肉桂酸合成文獻(xiàn)

1、Supplementary information.Structure Activity Relationship of trans-Substituted-Propenoic Acid Derivatives on the Nicotinic Acid Receptor HCA2 (GPR109A). J.P.D. van Veldhoven, C.C. Blad, C.M. Artsen, C. Klopman, D.R. Wolfram, M.J. Abdelkadir, J.R. Lane, J. Brussee and A.P. IJzermanDivision of Medicin

2、al Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands. 1. Chemistry. All reagents used were obtained from commercial sources and all solvents were of analytical grade. 1H and 13C NMR spectra were recorded on a Bruker AV 400 (1H NMR

3、, 400 MHz; 13C NMR, 100 MHz) spectrometer with tetramethylsilane as an internal standard. Chemical shifts are reported in (ppm) and the following abbreviations are used: s = singlet, d = doublet, dd = double doublet, t = triplet, m = multiplet, br = broad, ar = aromatic protons, ph = phenyl protons.

4、 Elemental analyses were performed by the Leiden Institute of Chemistry and are within 0.4% of the theoretical values unless otherwise stated or purity was confirmed by HPLC and High Resolution Mass Spectroscopy. HPLC was performed on a Gilson 306 system (detection at 254 nm) equipped with an analyt

5、ical C18 column (4.6 mm x 12.5 cm, 5 m) in combination with a gradient of mixture A: 1 MeCN / 9 H2O, B: 9 MeCN / 1 H2O. High resolution mass spectra were recorded by direct injection on a mass spectrometer (Thermo Finnigan LTQ Orbitrap) equipped with an electro spray ion source in positive mode (sou

6、rce voltage 3.5 kV, sheath gas flow 10, capillary temperature 250 C) with resolution R = 60000 at m/z 400 (mass range m/z = 1502000) and dioctyl phthalate (m/z = 391.2842) as a “l(fā)ock mass”. Reactions were routinely monitored on TLC using Merck silica gel F254 plates. Microwave reactions were perform

7、ed in an Emrys Optimizer (Biotage AB, formerly Personal Chemistry). Wattage was automatically adjusted so as to maintain the desired temperature. The yields of all products were not optimized. All the final products (Tables 1 and 2) were purified by column chromatography followed by recrystallizatio

8、n.Compounds 1-3, 25-46, 48, 50, 51, 55 and 56 were obtained from commercial sources.1.1. General procedures for the synthesis of the mono substituted fumarates.OHOOOROHOOOROHOOHOROHOHOOOR+a4,9-11, 131b33, 34 5-8, 12, 14-24 Scheme 1. Synthesis of fumaric/maleic acid esters: (a) N-methylmorpholine, ED

9、C*HCl, DMF; (b) DMF, microwave, 180 C.1.1.1. Method A 1:To a mixture of fumaric acid (8 mmol, 1.0 eq) and the appropriate alcohol (8 mmol, 1 eq.) in DMF (10 mL) was added N-methylmorpholine (8 mmol, 1 eq.) and EDC*HCl (8 mmol, 1 eq.) while cooled on ice. The reaction mixture was stirred overnight af

10、ter which the DMF was removed in vacuo. A 1M HCl solution was added and the product was extracted 3 times with DCM. The combined organic layers were dried over MgSO4 and concentrated to dryness. The crude product consisting of the di-ester, the trans- and cis product was purified by column chromatog

11、raphy on SiO2, eluting with a tetrahydrofuran/ petroleum ether solvent system and crystallization from benzene1.1.2. Method B 2:A neat mixture of fumaric acid (14 mmol, 1 eq.) and the alcohol (14 mmol, 1 eq.) was heated in the microwave at 180 oC for 40 minutes. To the reaction mixture 100 mL of dic

12、hloromethane was added and the non dissolved fumaric acid was filtered off. The filtrate was concentrated in vacuo, consisting of the diester and the trans product, and purified by column chromatography using dichloromethane and a 2% methanol/dichloromethane mixture. The crystalline product was obta

13、ined after crystallization from benzene.1.1.3. Propyl fumarate (4)Method A, yield 7%, 100 mg; white solid. 1H-NMR (DMSO): 6.69 (s, 2H, acrylic-H), 4.11 (t, 2H, J = 6.8 Hz, OCH2CH2CH3), 1.66 1.61 (m, 2H, OCH2CH2CH3 ), 0.90 (t, 3H, J = 7.2 Hz, OCH2CH2CH3). 13C-NMR (DMSO): 165.8, 164.7, 134.6, 132.7, 6

14、6.4, 21.5, 10.2. Anal. Calcd for C7H10O4: C, 53.16; H, 6.37. Found C, 52.95; H, 6.67. 1.1.4. Isopropyl fumarate (5)Method B, yield 4%, 170 mg; white solid. 1H NMR (DMSO): 13.15 (s br, 1H, COOH), 6.66 (d, J = 19.2 Hz, 2H, acrylic-H), 4.99 (sept, J = 6.4 Hz, 1H, CH(CH3)2), 1.24 (d, J= 6.4 Hz, 6H, CH(C

15、H3)2). 13C-NMR (DMSO): 165.7, 164.1, 134.4, 133.0, 68.6, 21.5. Anal. Calcd for C7H10O4: C, 53.16; H, 6.37. Found C, 53.60; H, .5. Butyl fumarate (6)Method B, yield 8%, 87 mg; white solid. 1H NMR (CDCl3): 6.95 (d, J = 15.6 Hz, 1H, acrylic-H), 6.85 (d, 1H, acrylic-H), 4.22 (t, J = 6.8 Hz, 2H,

16、CH2), 1.71-1.64 (m, 2H, CH2-), 1.46-1.37 (m, 2H, CH2), 0.95 (t, J = 7.6 Hz, 3H, CH3). 13C-NMR (CDCl3): 170.3, 164.9, 135.9, 132.7, 65.6, 30.6, 19.2, 13.7. Anal. Calcd for C8H12O4: C, 55.81; H, 7.02. Found C, 55.52; H, .6. Pentyl fumarate (7)Method B, yield 7%, 115 mg. off white solid. 1H NMR

17、 (CDCl3): 10.93 (s br, 1H, COOH), 6.96 (d, J = 15.6 Hz, 1H, acrylic-H), 6.85 (d, J = 16.0 Hz, 1H, acrylic-H), 4.22 (t, J = 6.8 Hz, 2H, CH2), 1.73-1.66 (m, 2H, CH2), 1.39-1.35 (m, 4H, CH2), 0.92 (t, J = 6.8 Hz, 3H, CH3). 13C-NMR (CDCl3): 170.3, 164.9, 136.1, 132.6, 65.9, 28.3, 28.1, 22.4, 14.1. Anal.

18、 Calcd for C9H14O4*0.04 C6H6: C, 58.72; H, 7.58. Found C, 58.72; H, .7. Hexyl fumarate (8)Method B, yield 24%, 190 mg; off white solid. 1H NMR (CDCl3): 10.45 (s br, 1H, COOH), 6.95 (d, J = 15.6 Hz, 1H, acrylic-H), 6.85 (d, J = 16.0 Hz, 1H, acrylic-H), 4.22 (t, J = 6.8 Hz, 2H, CH2), 1.72-1.66

19、 (m, 2H, CH2), 1.40-1.26 (m, 6H, CH2), 0.90 (t, J = 6.8 Hz, 3H, CH3). 13C-NMR (CDCl3): 169.9, 164.8, 136.1, 132.5, 65.9, 31.5, 28.6, 25.7, 22.7, 14.1. Anal. Calcd for C10H16O4: C, 59.98; H, 8.05. Found C, 60.37; H, 8.0. Cyclohexyl fumarate (9)Method A, yield 3%, 72 mg; white solid. 1H-NMR (DM

20、SO): 6.66 (s, 2H, acrylic-H), 4.80 4.74 (m, 1H, OCHcyclohexyl), 1.81 1.79 (m, 2H, CH2cyclohexyl), 1.68 1.65 (m, 2H, CH2cyclohexyl), 1.50 1.23 (m, 6H, CH2cyclohexyl). 13C-NMR (DMSO): 165.8, 164.0, 134.6, 133.1, 73.2, 30.9, 24.9, 23.1. Anal. Calcd for C10H14O4: C, 60.59; H, 7.12. Found C, 60.99; H, 7.

21、16. HRMS MH+: 199.09645 delta = 0.2 ppm. HPLC purity 99%.1.1.9. Phenyl fumarate (10)Method A, yield 6%, 160 mg; white solid. 1H-NMR (DMSO): 7.47 7.43 (m, 2H, Ar-H), 7.31 7.28 (m, 1H, Ar-H), 7.22 7.21 (m, 2H, Ar-H), 6.90 (s, 2H, acrylic-H ). 13C-NMR (DMSO): 165.6, 163.4, 150.1, 136.1, 131.9, 129.6, 1

22、26.3, 121.7. Anal. Calcd for C11H10O4: C, 62.50; H, 4.20. Found C, 62.46; H, .10. Benzyl fumarate (11)Method A, yield 5%, 90 mg; white solid. 1H-NMR (DMSO): 7.42 7.34 (m, 5H, Ar-H), 6.74 (s, 2H, acrylic-H ), 5.23 (s, 2H, CH2Ar). 13C-NMR (DMSO): 165.7, 164.5, 135.6, 137.1, 132.4, 128.6, 128.3

23、, 128.2, 66.5. Anal. Calcd for C11H10O4: C, 64.08; H, 4.89. Found C, 64.08; H, 4.98. HRMS MH+: 207.06522 delta = 0.1 ppm. HPLC purity 97%.1.1.11. (rac)-methylbenzyl fumarate (12)Method B, yield 20%, 534 mg; white solid. 1H NMR (DMSO) 13.21 (s br, 1H, COOH), 7.38-7.29 (m, 5H, Ar-H), 6.73 (s, 2H, acry

24、lic-H), 5.91 (q, J = 6.4 Hz, 1H, ArCHCH3). 13C-NMR (DMSO): 165.7, 163.8, 141.2, 134.9, 132.7, 128.5, 127.9, 125.9, 72.9, 22.1. Anal. Calcd for C12H12O4: C, 65.45; H, 5.49. Found C, 65.85; H, .12. Phenylethyl fumarate (13)Method A, yield 17%, 769 mg; white solid. 1H NMR (DMSO) 13.22 (s br, 1H

25、, COOH), 7.32-7.20 (m, 5H, Ar-H), 6.66 (s, 2H, acrylic-H), 4.36 (t, J = 6.8 Hz, 2H, ArCH2CH2O), 2.94 (t, J = 6.8 Hz, 2H, ArCH2CH2O). 13C-NMR (DMSO): 165.7, 164.4, 137.9, 134.7, 132.5, 128.9, 128.4, 126.5, 65.4, 34.2. Anal. Calcd for C12H12O4: C, 65.45; H, 5.49. Found C, 65.42; H, .13. Phenyl

26、propyl fumarate (14)Method B, yield 6%, 110 mg; white solid. 1H NMR (DMSO): 7.30-7.16 (m, 5H, Ar-H), 6.69 (s, 2H, acrylic-H), 4.14 (t, J = 6.4 Hz, 2H, CH2), 2.67 (t, J = 7.6 Hz, 2H, CH2), 1.97-1.90 (m, 2H, CH3). 13C-NMR (DMSO): 165.8, 164.6, 141.1, 132.6, 128.4, 128.3, 125.9, 64.3, 31.4, 29.6. Anal.

27、 Calcd for C13H14O4 * 0.06 C6H6: C, 67.29; H, 6.07. Found C, 67.28; H, .14. 2-Bromobenzyl fumarate (15)Method B, yield 6%, 66 mg; white solid. 1H NMR (CDCl3): 10.12 (s br, 1H, COOH), 7.60 (d, J = 7.6 Hz, 1H, Ar-H), 7.43 (d, J = 6.8 Hz, 1H, Ar-H), 7.34 (t, J = 7.2 Hz, 1H, Ar-H), 7.22 (t, J =

28、6.8 Hz, 1H, Ar-H), 7.01 (d, J = 15.6 Hz, 1H, acrylic-H), 6.91 (d, J = 16.0 Hz, 1H, acrylic-H), 5.34 (s, 2H, CH2). 13C-NMR (CDCl3): 170.1, 164.4, 135.3, 134.5, 133.4, 133.1, 130.3, 130.2, 127.7, 123.8, 66.9. Anal. Calcd for C11H9BrO4: C, 46.34; H, 3.18. Found C, 46.52; H, .15. 2-Methoxybenzyl

29、 fumarate (16)Method B, yield 4%, 34 mg; white solid. 1H NMR (CDCl3): 9.22 (s br, 1H, COOH), 7.33 (s, 2H, Ar-H), 7.00-6.85 (m, 4H, acrylic-H + Ar-H), 5.30 (s, 2H, CH2) 3.85 (s, 3H, CH3). 13C-NMR (CDCl3): 170.1, 164.7, 157.8, 135.9, 132.8, 130.2, 123.5, 120.6, 110.6, 63.0, 55.6. Anal. Calcd for C12H1

30、2O5: C, 61.02; H, 5.12. Found C, 61.20; H, .16. 3-Bromobenzyl fumarate (17)Method B, yield 10%, 336 mg; white solid. 1H NMR (DMSO) 13.25 (s br, 1H, COOH), 7.64 (m, 1H, Ar-H), 7.54 (d, J = 7.6 Hz, 1H, Ar-H), 7.42 (d, J = 7.6 Hz, 1H, Ar-H), 7.37-7.33 (m, 1H, Ar-H), 6.76 (s, 2H, acrylic-H), 5.2

31、2 (s, 2H, CH2). 13C-NMR (DMSO): 165.7, 164.4, 138.4, 135.1, 132.2, 131.6, 130.7, 127.1, 121.7, 65.5. Anal. Calcd for C11H9BrO4: C, 46.34; H, 3.18. Found C, 46.38; H, .17. 3-Chlorobenzyl fumarate (18)Method B, yield 11%, 119 mg; white solid. 1H NMR (CDCl3): 11.11 (s br, 1H, COOH), 7.38-7.14 (

32、m, 4H, Ar-H), 6.99 (d, J =15.6Hz, 1H, acrylic-H), 6.89 (d, J =15.6Hz, 1H, acrylic-H), 5.22 (s, 2H, CH2). 13C-NMR (CDCl3): 170.2, 164.4, 137.1, 135.4, 134.7, 133.4, 130.1, 128.9, 128.5, 126.5, 66.5. Anal. Calcd for C11H9ClO4: C, 54.90; H, 3.77. Found C, 55.21; H, .18. 3-Fluorobenzyl fumarate

33、(19)Method B, yield 11%, 112 mg; white solid. 1H NMR (CDCl3): 10.05 (s br, 1H, COOH), 7.38-7.32 (m, 1H, Ar-H), 7.15 (d, J = 7.6 Hz, 1H, Ar-H), 7.10-7.02 (m, 2H, Ar-H), 7.00 (d, J = 16.0 Hz, 1H, acrylic-H), 6.89 (d, J = 15.6 Hz, 1H, acrylic-H), 5.24 (s, 2H, CH2). 13C-NMR (CDCl3): 169.9, 164.3, 164.1,

34、161.6, 137.5, 137.4, 135.3, 133.3, 130.4 130.3, 123.8, 123.7, 115.7, 115.4, 115.3, 115.1, 66.4. Anal. Calcd for C11H9FO4*0.05 C6H6: C, 59.56; H, 4.12. Found C, 59.55; H, .19. 3-Methoxybenzyl fumarate (20)Method B, yield 6%, 52 mg; white solid. 1H NMR (CDCl3): 9.44 (s br, 1H, COOH), 7.31-7.27

35、 (m, 1H, Ar-H), 7.00-6.95 (m, 2H, Ar-H), 6.91-6.86 (m, 3H, acrylic-H + Ar-H), 5.22 (s, 2H, CH2), 3.83 (s, 3H, CH3). 13C-NMR (CDCl3): 169.9, 164.5, 159.8, 136.6, 135.4, 133.1, 129.8, 120.6, 114.1, 113.9, 67.2, 55.3. Anal. Calcd for C12H12O5: C, 61.02; H, 5.12. Found C, 61.43; H, .20. 4-Bromob

36、enzyl fumarate (21)Method B, yield 3%, 40 mg; white solid. 1H NMR (DMSO) 13.22 (s br, 1H, COOH), 7.59 (d, J = 8.4 Hz, 2H, Ar-H), 7.38 (d, J = 8.8 Hz, 2H, Ar-H), 6.74 (s, 2H, acrylic-H ), 5.20 (s, 2H, CH2Ar). 13C-NMR (DMSO): 165.6, 164.4, 135.1, 132.2, 131.4, 130.3, 121.4, 65.6. Anal. Calcd for C11H9

37、BrO4: C, 46.34; H, 3.18. Found C, 46.45; H, .21. 4-Chlorobenzyl fumarate (22)Method B, yield 10%, 90 mg; white solid. 1H NMR (DMSO): 13.28 (s br, 1H, COOH), 7.45 (s, 4H, Ar-H), 6.73 (s, 2H, acrylic-H), 5.21 (s, 2H, CH2). 13C-NMR (DMSO): 166.0, 164.8, 135.5, 135.1, 132.6, 130.4, 128.9, 65.9.

38、Anal. Calcd for C11H9ClO4: C, 54.90; H, 3.77. Found C, 54.59; H, .22. 4-Methylbenzyl fumarate (23)Method B, yield 3%, 30 mg; white solid. 1H NMR (DMSO): 13.19 (s br, 1H, COOH), 7.30 (d, J = 8.0 Hz, 2H, Ar-H), 7.19 (d, J = 8.0 Hz, 2H, Ar-H), 6.72 (s, 2H, acrylic-H), 5.18 (s, 2H, CH2), 2.30 (s

39、, 3H, CH3). 13C-NMR (CDCl3): 170.0, 164.6, 138.7, 135.8, 132.9, 132.2, 129.5, 128.8, 67.5, 21.4. Anal. Calcd for C12H12O4*0.03CH2Cl2: C, 64.73; H, 5.45. Found C, 64.71; H, .23. 4-Methoxybenzyl fumarate (24) Method B, yield 43%, 730 mg; white solid. 1H NMR (DMSO): 13.20 (s br, 1H, COOH), 7.35

40、 (d, J = 8.8 Hz, 2H, Ar-H), 6.93 (d, J = 8.4 Hz, 2H, Ar-H), 6.71 (s, 2H, acrylic-H), 5.15 (s, 2H, CH2), 3.75 (s, 3H, CH3). 13C-NMR (DMSO): 165.6, 164.5, 159.3, 134.8, 132.5, 130.2, 127.5, 113.9, 66.3, 55.1. Anal. Calcd for C12H12O5: C, 61.02; H, 5.12. Found C, 61.44; H, . General procedure f

41、or the synthesis of trans heterocyclic propenoic acidsHOXROHOOHOXOHOR+47 49,52-54,57,58X = NHX = OX = OX = OX = SX = SR = HR = 5-BrR = 5-MeR = 5-EtR = 3-BrR = 4-Bra495253545758Scheme 2. Synthesis of trans-heterocyclic-propenoic acids: (a) piperidine, pyridine, reflux 3 hours.Piperidine was added to

42、a solution of the appropriate aldehyde (4.97 mmol), malonic acid (5.96 mmol) and pyridine (10.94 mmol) under nitrogen atmosphere. The reaction mixture was heated to reflux under vigorous stirring for 3 hours, after which according to TLC the reaction was completed. The reaction mixture was allowed t

43、o cool and EtOAc (20 mL) was added, acidified to pH 5 with 1 M HCl, and the organic layer was sequentially washed with water, dried over MgSO4 and concentrated in vacuo. The pure desired product was obtained after purification by column chromatography.1.2.1. trans-3-(1H-Pyrrol-2-yl)propenoic acid (4

44、9)Yield 32%, 461 mg; brown solid. 1H NMR (DMSO) 11.95 (s br, 1H, COOH ), 11.47 (s br, 1H, NH), 7.38 (d, J = 16.0 Hz, 1H, acrylic-H), 6.99 (d, J = 1.2 Hz, 1H, Ar-H), 6.51 (s, 1H, Ar-H), 6.156.13 (m, 1H, Ar-H), 6.11 (d, J = 16.0 Hz, 1H, acrylic-H). 13C-NMR (DMSO): 168.8, 134.8, 128.6, 123.6, 114.8, 11

45、1.8, 110.3. HRMS ESI+H calc for C7H7NO2: 138.05496, found:138.05478 delta: 1.3ppm. HPLC 99%.1.2.2. trans-3-(5-Bromofuran-2-yl)propenoic acid (52)Yield 60%, 250 mg; yellow solid. 1H NMR (DMSO) 12.45 (s br, 1H, COOH ), 7.32 (d, J = 15.6 Hz, 1H, acrylic-H), 6.96 (d, J = 3.6Hz, 1H, Ar-H), 6.75 (d, J = 3

46、.6 Hz, 1H, Ar-H), 6.14 (d, J = 15.6 Hz, 1H, acrylic-H). 13C-NMR (DMSO): 167.1, 152.5, 129.7, 125.1, 117.7, 116.6, 114.8. HRMS ESI+H calc. for C7H5BrO3: 216.94948; found: 216.94953 delta: 0.2 ppm. HPLC 98%. 1.2.3. trans-3-(5-Methylfuran-2-yl)propenoic acid (53)Yield 46%, 670 mg; white solid. 1H NMR (

47、DMSO) 12.26 (s br, 1H, COOH ), 7.32 (d, J = 15.6 Hz, 1H, acrylic-H), 6.81 (d, J = 3.2 Hz, 1H, Ar-H), 6.26 (d, J = 3.2 Hz, 1H, Ar-H), 6.05 (d, J = 15.6 Hz, 1H, acrylic-H), 2.33 (s, 3H, CH2CH3). 13C-NMR (DMSO): 167.9, 155.7, 149.4, 131.3, 117.6, 114.7, 109.7, 14.0. HRMS ESI+H calc. for C8H8O3: 153.054

48、62; found: 153.05448 delta: 0.9 ppm. HPLC 99%.1.2.4. trans-3-(5-Ethylfuran-2-yl)propenoic acid (54)Yield 74%, 300 mg; white solid. 1H NMR (DMSO) 12.27 (s br, 1H, COOH ), 7.32 (d, J = 15.6 Hz, 1H, acrylic-H), 6.82 (d, J = 3.2 Hz, 1H, Ar-H), 6.28 (d, J = 2.8 Hz, 1H, Ar-H), 6.06 (d, J = 15.6 Hz, 1H, ac

49、rylic-H), 2.67 (q, J = 7.6 Hz, 2H, CH2CH3), 1.20 (t, J = 7.6 Hz, 3H, CH2CH3). 13C-NMR (DMSO): 167.9, 161.0, 149.3, 131.3, 117.4, 114.8, 108.2, 21.5, 12.2. HRMS ESI+H calc. for C9H10O3: 167.07027; found: 167.07015 delta: 0.7 ppm. HPLC 97%.1.2.5. trans-3-(3-Bromothiophen-2-yl)propenoic acid (57)Yield

50、60%, 700 mg; yellow solid. 1H NMR (DMSO) 12.66 (s br, 1H, COOH ), 7.84 (d, J = 5.2 Hz, 1H, Ar-H), 7.62 (d, J = 15.6 Hz, 1H, acrylic-H), 7.24 (d, J = 5.2 Hz, 1H, Ar-H), 6.28 (d, J = 15.6 Hz, 1H, acrylic-H). 13C-NMR (DMSO): 167.4, 124.3, 133.8, 131.8, 130.6, 120.2, 115.9. HRMS ESI+H calc for C7H5BrO2S

51、: 232.92664; found: 232.92673 delta: 0.4 ppm. HPLC 93%.1.2.6. trans-3-(4-Bromothiophen-2-yl)propenoic acid (58)Yield 32%, 385 mg; brown solid. 1H NMR (DMSO) 12.53 (s br, 1H, COOH ), 7.82(d, J = 1.2 Hz, 1H, Ar-H), 7.69 (d, J = 16.0 Hz, 1H, acrylic-H), 7.58 (d, J = 1.2 Hz, 1H, Ar-H), 6.28 (d, J = 16.0

52、 Hz, 1H, acrylic-H). 13C-NMR (DMSO): 167.5, 140.7, 135.9, 133.1, 127.1, 119.5, 110.4. HRMS ESI+H calc for C7H5BrO2S: 234.92455; found: 234.92466 delta: 0.2 ppm. HPLC 98%.2. Pharmacology2.1. Membrane isolationHEK293T cells stably expressing human HCA2 (GPR109A) were harvested by scraping in PBS. The

53、cells were centrifuged and resuspended in 50 mM Tris-HCl buffer, pH 7.4. Then a DIAX 900 homogenizer (Heidolph, Schwabach, Germany) was used for cell lysis. The suspension was centrifuged at 225,000 g for 20 min and the supernatant was discarded. The pellet was resuspended in 50 mM Tris-HCl (pH 7.4), and the homogenization and centrifugation steps were repeated. The membranes were resuspended in assay buffer (50 mM Tris HCl, 1 mM