3-Methylene-4-pentenyl tosylate (4-OTs)
To a solution of 93 mg (0.50 mmol) of p-toluenesulfonyl chloride and 73 mg (0.60 mmol) of 4-(N,N-dimethylamino)pyridine (DMAP) in 4 mL of CH2Cl2 was added 47 mg (0.48 mmol) of 4-OH. After 2 h, the mixture was poured into hexane, and the resulting precipitates were removed by filtration. Solvent was removed at reduced pressure, and the residue was chromatographed on silica gel (40/60 = diethyl ether/hexane) to give 113 mg (92%) of a clear colorless oil; IR (neat) 3100, 3043, 3015, 2959, 2917, 1650, 1603, 1358, 1309, 1293, 1230, 1188, 1176, 1096, 970 cm−1; UV (CHCl3) _max 222 (_ 18,600), 197 (_13,800) nm; 1H NMR (CDCl3) _ 2.45 (3H, s), 2.59 (2H, td, JH,H = 7.2, 0.9 Hz), 4.14 (2H, t, JH,H = 7.2 Hz), 5.0–5.15 (4H, m), 6.29 (1H, dd, JH,H = 17.6, 10.5 Hz), 7.35 (1H, d, JH,H = 8.4 Hz), 7.79 (1H, d, JH,H = 8.4 Hz); 13C NMR (CDCl3) _ 21.9, 31.1, 68.9, 114.0, 118.6, 128.0, 129.9, 130.0, 133.2, 138.0, 140.7, 144.9; HRMS (EI at 70 eV) calcd for C13H16O3S 252.0816, found 252.0797.
General procedure for synthesis of diphosphates
The procedure of Davission and coworkers26
was followed with the exception that HPLC was used in the final purification step instead of flash chromatography on cellulose. To a solution of 1.5–3.0 equiv of tris(tetra-n
-butylammonium) hydrogen pyrophosphate in CH3
CN was added a solution of tosylate, chloride, or bromide (0.25–0.50 mM) in CH3
CN. The resulting mixture was allowed to stir for 2–3 h at room temperature. Solvent was removed at reduced pressure, and the resulting residue was dissolved in a minimal volume of 25 mM NH4
(ion-exchange buffer). The clear solution was passed through DOWEX AG 50W-X8 (100–200 mesh, ammonium form) that had been equilibrated with two column volumes of the ion exchange buffer at a flow rate of one column volume/15 min. The resulting clear solution was concentrated by rotary evaporation and lyophilized. The residue was then transferred to a centrifuge tube and dissolved in 100 mM NH4
. The resulting solution was extracted two or three times with a 1:1 mixture of CH3
CN and isopropanol. CH3
CN and isopropanol were removed at reduced pressure and the aqueous layer was lyophilized. The residue was by HPLC, and the purified diphosphate was stored at −78 °C.
(E)-3-Methyl-2,4-pentadienyl diphosphate (E-2-OPP)
A solution of 90 mg (0.92 mmol) of E-2-Br in CH3CN was treated with 2.74g (3.00 mmol) of tris(tetra-n-butyl-ammonium) hydrogren pyrophosphate. The ammonium form of the diphosphate was purified by HPCL on an Asahipak ODP-90 HPLC column with gradient elution (A = 100 mM NH4HCO3, solvent B = CH3CN; 0–20 min (A) 20–35 min 1:1 (A/B), 35–45 min, 1:1 (A/B)); to give 120 mg (42%) of a white solid; UV (25 mM NH4HCO3) _max 228 (_ 14,800), 197 (_ 10,600) nm; 1H NMR (D2O/ND4OD, pH = 8.0) _ 1.60 (3H, s), 4.40 (2H, dd, JH,H = JH,P = 6.6 Hz), 4.94 (1H, d, JH,H = 11.1 Hz), 5.26 (1H, d, JH,H = 17.7 Hz), 5.53 (1H, t, JH,H = 6.6 Hz), 6.29 (1H, dd, JH,H = 17.7, 10.8 Hz); 13C NMR (D2O/ND4OD, pH = 8.0) _ 14.1, 65.3 (d, JC,P = 5.2 Hz), 112.7, 129.9 (d, JC,P = 7.9 Hz), 140.9, 143.2; 31P NMR (D2O/ND4OD, pH = 8.0) _ −1.90 (1P, d, JP,P = 22 Hz), −4.76 (1P, d, JP,P = 22 Hz); HRMS ((−)-FAB) calcd for C6H11P2O7 256.9980, found 256.9966.
(Z)-3-Methyl-2,4-pentadienyl diphosphate (Z-2-OPP)
Following procedure described for E-2-OPP, 63 mg (0.54 mmol) of Z-2-Cl was treated with 0.901 g (1.0 mmol) of tris(tetra-n-butylammonium) hydrogen pyrophosphate to give 87 mg (52 %) of a white solid; UV (25 mM NH4HCO3) _max 229 (_ 8,9000), 196 (_ 6,800), nm; 1H NMR (D2O/ND4OD, pH = 8.0) _ 1.72 (3H, s), 4.44 (2H, dd, JH,H = JH,P = 7.0 Hz), 5.13 (1H, d, JH,H = 11.1 Hz), 5.25 (1H, d, JH,H = 17.4 Hz), 5.50 (1H, t, JH,H = 7.3 Hz), 6.71 (1H, dd, JH,H = 17.2, 10.9 Hz); 13C NMR (D2O/ND4OD, pH = 8.0) _ 21.6, 64.2 (d, JC,P = 5.2 Hz), 119.3, 127.7 2 (d, JC,P = 7.8 Hz), 135.6, 140.1; 31P NMR (D2O/ND4OD, pH = 8.0) _ −1.83 (1P, d, JP,P = 21 Hz, P1), −4.83 (1P, d, JP,P = 21 Hz, P2), HRMS ((−)-FAB) calcd for C6H11P2O7 256.9980, found 256.9959.
3-Methylene-4-pentenyl diphosphate (4-OPP)
Following the procedure described for E-2-OPP, 230 mg (1.10 mmol) of 4-OTs was treated with 2.98 g (3.30 mmol) of tris(tetra-n-butyl) ammonium pyrophosphate to give 147 mg (43 %) of a white solid; UV (25 mM NH4HCO3) _max 224 (_ 16,600) nm; 1H NMR (D2O/ND4OD, pH = 8.0) _ 2.51 (1H, dd, JH,H = 18.0, 10.5 Hz), 3.98 (2H, dd, JH,H = 7.2 Hz), 5.03–5.31 (4H, m), 6.37 (1H, dd, JH,H = 18.0, 10.5 Hz); 13C NMR (D2O/ND4OD, pH = 8.0) _ 34.5 (d, JC,P = 5.6 Hz), 67.7 (d, JC,P = 4.8 Hz), 116.9, 120.5, 141.1, 145.3; 31P NMR (D2O/ND4OD, pH = 8.0) _ −0.46 (1P, d, JP,P = 22 Hz), −4.63 (1P, d, JP,P = 22 Hz); HRMS ((−)-FAB) calcd for C6H11P2O7 256.9980, found 256.9972.
Assay for IPP isomerase Schizosaccharomyces pombe27
and Escherichia coli28
IPP isomerase for were obtained as described previously. The activity of the enzymes was measured by the acidliability protocol9
in a total volume 200 _l of 50 mM HEPES buffer, pH 7.0, containing10 mM MgCl2
, 200 mM KCl, 0.5 mM DTT, 1 mg/ml BSA, 400 _M [1-14
C] IPP (10 _Ci/_mol). The mixture equilibrated at 37 °C and initiated by addition of 10 _l of a solution of enzyme in assay buffer. Addition of a His6
tag to E. coli
IPP isomerase did not significantly alter its kinetic properties.
Time dependent inactivation of the enzyme was measured as follows. Assay buffer containing varying concentrations of inhibitor was equilibrated at 37 °C before a 20-fold higher concentration of enzyme than required for the standard assay was added to a final volume of 200 _l. Samples (10 _l) were removed at different times and added to assay buffer containing 400 _M [14C]IPP (10 _Ci/_mol) to a final volume of 200 _l.
Crystallization of E. coli IPP isomerase
Crystals of recombinant (C-terminal His-tagged) E. coli
IPP isomerase were grown at room temperature by the hanging drop method as described by Oudjama et al.29
. Briefly, protein (10 mg/ml) was equilibrated against a reservoir containing PEG2000 (16%), ammonium sulfate (100 mM) and MnCl2
(10 mM) buffered with Tris/maleate at pH 5.5. Complexes with 4-OPP
were obtained by soaking crystals of the enzyme in a (25 mM) solution of the inhibitor in Tris/maleate (100 mM, pH 5.5), PEG2000 (16%), ammonium sulfate (100 mM), MnCl2
(10 mM), and glycerol (25 %). These conditions were identical to those used to crystallize the wild type enzyme. After soaking, a crystal was flash frozen and diffraction data were collected with a Mar345 imaging plate system from Marresearch equipped with Osmic optics and running on an FR591 rotating anode generator. Diffraction data were processed with the MarFLM suite. In the presence of metal, the protein crystallizes, with two molecules in the asymmetric unit, in orthorhombic space group P21
with cell parameters a = 69.3, b = 72.6 and c = 92.5 Å. A data set was recorded at 2.05 Å resolution. Refinement was performed with the program Shelxl97 using the structure of unliganded IPP isomerase (PDB reference code : 1hxz) as starting model.