 , 396 ; 1HNMR (DMSO/500 MHz): 2.48 , 7.37 benzoyl)hydrazinyl)-4-oxobutanoic acid (6c)J = 8.7 Hz), 7.80 , 8.00 , 10.28 , 10.40 , 10.57 , 12.10 .Yield: 75%; white crystalline powder; mp: 168 °C, IR (KBr): υ (cm-1) 2965-3492 (OH), 3297, 3043 (NH), 1686, 1644 (C=O); LC-MS (ESI) m/z = 390 ; 1HNMR (DMSO/500 MHz): 2.38 , 7.27 benzoyl)hydrazinyl)-4-oxobutanoic acid (6d)J = 8.7 Hz), 7.50 , 7.95 , 10.20 , 12.00 .Yield: 80%; white crystalline powder; mp: 160 °C, IR (KBr): υ (cm-1) 2942-3501 (OH), 3312, 3273 (NH), 1702, 1661 (C=O); LC-MS (ESI) m/z = 370 ; 1HNMR (DMSO/500 MHz): 2.39 , 2.48 , 7.07 benzoyl)hydrazinyl)-4-oxobutanoic acid (6e)J = 8.3 Hz), 7.30 , 7.75 , 10.70 , 12.00 .Yield: 72%; white crystalline powder; mp: 165 °C, IR (KBr): υ (cm-1) 2981-3513 (OH), 3323, 3284 (NH), 1709, 1667 (C=O); LC-MS (ESI) m/z = 386 , 408 ; 1HNMR (DMSO/500 MHz): 2.38 , 3.84 , 7.00  complexed with 4-cyano-N-[-2-phenylcyclopropyl]benzamide was retrieved from RCSB Protein Data Bank. The structures of compounds were investigated using the Lamarckian genetic algorithm search method implemented in AutoDock 4.0 software. The receptors were kept rigid, and ligands were allowed to be flexible. Polar hydrogens and Kollman united atom partial charges were added to the individual protein atoms. Each structure was energy minimized under MM+ method in HyperChem8 software and converted to pdbqt format file using AutoDockTools 4.0 version1.5.4. A docking grid box was built with 40, 40 and 40 points in 25.8460, 24.0730 and 114.8150 directions in the catalytic site of protein and the number of generations and maximum number of energy evaluations was set to 100 and 2,700,000, respectively. Docking results were clustered with a root mean square deviation (RMSD) of 0.5 Å and evaluated by Pymol software.In-vitro biological activityBiological evaluation was performed by Cayman fluorescence-based human soluble epoxide hydrolase assay kit (item number 10011671). The enzyme and inhibitors were incubated for 15 min in 25 mM Bis-Tris/HCl buffer  at 30 °C. 3-phenyl-cyano  methyl ester-2-oxiraneacetic acid (PHOME) was used as the substrate for assay. The reference inhibitor for assay is AUDA, one of the most effective inhibitors of sEH with 50% inhibitory activity in 1 nM concentration. Test samples were dissolved in DMSO and tested in 1 nM concentration in duplicate to the determination of the inhibitory activity.The designed compounds were synthesized in good yield according to According to


Cyclam was attached to 1-, 2- and 3-pyrrole lexitropsins for the first timethrough a synthetically facile copper-catalyzed “click” reaction.The corresponding copper and zinc complexes were synthesized and characterized.The ligand and its complexes bound AT-rich DNA selectively over GC-rich DNA, andthe thermodynamic profile of the binding was evaluated by isothermal titrationcalorimetry. The metal, encapsulated in a scorpion azamacrocyclic complex, didnot affect the binding, which was dominated by the organic tail. The sequence-selective binding of small molecules to DNA is an important area ofresearch because through such binding it may be possible to control gene expression,which has significant implications for new therapeutics.Many naturally-occurring small molecules are known to bind DNA with sequenceselectivity, most notably the polyamide class of minor groove binders that includesdistamycin and netropsin, known generically as the lexitropsins.With such a well-evolved scaffold for interaction with DNA, it is unsurprising thatthere has been a great deal of interest in tailoring the basic design to build ingreater sequence-selectivity and adapt these structures to develop new types ofdrugs.N-C-N-terminal peptide andC-terminal intercalator.in situ. This report addresses theseareas.There has been much interest in the attachment of chemically active groups such asalkylating agents to lexitropsins in the hope of targeting reactive chemicalfunctionality to the double helix.We recently became interested in the attachment of azamacrocycles to motifs thatrecognize biological molecules. We have previously demonstrated that it is possibleto influence an azamacrocycle's interaction with DNA by changing the nature ofan amino acid appended to the macrocycle,4a–c  attachment of an unprecedented group (cyclam) to the C terminus, c)inclusion of an alkyl spacer between the azamacrocycle and the recognition motifand d) complexation of metal ions (copper and zinc). It was anticipated thatthese features would combine to provide structures capable of binding DNA, andthe influence of each feature is discussed in more detail below.The targets of the synthesis were lexitropsin-cyclam conjugates4a–c , formed a–c 1a–c. Four as1a–c were coupled withcommercially-available 3-aminopropyl azide to give2a–c which were coupled to the protectedpropargyl cyclam in good yields. Removal of the Boc groups to give the freeamines proceeded smoothly. It was noted that intermediates in the synthesisof 1 containing deprotected amines  decomposed after a few hours at roomtemperature, and were therefore typically used immediately after isolation.Compounds 2 and 3 were found to be hygroscopic,but were effectively handled (and weighed) as ethereal solutions.The pyrrole acids 4a, containing a single pyrrole in the sidechain, was employed for these studies as representative of the othercompounds. Titration with copper(II) chloride in methanol led to theappearance of a peak in the UV-visible spectrum that reached a maximumabsorbance with the addition of one equivalent of CuCl2,indicating the formation of a well-defined complex  chloride in CD3OD bythe addition of the metal salt in 0.2-equivalent increments to a solution of4a up to a maximum of 1.2 equivalents 2 (oligo I) andd-(GGGCGGCCGCCC)2 (oligo II). The GCrich ends were chosen to stabilise the DNA duplex and encourage annealing; thesesequences have melting temperatures of 36°C and 48°C respectively,meaning that they are duplexes under the conditions of the ITC experiments(25°C). The middle section of the oligonucleotide sequences was designed toprobe for AT vs. GC selectivity, and the question arose as towhether the exact sequence of the bases in the variable region is important.Netropsin binds less well to alternating AT sequences than continuous runs (2 ormore) of the two bases.The DNA binding characteristics of cyclam-lexitropsin conjugates DNA binding studies with small molecules are very sensitive to the saltconcentration of the solution.−1,ΔH = −44.8 kJ mol−1,ΔS = −56.9 J mol−1K−1) are in broad agreement with those in the literaturefor the titration between ethidium bromide and the relatedpoly[d(A-T)]-poly[d(A-T)],ca. 0.7×105M−1).The concentrations of oligonucleotide and complex were 10 µM and 1000µM respectively. Each injection (2 µL) by the calorimeter contained1 equivalent of ligand with respect to the oligonucleotide. Control titrationswere performed with ethidium bromide to validate this experimental method. EtBrwas chosen for convenience; despite being an intercalator, it was important toverify correspondence between experimental and literature ITC values. The valuesobtained for coordination of ethidium bromide with the AT-rich oligonucleotide with a benzyl sidechain in place of the oligopyrrolemoiety.The data obtained gave Klculated. Nodete4cand both of its metal complexes 5c and 6c with theAT-rich oligonucleotide I ].poly[d(GC)] with 38% of the enthalpychange with which it binds poly[d(AT)].poly[d(AT)].While compound The results above clearly show that three pyrroles are required for syntheticlexitropsins of this type to bind to AT-rich DNA, a figure that is consistentwith the literature for related compounds.The greatest variation in the structure of these new lexitropsins compared toknown analogs is the addition of cyclam  to the C-terminus.A C-terminal methylene spacer between the pyrrole rings and the cyclam wasemployed in the design, since methylene groups form favourable van der Waalsinteractions with terminal A/T base pairs,Cyclam is an important modification because the nature of the C-terminalalkylamine can significantly alter lexitropsin binding strength. Apparentlytrivial changes to the alkylamine tail of lexitropsins can change their bindingaffinity for their cognate sequence by up to two orders of magnitude , it should be remembered thatthe unmetallated cyclam ring, drawn as neutral in 4c–6c.Interestingly C-terminal alkylamine tails on other minor groove binders can actas a GC-directing motif, for example the piperazine ring in the compound Hoechst33258, which exerts this change essentially on the steric grounds of requiring awider minor groove.N-formamido moiety from lexitropsins cansignificantly reduce their binding affinity for DNA,N-formamido moiety is removed issmaller than for other lexitropsins  that was only slightly lower than that for themetalated version (2.26×105 M−1) and a similar“shielding” of the metal from the DNA backbone by bulky ligandsubstituents was proposed.et al. recentlyreported a Zn-lexitropsin conjugate based on thebis(2-benzimidazolyl-methyl)amine scaffold, in which the metalis available for coordination, and which exhibited a 3-fold enhancement ofaffinity for AT-rich oligonucleotides compared to the metal-free ligand.The cyclam-lexitropsin conjugates described here show essentially the samebinding characteristics whether the cyclam is unmetalated 4c . Theinteraction was modeled by taking the geometry-optimized DNA oligonucleotide andafter inserting an optimized dimer of cyclam ligands into the minor groove, theresulting DNA-dimer complex was then subjected to geometry optimization. Theresults of this procedure can be seen in 6c) was modeled in this way.To verify whether the cyclam in the ligand is well placed to form favourableinteractions with the phosphate backbone, molecular modeling was carried out onthe complex formed between the AT-rich oligonucleotide and compoundA consideration of both binding enthalpy and entropy is important, rather thansolely the binding free energy, since enthalpic and entropic changes in smallmolecule-DNA binding can compensate for one another to give a misleading freeenergy change.4c–6c do not show theenthalpy-entropy compensation that is expectedvs. the natural ligands.The lexitropsins The compounds in the present work bind with a 2∶1 stoichiometry to AT-richoligonucleotides, despite being pyrrole rich and being potentially multiplycharged under the conditions employed. It is known that lexitropsins can bind toDNA and oligonucleotides with either a 1∶14c–6c to oligo I, it might beexpected that the association constants for the first and second binding eventscould be deconvoluted, or that the two binding events would be clear from adiscontinuity in the ITC data. However since there is no such discontinuity,binding is either statistical (no cooperativity) or there is cooperativity buttwo molecules of the lexitropsin bind simultaneously to a singleoligonucleotide, rather than in a statistical 1∶1 binding.CGCATATATGCG)2.a should formally be thought of as a combination of thetwo contributing binding events, i.e.(K1K2)1/2.Given the 2∶1 binding stoichiometry of lexotropsinsa values observed for the three conjugates that exhibit bindingare high, with complete selectivity for the AT-rich oligonucleotide over theGC-rich sequence. The data (and modeling) show that in the cases studied, therewas little influence of the nature of the cyclam and coordinated metal on thedegree of DNA binding. This arises because once the lexitropsin binds as a dimerin the minor groove, the cyclam is positioned beyond the backbone of the DNAhelix.The magnitude and selectivity of the binding exhibited by these cyclam-polyamidecompounds is gratifying for the reasons detailed above. Despite lacking aterminal formamide, not necessarily incorporating an optimized DNA sequence forbinding, and in the face of literature precedent showing that unoptimisedalkylamines can significantly reduce the binding efficiency of lexitropsins, theKThere is considerable scope for modifying these structures to optimize binding,and to position the cyclam and its complexes for interaction with the DNAbackbone. Of particular interest will be to vary the structure of the scorpionligand to facilitate metal interaction with the DNA helix upon binding, so as topermit the future development of sequence-specific DNA cleavage. Future study ofthe potential nuclease activity of the metal centre would likely employ therelated azamacrocycle cyclen, the metal complexes of which are known to promotephosphodiester cleavage in model systemsAnother future application of complexes of this type is as imaging agents for thepresence of specific DNA sequences using complexes whose optical propertieschange upon binding. The attachment of cyclams also offers potentialimprovements in the cell permeability of the resulting lexitropsins: it is knownthat zinc sensors based on related triazole-cyclam motifs arecell-permeable,t as the coupling reagents. Theoligonucleotides d-(GGGATATATCCC)2 and d-(GGGCGGCCGCCC)2 and werepurchased from Geneworks . Reagents wereobtained from Sigma Aldrich, Fluka, Novabiochem or Alfa Aesar and useddirectly without further purification. Milli-Q water was used in allphysical measurements. NMR spectra for novel compounds are provided alongwith the .dx files complex titration experiment, measurements were taken of cyclam complex (1.0eq) dissolved in methanol (1 mL). Copper(II) chloride (73.4 mM) was added in0.2 eq aliquots until 2 eq had been added. Measurements were taken after 30s of stirring. For the Job plot a series of metal and ligand mixtures wasprepared, such that the total molarity was the same while changing the metaland ligand ratio at 0.2 eq intervals.1H and 13C Nuclear Magnetic Resonance spectroscopy wasperformed on either a Bruker Avance DPX 200 Spectrometer or a Bruker AvanceDPX 300 Spectrometer. For the zinc titration experiment, the cyclam ligandswere dissolved in CD3OD (to 5.6 mM) and a solution of zinc(II)chloride in CD3OD (73.4 mM) was titrated to 1.2 eq in 0.2 eqincrements.DNA binding studies were performed on an iTC200 Microcalorimeter made ofHastelloy® Alloy C-276. The system was operated at 25°C with a coincell design with a capacity of 200 µL and a titration syringe with acapacity of 40 µL. The amount injected was 2 µL per 150 secondswith a stirring rate of 1000 rpm. The stock solution of DNA in thecalorimeter chamber was 10 µM in 10 mM HEPES buffer containing 100 mMNaCl and the ligand. The stock ligand solution (1000 µM) was dilutedto a concentration of 10 µM with the buffer solution prior to ITCexperiments and was titrated into the DNA solution. Single stranded DNAoligos were supplied by Geneworks and dissolved in buffer  and shaken gently at 25°C for 2 days toyield double stranded oligonucleotides to a stock concentration of 100µM determined using a Nanodrop 1000 spectrophotometer (Thermoscientific version 3.6.0). A correction was made for the heat of dilution ofthe ligands, estimated from the peaks obtained from injections at the end ofa given ITC experiment (following saturation).Metal complex synthesis for ITC experiments: to the ligand (1 eq) was added asolution of copper(II) chloride solution  in methanol orzinc(II) chloride solution  in methanol. The methanol wasremoved under reduced pressure and HEPES buffer (10 mM with 100 mM NaCl) wasadded to obtain a final stock ligand concentration of 1000 µM whichwas kept at 0°C. These complexes were used directly in DNA bindingstudies.ca. 125 mM in acid) were addedEDC·HCl (1.2 eq), HOBt (1.2 eq) andN,N-diisopropylethylamine (3.0 eq).The reaction mixture was stirred at rt under nitrogen for 12 h. Sodiumbicarbonate solution (10% w/v) was added dropwise to the reactionmixture until pH 10 was reached and the reaction mixture was extracted withdichloromethane (3 times). The combined organic phases were dried(Na2SO4) and concentrated under reducedpressure.To the carboxylic acid (1.0 eq) and amine (1.3 eq) in anhydrousdichloromethane (solution is 2SO4) and concentrated underreduced pressure.Alkyne (0.93 eq) and azide (1.0 eq) were dissolved in a mixture ofwater/tert-butanol  and stirredat 27°C under nitrogen. A solution of copper(II) sulfate pentahydrate and sodium ascorbate  in water (togive a solution that was 125 mM in copper) was added to the reaction mixtureand stirring was continued for 16 h. The reaction was quenched withsaturated sodium bicarbonate solution until pH 10 was reached and themixture extracted with dichloromethane (3 times). The combined organicphases were dried (Na2SO4) and concentrated under reducedpressure.To the Boc-protected compound (1.0 eq) in anhydrous dichloromethane (300 mM)was added trifluoroacetic acid (10 eq) dropwise and stirring was continuedat rt for 6 h. The reaction was cooled to 0°C before the addition ofwater (same volume as dichloromethane). Sodium hydroxide (1 M) was addeddropwise until pH 10 was reached. The mixture was extracted with chloroform(3 times). The combined organic phases were dried(Na2SO4) and concentrated under reduced pressure.To the ester-protected compound (1.0 eq) in a mixture of water/methanol was added sodium hydroxide  and thesolution was heated at reflux for 3 h under nitrogen. The reaction mixturewas washed with ethyl acetate (2 times) and the aqueous phase was acidifiedto pH 3 with hydrochloric acid (1 M) and was extracted with ethyl acetate (3times). The combined organic phases were dried was added copper(II)chloride or zinc(II) chloride solution in methanol  andstirring was continued at rt for 10 min. Methanol was evaporated invacuo and HEPES buffer (10 mM containing 100 mM NaCl) was addedto give a final ligand concentration of 1000 µM.To GGGATATATCCC)2 was constructed as the B-form regular helixusing the Maestro 9.1  graphical userinterface. Cyclam ligand structures were built, manipulated and adjusted forchemical correctness using Maestro, employing MacroModel 9.8 . Geometry minimizations were performed on all cyclamligands using the OPLS_2005 (MacroModel) force field and the Truncated NewtonConjugate Gradient (TNCG). Optimizations were converged to a gradient RMSD below0.05 kJ/mol or continued to a maximum of 1000 iterations, at which point therewere negligible changes in RMSD gradients.DNA oligonucleotide d-and 3-azidopropylamine  were coupled using generalprocedure A with purification by flash column chromatography  yielding2a  as a light yellow oil;IR (ATR) 2091, 1631 cm−1;1H NMR  δ6.69–7.72 , 6.51 , 6.07 , 5.96–6.05, 3.94 , 3.46 , 3.41 , 1.86  δ 161.9 (C = O), 127.4 (Ar), 125.2 (Ar), 111.5(Ar), 106.7 (Ar), 48.8, 36.2, 36.0, 28.6 (MS (APCI) m/z 108.0, 208.0; HRMS (APCI) calcd forC9H14N5O+ 208.11984found 208.11929 (MH+).1-Methylpyrrole-2-carboxylic acid  Hz, H2) ; 13C NMR.0, 28.6 ; MS and 3-azidopropylamine  were coupled according toprocedure A, with purification by flash column chromatography  yielding 2b  as a light yellow oil; IR (CHCl3)3326, 2096, 1640, 1535 cm−1; 1H NMR δ 7.74 , 7.09–7.11 , 6.73–6.77 , 6.66–6.70 ,6.55–6.57 , 6.05–6.15 , 3.95 , 3.86 , 3.35–3.55 ,2.74–3.25 , 1.77–1.88 δ 161.8 (C = O), 159.4(C = O), 128.5 (Ar), 125.4 (Ar), 123.2 (Ar), 121.3(Ar), 118.9 (Ar), 112.0 (Ar), 107.4 (Ar), 103.5 (Ar), 49.4, 36.9, 36.8,36.5, 28.9 (HRMS (APCI) calcd forC15H19N7NaO2+352.14979 found 352.14924 (MNa+).Methylpyrrole amide carboxylic acid 2CH2N3); 13C NMR.5, 28.9 ; HRMS  and3-azidopropylamine  were coupled using procedureA. The residue was purified by flash column chromatography  yielding 2c  as a light yellow oil; 1H NMR  δ 8.46 , 8.15 , 8.05, 6.88–6.94 , 6.80–6.82 ,6.68–6.71 , 6.61–6.67 , 6.21–6.25, 4.09 , 3.98 ,3.95 , 3.42–3.70 ,1.93–2.10 δ 161.9 (C = O), 159.6(C = O), 159.0 (C = O), 128.4(Ar), 125.3 (Ar), 123.2 (Ar), 122.9 (Ar), 121.5 (Ar), 121.3 (Ar), 119.5(Ar), 119.0 (Ar), 112.3 (Ar), 107.3 (Ar), 104.0 (Ar), 103.5 (Ar), 49.2,36.8, 36.7, 36.4, 36.4, 28.8 (MS(ESI) m/z 452.1 ,474.3 ; HRMS (ESI) calcd forC21H25N9NaO3+474.19781 found 474.19726 (MNa+).Pyrrole amide carboxylic acid 2CH2N3); 13C NMR.4, 28.8 ; MS were reacted together accordingto procedure B, giving a white gum which was purified by flash columnchromatography  yielding3a  as a white solid; mp72–74°C; IR (ATR) 3366, 1687, 1544cm−1; 1H NMR  δ 7.51 , 6.69–6.72, 6.59–6.66 , 6.30–6.57 ,6.05–6.09 , 4.44 , 3.93 , 3.74 , 3.68–3.85 , 3.20–3.50, 2.58–2.70 ,2.40–2.50 , 2.16–2.36 , 1.80–2.00 , 1.65–1.80, 1.44 3) δ 162.0, 155.4, 155.1, 127.5, 125.1, 122.6, 111.8, 79.1, 77.6, 77.0,76.4, 59.9, 46.0–47.5 (multiple peaks), 36.3, 35.9, 30.1, 28.1 (MS (ESI) m/z 746.3 , 768.3 , HRMS(ESI) calcd forC37H64N9O7+746.49287 found 746.49162 (MH+).Tri-Boc propargyl cyclam  and azideC(CH3)3) ; 13C NMR.1, 28.1 ; MS  were reacted togetheraccording to general procedure B yielding a light yellow oil, which waspurified by flash column chromatography  yielding 3b  as a hygroscopic white gum; IR (ATR) 3337, 2974,1681 cm−1; 1H NMR  δ 8.05 , 7.50 ,6.55–6.80 , 6.08 , 4.38–4.50 , 3.95 ), 3.88 ), 3.68–3.78 ,3.20–3.45 , 2.57–2.65 , 2.36–2.52 , 2.12–2.23, 1.80–1.96 ,1.63–1.79 , 1.43 3)  δ 162.1, 159.3, 155.8,155.6, 128.3, 125.5, 122.9, 121.6, 119.1, 111.9, 107.3, 103.6, 79.6,46.0–47.8 , 36.8, 36.5, 36.3, 29.9, 28.5 (MS (ESI) m/z 868.4 , 890.6 ; HRMS(ESI) calcd forC43H70N11O8+868.54088 found 868.54034 (MH+).Tri-Boc propargyl cyclam  and azideC(CH3)3) ; 13CNMR.9, 28.5 ; MS  were reacted togetheraccording to procedure B yielding a light yellow oil, which was purified byflash column chromatography  yielding 3c  as a hygroscopic whitegum; IR (ATR) 3316, 2975, 2935, 1685 cm−1;1H NMR  δ8.07–8.55 , 8.25–8.56 , 7.52 , 6.70–6.90 , 6.46–6.54 , 6.02–6.08 , 4.39 , 3.92 , 3.86 , 3.82 , 3.62–3.80 ,3.00–3.60 , 2.52–2.65 , 2.30–2.50 , 2.10–2.22, 1.80–1.95 ,1.60–1.75 , 1.42 3)  δ 162.1, 159.5, 159.0,155.8, 128.3, 125.5, 123.0, 122.8, 121.7, 119.4, 119.2, 112.2, 107.3, 104.0,103.6, 79.6, 44–50 , 36.7, 36.6, 30.0, 28.4 (MS (ESI) m/z 1012.8 ; HRMS (ESI) calcd forC49H76N13O9+990.58890 found 990.58835 (MH+).Tri-Boc propargyl cyclam  and methylpyrrole azideC(CH3)3) ; 13CNMR.0, 28.4 ; MS  was deprotected according to general procedure C yielding4a as a colourless oil. The product was purified by reversephase HPLC ) to yield compound 4a as a white foam; IR (ATR) 3272, 1634, 1546cm−1; 1H NMR  δ 7.51 , 6.95 , 6.48–6.55 , 6.40–6.48 ,5.80–5.90 , 4.25 , 3.75 , 3.63 , 3.16 , 2.20–2.70 , 1.87–2.10 ,1.58–1.66 , 1.60–1.58   δ 162.0, 143.8, 127.4,125.1, 122.6, 111.7, 106.7, 54.1, 52.2, 50.4, 49.0, 48.8, 48.3, 47.6, 47.4,46.8, 46.7, 36.2, 35.7, 30.1, 28.4, 25.7 (MS (ESI) m/z 446.3 , HRMS (ESI) calcd forC22H40N9O+ 446.33558found 446.33463 (MH+).Monomethylpyrrole tri-Boc-protected cyclam , m,He) ; 13CNMR.4, 25.7 ; MS  was deprotected according to general procedure C yielding compound4b  as a white gum without any furtherpurification; IR (ATR) 3288, 2935, 1641 cm−1;1H NMR  δ8.75 , 7.65 , 7.32–7.36 , 6.80–6.90 , 6.70–7.76 ,6.57–6.68 , 6.05–6.13 , 4.40–4.50, 3.96 , 3.87 , 3.65–3.70 ,3.36–3.43 , 2.70–2.85 , 2.64–2.70 , 2.50–2.63, 2.15–2.25 ,1.80–1.90 , 1.65–1.75   161.7, 159.4, 144.4, 128.0,125.4, 123.0, 121.7, 119.1, 122.2, 106.9, 104.6 103.5, 54.5, 53.4, 53.1,52.6, 50.2, 49.0, 48.4, 48.0, 47.2, 46.6, 36.4, 36.2, 29.4, 27.1, 25.0(MS (ESI) m/z 568.3 ; HRMS (ESI) calcd forC28H46N11O2+568.38359 found 568.38305 (MH+).Bismethylpyrrole tri-Boc-protected cyclam , m,He) ; 13CNMR.1, 25.0; MS  was deprotected according to general procedure C yieldingcompound 4c  as a pale yellow gum withoutany further purification; IR (ATR) 3267, 2924, 1635cm−1; 1H NMR  δ 9.35 , 7.67, 7.51 ,7.42–7.47 , 7.38–7.42 , 7.05–7.16, 6.88–6.95 , 6.81–6.88 ,6.65–6.75 , 5.97–6.06 , 5.00–5.85, 4.30–4.40 , 3.93 , 3.88 , 3.81 , 3.48–3.58 ,3.27–3.40 , 2.65–2.90 , 2.51–2.62 , 2.43–2.51, 2.10–2.20 ,1.72–1.80 , 1.60–1.72   δ 162.0, 159.3, 158.9,144.1, 128.1, 125.2, 123.3, 122.3, 122.2, 122.0, 121.8, 119.3, 118.8, 114.7,112.8, 107.1, 103.6, 54.3, 50.0, 48.8, 48.5, 48.2, 47.7, 47.2, 46.1, 45.3,36.9, 36.6, 36.5, 29.6, 29.2, 23.9 (MS (ESI) m/z690.3 , HRMS (ESI) calcd forC34H52N13O3+690.43161 found 690.43152 (MH+).Three-methylpyrrole tri-Boc-protected cyclam , m,He) ; 13CNMR.2, 23.9 ; MS (ESI5a. Copper(II) chloride was complexed with4a  according to generalprocedure D. The solution was made up to 3 mL in methanol to a finalconcentration of 1.50 mM; UV-vis (MeOH)λmax = 590 nm,ε = 414 M−1 cm−1;MS (ESI) m/z 543.0.Complex 6a. Zinc(II) chloride was complexed with 4a according to general procedure D.MS (ESI) m/z 581.0 (multiplet). 1H NMR spectrumshown as Complex 5b. Copper(II) chloride was complexed with4b  according to procedureD. The solution was made up to 3 mL in methanol to a final concentration of5.2 mM; UV-vis (MeOH)λmax = 615 nm,ε = 113.8 M−1cm−1; MS (ESI) m/z 665.3, 667.3; HRMS (ESI) calcd forC28H4535ClCuN11O2+665.27422 found 665.27305 ((M-Cl)+), calcd forC28H4537ClCuN11O2+667.27242 found 667.27176 ((M-Cl)+).Complex 6b. Zinc(II) chloride was complexed with 4b according to procedure D. MS(ESI) m/z 583.3 (100%). 1H NMR spectrum shown as Complex 5c. Copper(II) chloride was complexed with4c  according toprocedure D. The solution was made up to 3 mL in to a final concentration of0.45 mM; UV-vis (MeOH)λmax = 615 nm,ε = 162.7 M−1cm−1; IR (ATR) 3446, 2925, 1640, 1548,1414, 1254, 1114, 742 cm−1; MS (ESI) m/z 543.0.Complex 6c. Zinc(II) chloride was complexed with 4c according to procedure D. MS(ESI) m/z 876.0 (96%), 875.1 (100%), 797.4 (93%), 795.3(82%). 1H NMR spectrum shown as Complex Scheme S1Synthetic Scheme for Supporting Information Compounds.(TIF)Click here for additional data file.Figure S13, 400 MHz 1H NMR spectrum ofN-(3-azidopropyl)-1-methylpyrrole-2-carboxamide(2a).CDCl(TIFF)Click here for additional data file.Figure S23, 50.3 MHz 13C NMR spectrum ofN-(3-azidopropyl)-1-methylpyrrole-2-carboxamide(2a).CDCl(TIFF)Click here for additional data file.Figure S33, 300 MHz 1H NMR spectrum ofN-(3-azidopropyl)-1-methyl-4-(1-methyl-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamide(2b).CDCl(TIFF)Click here for additional data file.Figure S43, 75.5 MHz 13C NMR spectrum ofN-(3-azidopropyl)-1-methyl-4-(1-methyl-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamide(2b).CDCl(TIFF)Click here for additional data file.Figure S53, 300 MHz 1H NMR spectrum ofN-(3-Azidopropyl)-1-methyl-4-(1-methyl-4-(1-methyl-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamide(2c).CDCl(TIFF)Click here for additional data file.Figure S63, 75.5 MHz 13C NMR spectrum ofN-(3-azidopropyl)-1-methyl-4-(1-methyl-4-(1-methyl-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamide(2c).CDCl(TIFF)Click here for additional data file.Figure S73, 300 MHz 1H NMR spectrum oftri-tert-butyl11-((1-(3-(1-methyl-1H-pyrrole-2-carboxamido)propyl)-1H-1,2,3-triazol-4-yl)methyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylate(3a).CDCl(TIFF)Click here for additional data file.Figure S83, 50.3 MHz 13H NMR spectrum ofTri-tert-butyl11-((1-(3-(1-methyl-1H-pyrrole-2-carboxamido)propyl)-1H-1,2,3-triazol-4-yl)methyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylate(3a).CDCl(TIFF)Click here for additional data file.Figure S93, 300 MHz 1H NMR spectrum oftri-tert-butyl11-((1-(3-(1-methyl-4-(1-methyl-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)propyl)-1H-1,2,3-triazol-4-yl)methyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylate(3b).CDCl(TIFF)Click here for additional data file.Figure S103, 50.3 MHz 13C NMR spectrum oftri-tert-butyl11-((1-(3-(1-methyl-4-(1-methyl-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)propyl)-1H-1,2,3-triazol-4-yl)methyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylate(3b).CDCl(TIFF)Click here for additional data file.Figure S113, 300 MHz 1H NMR spectrum oftri-tert-butyl11-((1-(3-(1-methyl-4-(1-methyl-4-(1-methyl-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)propyl)-1H-1,2,3-triazol-4-yl)methyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylate(3c).CDCl(TIFF)Click here for additional data file.Figure S123, 50.3 MHz 13C NMR spectrum oftri-tert-butyl11-((1-(3-(1-methyl-4-(1-methyl-4-(1-methyl-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)propyl)-1H-1,2,3-triazol-4-yl)methyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylate(3c).CDCl(TIFF)Click here for additional data file.Figure S133, 200 MHz 1H NMR spectrum ofN-methyl)-1H-1,2,3-triazol-1-yl)propyl)-1-methyl-1H-pyrrole-2-carboxamide(4a).CDCl(TIFF)Click here for additional data file.Figure S143, 75.5 MHz 13C NMR spectrum ofN-methyl)-1H-1,2,3-triazol-1-yl)propyl)-1-methyl-1H-pyrrole-2-carboxamide(4a).CDCl(TIFF)Click here for additional data file.Figure S153, 200 MHz 1H NMR spectrum ofN-methyl)-1H-1,2,3-trizol-1-yl)propyl)-1-methyl-4-(1-methyl-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamide(4b).CDCl(TIFF)Click here for additional data file.Figure S163, 50.3 MHz 13C NMR spectrum ofN-methyl)-1H-1,2,3-trizol-1-yl)propyl)-1-methyl-4-(1-methyl-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamide(4b).CDCl(TIFF)Click here for additional data file.Figure S173, 300 MHz 1H NMR spectrum ofN-methyl)-1H-1,2,3-triazol-1-yl)propyl)-1-methyl-4-(1-methyl-4-(1-methyl-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamide(4c).CDCl(TIFF)Click here for additional data file.Figure S183, 75.5 MHz 13C NMR spectrum ofN-methyl)-1H-1,2,3-triazol-1-yl)propyl)-1-methyl-4-(1-methyl-4-(1-methyl-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamide(4c).CDCl(TIFF)Click here for additional data file.Figure S191H NMR spectrum of mono-pyrrole zinc chloridecyclam complex (6a).300 MHz, MeOD, (TIFF)Click here for additional data file.Figure S201H NMR spectrum of di-pyrrole zinc chloride cyclamcomplex (6b).300 MHz, MeOD, (TIFF)Click here for additional data file.Figure S211H NMR spectrum of tri-pyrrole zinc chloride cyclamcomplex (6c).300 MHz, MeOD, (TIFF)Click here for additional data file.Figure S224a.(TIFF)Click here for additional data file.Figure S232 withcompound 4c in methanol .UV-vis spectrum for the titration of a solution of CuCl(TIFF)Click here for additional data file.Figure S244c to 10 µM GC oligo(oligo II).Example ITC curve for GC-rich oligonucleotide illustrating no observablebinding; titration of 1000 µM (TIFF)Click here for additional data file.Spreadsheet S1Error calculations for (XLS)Click here for additional data file.Text S1Procedures for preparation of known compounds, and description of entropyerror calculations.(DOC)Click here for additional data file.Table S14cvs. selected literature compounds.Effect of structural modifications of lexitropsins on binding affinities forcompound (DOC)Click here for additional data file.NMR Data S12–4(1H and 13C) and 6(1H).Raw NMR data files (.dx) for compounds (ZIP)Click here for additional data file.


O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3′-blocking moiety and a linker-dye system that is removable under the same fluoride-based treatment. Each nucleotide analogue has a different fluorophore attached to the base through a fluoride-cleavable linker and a 2-cyanoethyl moiety as the 3′-blocking group, which can be removed by using a fluoride treatment as well. Furthermore, we identified a DNA polymerase, namely, RevertAid M-MuLV reverse transcriptase, which can incorporate the four modified reversible terminators. The synthesised nucleotides and the optimised DNA polymerase were used on CodeLink slides spotted with hairpin oligonucleotides to demonstrate their potential in a cyclic reversible terminating approach.Fluorescent 2′-deoxynucleotides containing a protecting group at the 3′- The completion of the two Human Genome Projects in 2001 implemented the ultimate goal of sequencing the whole human genome. However, the demands these molecules have to meet are challenging, and therefore implementation is difficult. The general structure of a reversible terminator 1 is shown in This idea of using 3′-reversibly blocked nucleotides for sequencing was proposed in the beginning of the 1990s. The first examples of potential reversible terminators were reported in 1994 by Metzker and co-workers and CanaO-(2-nitrobenzyl) group investigated by Metzker and co-workers  O- group as a 3′-OH blocking moiety (0.1 equiv) were added. In a separate heat-gun-dried Schlenk flask, alkyne 23 (1.2–2.0 equiv) was dissolved in dry DMF (250 μL) and added in two portions to the reaction mixture with a syringe. One half portion was added immediately and the other half portion after 1 h. The reaction mixture was stirred for 3.5–5 h at room temperature in the dark. The reaction mixture was evaporated to dryness, the crude product redissolved in CH2Cl2 (10 mL) and 5 % ethylenediaminetetraacetate  solution, and stirred vigorously for 10 min. The layers were separated and the organic layer was washed with 5 % EDTA solution (5 mL), dried over sodium sulphate, and concentrated in vacuum. The residue was purified by flash-column chromatography. For example, the synthesis of 5-(prop-2-ynyl)carbamic acid-1-{2-[2-(2-tert-butoxycarbonylaminoethoxy)ethoxy]ethoxymethyl}-2-cyanoethyl ester-3′-O-(2-cyanoethyl)-2′-deoxyuridine (24).O-(2-cyanoethyl)-2′-deoxyuridine  with alkyne 23  gave, after flash-column chromatography (CH2Cl2/MeOH 99:1→90:10), 24 as a slightly yellow oil . RF=0.34 (CH2Cl2/MeOH 90:10); 1H NMR : δ=1.37 , 2.16–2.31 , 2.77 , 2.88 , 3.06 , 3.37 , 3.48–3.65 , 3.93–3.97 , 4.02 , 4.14 , 4.95 , 5.13–5.19 , 6.07 , 6.68–6.77 , 7.93 , 8.14 , 11.64 ppm ; 13C NMR : δ=18.23 (25-C), 19.72 (12-C), 28.21 (23-C), 30.68 (9-C), 36.90 (2′-C), 39.69 (20-C), 61.14 (5′-C), 63.56 (24-C), 67.91 (11-C), 69.17 (19-C), 69.49, 69.60, 69.73, 70.21, 70.30 , 74.49 (7-C), 77.58 (22-C), 79.08 (3′-C), 84.64 (1′-C), 84.89 (4′-C), 89.57 (8-C), 98.23 (5-C), 117.64 (13-C), 119.26 (26-C), 143.40 (2-C), 143.56 (6-C), 154.84 (10-C), 155.54 (21-C), 161.49 ppm (4-C); ESI+-MS (m/z): calcd: 692.71 [C31H44N6O12]; found: 710.5 [M+H2O]+, 715.4 [M+Na]+; elemental analysis (%) calcd for C31H44N6O12: C 53.75, H 6.40, N 12.12; found: C 53.53, H 6.51, N 11.88.Following GP2, the reaction of 5-iodo-3′-General procedure for the synthesis of N-deprotected triphosphates (GP3): The N-Boc protected nucleoside was vacuum dried in a Schlenk flask for 2 days, the flask was flushed with argon, and the nucleoside (0.1 mmol) dissolved in dry pyridine (100 μL) and dry dioxane (300 μL). A freshly prepared solution of 2-chloro-4H-1,2,3-benzodioxaphosphorin-4-one  in dry dioxane was added with a syringe and the reaction mixture stirred for 15 min at room temperature. Dry DMF (150 μL), bis(tributylammonium)pyrophosphate , and tributylamine  were added simultaneously with syringes. The reaction mixture was stirred for 20 min before a solution of iodine in pyridine/water  was added (1 mL). The reaction mixture was left for a further 20 min. Afterwards, the excess of iodine was quenched with 5 % Na2SO3 solution, and the mixture was evaporated to dryness. The crude triphosphate derivatives (0.1 mmol) were dissolved in water (5 mL) containing TFA (40 equiv). The solution was stirred for 5–6 h at room temperature and subsequently concentrated in vacuum. For the ion-exchange FPLC purification, the deprotected triphosphate derivatives were dissolved in Millipore water (8–12 mL) and filtered through a 0.45 μm syringe filter. The following gradient was used for the separation at a flow rate of 4 mL min−1: 0 % B (0 mL)→50 % B (500 mL)→100 % B (650 mL). The combined fractions were lyophilised, dissolved in 1–2 mL of Millipore water, and filtered through a 0.45 μm syringe filter before the second purification step by preparative RP-HPLC. The following gradient was used at a flow rate of 6 mL min−1: 0 min: 10:90:0 A/B/C, 15 min: 10:60:30 A/B/C; 16 min: 0:0:100 A/B/C; 22 min: 0:0:100 A/B/C; 23 min: 10:90 A/B/C, 30 min: 10:90:0 A/B/C. The final yields were calculated from the 1H NMR spectrum of the HPLC purified triphosphate derivatives because the products contained traces of triethylammonium acetate after RP-HPLC purification .N-Boc-protected nucleoside 24  yielded triphosphate 28 as a colourless oil . RF=0.39 (iPr/H2O/EtMe2N 6:3.8:0.2); IE-FPLC: elution concentration=0.29 m TEAB/buffer (32 % B); RP-HPLC: retention time=14.92 min; 1H NMR : δ=2.32 , 2.55 , 2.81 , 2.89–2.99 , 3.25 , 3.67–3.79 , 3.84 , 4.12–4.26 , 4.37 , 4.45–4.49 , 5.12–5.23 , 6.26 , 8.17 ppm ; 13C NMR : δ=18.91 (22-C), 20.21 (12-C), 31.49 (9-C), 37.28/37.30 (2′-C), 39.67 (21-C), 64.43 (21-C), 66.31 , 66.95, 70.09, 70.12, 70.19, 70.84, 71.18 , 69.30 (11-C), 73.98/74.01 (7-C), 80.45/80.47 (3′-C), 84.31 , 86.50 (1′-C), 90.93 (8-C), 99.77 (5-C), 118.78 (13-C), 120.45 (23-C), 145.34/145.35 (6-C), 151.03 (2-C), 157.27/157.29 (10-C), 164.99 ppm ; 31P NMR : δ=−10.97 , −11.69 , −23.43 ppm ; ESI−-MS (m/z): calcd for [C26H39N6O19P3]: 832.54; found: 831.4 [M−H]−, 849.6 [M−H+H2O]−.Following GP3, the reaction of the Synthesis of 2: KHCO3  was added to a solution of triphosphate 28  in dry DMF (500 μL). The reaction mixture was cooled to 0 °C, and 5- and 6-carboxyfluorescein-(N-hydroxysuccinimidyl)ester  was dissolved in dry DMF (250 μL) and added in two portions to the reaction mixture, one half immediately and the other after 1 h at 0 °C. The reaction mixture was stirred for 20 h at 0 °C. After evaporation of the solvent, the crude product was purified by RP-FPLC at 4 °C and preparative RP-HPLC (see GP4 in the Supporting Information). The triethylammonium salt of the labelled triphosphate 2 was obtained as a yellow solid  as a mixture of the two regioisomers, each consisting of two diastereomers. RF=0.65 (H2O/CH3CN 80:20); RP-FPLC: elution concentration=2.5→6 % B; RP-HPLC: retention time=16.49 min isomer A, 16.90 min isomer B (UV detection), 16.53 min isomer A, 16.94 min isomer B (fluorescence detection); UV absorption: λ=494, 277, 235 nm; fluorescence: λex=492, λem=512 nm; 1H NMR : δ=2.15–2.37 , 2.44–2.59 , 2.81–2.93 , 3.57–3.91 , 4.00 , 4.10–4.32 , 4.10–4.32 , 4.99–5.14 , 6.04–6.25 , 6.69–7.04 , 7.09–7.32 , 7.56–8.46 ppm ; 31P NMR : δ=−10.63 , −11.56 , −23.09 ppm ; ESI−-MS (m/z): calcd for [C47H49N6O25P3]: 1190.85; found: 594.6 [M−2 H]2−, 1110.8 [M−PO3-H]−, 554.8 [M−PO3-2 H]2−, 1190.4 [M−H]−.Solution-based primer-extension experiments: The oligodeoxynucleotides (shown in O-(2-cyanoethyl)-modified nucleotides and an extension experiment using fluorescence-labelled reversible terminators 2–5. The primer strand was 5′-radiolabelled and annealed to the complementary (template) strand by heating for 5 min to 95 °C and gradually cooling to room temperature over 2 h. For the primer extension, 10 nm DNA duplex, polymerases in at least 10-fold molar excess, and 50 μm of the 3′-modified nucleotides was used. The 3′-modified nucleotides were additionally purified by enzymatic depletion of the natural nucleotide counterpart before use.crown-6 (1 %), 2–5 (100 μm each), and RevertAid M-MuLV reverse transcriptase (10 U μL−1). 3) Blocking of the starting-material oligonucleotide primers on the array was accomplished with TdT  and ddATP by incubating the arrays for 20 min at 37 °C. The TdT reaction mixture consisted of the reaction mixture with an aliquot (40 μL) of potassium cacodylate (200 mm), Tris (25 mm), Triton X-100 , CoCl2 , ddATP (1 mm), and TdT enzyme (80 U). 4) The reactions were stopped by washing with deionized water at 95 °C. The slides were dried, covered with a droplet of Slowfade light antifade reagent , and imaged with Genorama QuattroImager detector (Asper Biotech Ltd.). The fluorescence signal intensities were extracted and analysed with BaseCaller module of the Genorama genotyping software package (Asper Biotech Ltd.). 5) Cleavage of the fluorescence dye and the 3′-O-(2-cyanoethyl) group from the incorporated fluorescence labelled 3′-O-(2-cyanoethyl)-blocked nucleotides was accomplished with TBAF (1 m) in THF. Before the unblocking reaction, the DNA arrays were washed three times with THF. The unblocking reaction was carried out by immersing the DNA arrays for 15 min at 45 °C into a 1:1 solution of TBAF/THF  and dry DMF. 6) The DNA arrays were washed with deionized water, dried, and read with the Genorama QuattroImager detector to be sure that the unblocking reaction was successful. 7) Before the second primer-extension reaction, the slides were blocked again with blocking solution  for 10 min at 37 °C. 8) The second primer-extension reaction using the fluorescently labelled 3′-O-(2-cyanoethyl)-dNTP derivatives 2–5 and the reverse transcriptase was carried out under the reaction conditions described for the first extension step (step 2). 9) The reactions were stopped by washing with deionized water at 95 °C. The slides were dried, read, and analysed as described above for first primer-extension reaction.Spotting of DNA arrays and APEX reactions on CodeLink arrays with fluorescently labelled 3′-


A combined experimental and computational study on regioselective gold-catalyzed synthetic routes to 1,3-oxazinan-2-ones  and 1,3-oxazin-2-one derivatives  from easily accessible allenic carbamates has been carried out. N-Boc-3-butyn-1-amines to afford six-membered 2-oxazinones, namely, 6-methylene-1,3-oxazinan-2-ones involving a 6-exo-dig cyclization /AgOTf was an excellent catalyst for our purpose. To our delight, the reaction of allenic carbamate 2a at room temperature afforded 3-benzyl-6-methylene-1,3-oxazinan-2-one (3a) bearing an exocyclic double bond as the sole product  2g also undergoes this interesting transformation to give bis 3g through a two-fold cyclization. This product particularly underlines the power of the present cyclization reaction, as none of the conventional methods would allow its synthesis with such great ease.As revealed in 2a in dichloromethane at 90 °C, afforded 1,3-oxazin-2-one 4a bearing an endocyclic double bond as the major component, and 1,3-oxazinan-2-one 3a was also isolated as a minor component. Notably, starting from allenic carbamates 2a–j and performing the reaction in dichloromethane at 130 °C, a series of 6-methyl-3-substituted 3,4-dihydro-2H-1,3-oxazin-2-ones 4a–j were exclusively formed (3 (arising from 6-endo-dig cyclization) or 3,4-dihydro-2H-1,3-oxazin-2-ones 4 (arising from 6-exo-dig cyclization) were achieved, with the nucleophilic oxygen attacking the central allene carbon atom in each case. This is an interesting result, because the available examples on related metal-catalyzed allene heterocyclizations usually lead to 5-exo-trig cyclization .–68.2a inlization –70; onlylization .4 is the thermodynamically controlled product while heterocycle 3 is the kinetically controlled product D is given in 10−1 deg cm2 g−1 at 20 °C, and the concentration (c) is expressed in grams per 100 mL. All commercially available compounds were used without further purification.3] (0.00475 mmol), AgOTf (0.00475 mmol), and p-toluenesulfonic acid (0.019 mmol) were sequentially added to a stirred solution of the allenic carbamate 2a  in dichloromethane (1.9 mL). The resulting mixture was heated in a sealed tube at 130 °C until disappearance of the starting material . The reaction was allowed to cool to room temperature and filtered through a pack of celite. The filtrate was extracted with dichloromethane (3 × 5 mL), and the combined extracts were washed twice with brine. The organic layer was dried (MgSO4), concentrated under reduced pressure, and purified by flash column chromatography on silica gel (hexanes/ethyl acetate 4:1) to afford product 4a  as a colorless oil. 1H NMR  δ 7.33 , 4.76 , 4.58 , 3.68 , 1.86 ; 13C NMR  δ 150.9, 148.1, 135.6, 128.7, 128.2, 127.9, 94.4, 52.1, 44.6, 18.3; IR (CHCl3) ν: 1685 cm−1; HRMS–ES (m/z): [M]+ calcd for C12H13NO2, 203.0946; found, 203.0952.[AuClPPhFile 11H NMR and 13C NMR spectra and computational details.Experimental details, analytical data of new compounds, copies of