General Methods and Materials
Dimethyl Geranylphosphonate (9)
A mixture of 8 (35 g, 0.16 mol) and trimethyl phosphite (22 g, 0.18 mol) was heated to reflux for 4 h. After cooling to rt, the mixture was chromatographed on silica gel (hexanes/ethyl acetate, 5:1 to 1:2) to give the product as a colorless oil (36 g, 91%). 1H-NMR (CDCl3, 300 MHz): 5.15 (1H, m), 5.03 (1H, m), 3.72 (3H, s), 3.69 (3H, s), 2.55 (2H, dd, J = 7.8, 21.9 Hz), 2.03 (4H, m), 1.64 (3H, s), 1.63 (3H, d, J = 5.7 Hz) and 1.57 (3H, s). 13C-NMR (CDCl3, 75 MHz): 140.4 (d, J = 14.3 Hz), 131.6, 123.8, 112.0 (d, J = 11.2 Hz), 52.6, 52.5, 39.6 (d, J = 2.9 Hz), 25.6, 25.4 (d, J = 142.0 Hz), 17.6, 16.2 and 16.1. 31P-NMR (CDCl3, 121 MHz): 31.7. HRMS (ESI) calcd for C12H24O3P [M + H]+ 247.1463; found 247.1453.
Dimethyl (8-Hydroxy-3,7-dimethylocta-2,6-dienyl)phosphonate (10)
To a suspension of SeO2 (31 mg, 0.27 mmol), salicylic acid (190 mg, 1.3 mmol) and 90% t-Bu-OOH (4.5 ml) in CH2Cl2 (20 ml) was added 9 (2.5 g, 10 mmol) in CH2Cl2 (5.0 mL) at rt. The mixture was diluted with Et2O after being stirred for 21 h, washed with dilute Na2SO3 solution and brine, then dried over Na2SO4. The solvents were evaporated and the residue was chromatographed on silica gel (hexanes/ethyl acetate, 1:1 to 0:1) to give the alcohol as a colorless oil (1.3 g, 78% yield based on the converted starting material), together with the recycled starting material (0.95 g). 1H-NMR (CDCl3, 300 MHz): 5.26 (1H, m), 5.08 (1H, m), 3.91 (2H, s), 3.71 (3H, s), 3.67 (3H, s), 2.98 (1H, br s), 2.51 (2H, dd, J = 8.1, 21.9 Hz), 2.11 (4H, m), 1.60 (3H, s), 1.59 (3H, m). 13C-NMR (CDCl3, 75 MHz): 139.9 (d, J = 14.6 Hz), 135.5, 124.6, 112.4 (d, J = 11.4 Hz), 68.6, 52.6, 52.5, 39.1 (d, J = 3.1 Hz), 25.2 (d, J = 3.9 Hz), 25.1 (d, J = 140.3 Hz), 15.9 (d, J = 2.9 Hz), 13.6. 31P-NMR (CDCl3, 121 MHz): 31.8. IR (NaCl, cm−1): 3400 (br), 2955 (m), 1719 (m), 1683 (m). HRMS (ESI) calcd for C12H24O4P [M + H]+ 263.1412; found 263.1419.
Dimethyl (E,E) ((8-(3-Benzoyl)benzoyloxy)-3,7-dimethylocta-2,6-dienyl)phosphonate (11)
To a solution of 10 (2.2 g, 8.3 mmol), 3-benzoyl-benzoic acid (2.3 g, 10 mmol) in anhydrous CH2Cl2 (100 mL) was added DCC (2.1 g, 10 mmol) and DMAP (50 mg, 0.40 mmol) at rt. The resulting suspension was stirred for 20 h at room temperature and then filtered through Celite. The filtrate was concentrated and chromatographed on silica gel (hexanes/ethyl acetate, 2:1 to 1:2) to give the ester as a viscous oil (3.3 g, 82%). 1H-NMR (CDCl3, 300 MHz): 8.43 (1H, m), 8.24 (1H, dt, J = 7.8, 1.8 Hz), 7.97 (1H, dt, J = 7.8, 1.5 Hz), 7.79 (1H, m), 7.77 (1H, m), 7.45–7.62 (4H, m), 5.50 (1H, m), 5.17 (1H, m), 4.70 (2H, s), 3.72 (3H, s), 3.69 (3H, s), 2.55 (2H, dd, J = 7.8, 21.9 Hz), 2.16 (2H, m), 2.09 (2H, m), 1.70 (3H, s), 1.65 (3H, d, J = 3.3 Hz). 13C-NMR (CDCl3, 75 MHz): 195.8, 165.6, 139.9 (d, J = 14.3 Hz), 137.9, 137.0, 134.0, 133.2, 132.8, 131.0, 130.6, 130.1, 130.0, 129.4, 128.5, 128.4, 112.5 (d, J = 11.1 Hz), 70.9, 52.6, 52.5, 38.9 (d, J = 2.6 Hz), 26.1 (d, J = 3.4 Hz), 25.4 (d, J = 140.0 Hz), 16.2 (d, J = 2.4 Hz), 14.0. 31P-NMR (CDCl3, 121 MHz): 31.6. IR (NaCl, cm−1): 3065 (w), 2955 (s), 2854 (m), 1731 (s), 16675 (s) 1602 (s), 1579 (m). HRMS (ESI) calcd for C26H32O6P [M + H]+ 471.1936; found 471.1945.
Ammonium (E,E) 8-(3-Benzoylbenzoyloxy)-3,7-dimethylocta-2,6-dienylphosphonate (12)
To a solution of 11 (1.0 g, 2.1 mmol) in anhydrous CH2Cl2 (30 ml) was added allyltrimethylsilane (0.50 ml, 3.1 mmol) and bromotrimethylsilane (0.90 ml, 6.8 mmol) at rt. The mixture was stirred for 38 h and concentrated in vacuo. The residue was dissolved in H2O (4.0 mL), neutralized with NH4HCO3, then lyophilized to afford the phosphonic acid in its ammonium form as a white powder (0.87 g, 92%). 1H-NMR (25 mM NaHCO3 in D2O, 500 MHz): 7.80 (1H, s), 7.60 (1H, s), 7.27 (1H, d, J = 6.0 Hz), 7.15 (2H, m), 7.00 (2H, m), 6.89 (2H, m), 5.06 (1H, s), 5.01 (1H, d, J = 5.5 Hz), 4.15 (2H, s), 2.10 (2H, d, J = 15.0 Hz), 1.67 (4H, m), 1.28 (3H, s), 1.17 (3H, s). 13C-NMR (25 mM NaHCO3 in D2O, 75 MHz): 195.1, 165.3, 136.8, 136.4 (d, J = 13.0 Hz), 136.0, 133.8, 132.7, 129.9, 129.8, 129.6, 129.2, 128.1, 117.1, 70.9, 38.6, 28.9 (d, J = 131.2 Hz), 25.9, 15.3, 13.2. 31P-NMR (25 mM NaHCO3 in D2O, 121 MHz): 22.8. IR (NaCl, cm−1): 2971 (w), 2922 (w), 1720 (s), 1655 (s), 1602 (w). HRMS (ESI) calcd for C24H26O6P [M − H]− 441.1473; found 441.1481. The ammonium counter-ions within this compound are not observed during mass spectral analysis.
Ammonium (E,E) P-8-(3-Benzoylbenzoyloxy)-3,7-dimethylocta-2,6-dienylphosphonophosphate (3b)
A mixture of the phosphonate 12 (100 mg, 230 μmol) and 1,1′-carbonyldiimidazole (60 mg, 370 μmol) in anhydrous DMF (4.0 mL) was stirred for 5 h at rt. To this clear solution was added directly 98% phosphoric acid solid (~ 50 mg, 510 μmol). The solvent was removed under high vacuum after being stirred for 27 h. The residue was dissolved in H2O (1.0 mL), and loaded on an octyl-functionalized silica gel column (18 cm × 1 cm). The column was washed with a step gradient of H2O and MeOH (10 mL per step, 10% MeOH per step) from 0% to 70% MeOH. Fractions were collected and those containing product as determined by TLC were pooled and lyophilized. The lyophilized powder was dissolved in H2O, passed through a short, strong acidic ion exchange column, then washed with H2O. The resulting solution was neutralized with NH4HCO3 and lyophilized to give the product in it’s ammonium form as a white powder (28 mg, 23%). 1H-NMR (D2O, 300 MHz): 7.77 (1H, s), 7.75 (1H, d, J = 7.5 Hz), 7.50 (1H, d, J = 7.5 Hz), 7.09–7.31 (6H, m), 5.16 (1H, m), 5.03 (1H, m), 4.26 (2H, s), 2.33 (2H, dd, J = 7.5, 21.5 Hz), 1.78 (4H, m), 1.39 (3H, d, J = 3.3 Hz) and 1.31 (3H, s). 13C-NMR (D2O, 75 MHz): 197.5, 166.4, 138.2 (d, J = 14.0 Hz), 136.6, 135.7, 134.3, 133.4, 130.6, 129.8, 129.5, 129.4, 128.7, 128.4, 115.5 (d, J = 9.7 Hz), 71.1, 38.5, 28.4 (d, J = 135.2 Hz), 25.7, 15.4, 13.1. 31P-NMR (D2O, 121 MHz, pD 7.0, NH4+): 16.4 (d, J = 26.4 Hz), −9.6 (d, J = 26.4 Hz). IR (NaCl, cm−1): 2925 (w), 1715 (m), 1655 (m), 1101 (m). (HRMS (ESI) calcd for C24H27O9P2 [M − H]− 521.1136; found 521.1174. The ammonium counter-ions within this compound are not observed during mass spectral analysis.
(E,E) β-P-(3-Benzoylbenzoyloxy)-3,7-dimethylocta-2,6-dienylphosphonophosphate ([32P]3b)
A mixture of phosphonate 1 (4.0 mg, 8.0 μmol) and 1,1′-carbonyldiimidazole (4.0 mg, 24 μmol) in anhydrous DMF (250 μL) was stirred for 12 h at rt. 32P-labeled phosphoric acid was then prepared by lyophilizing 250 μL of 1% (v/v) phosphoric acid and 250 μL of 10 mCi/mL 32P-phosphoric acid over P2O5 overnight. Activated phosphonate in DMF was then added via syringe to the flask containing the dry 32P-labeled phosphoric acid (4.0 mg, 40 μmol) and left to stir for 24 h at rt, and monitored by TLC (6:3:1 2-propanol/NH4OH/H2O). Solvent was then removed under a stream of N2(g). The residue was dissolved in 25 mM NH4HCO3 (3 mL) and applied to a Sep-Pak® column equilibrated in the same solution. The column was washed with a step gradient of 25 mM NH4HCO3 and increasing CH3CN (10% increase per step, 2 mL per step). Fractions were collected and analyzed by TLC (6:3:1 2-propanol/NH4OH/H2O). Under these conditions, the desired product 2 eluted at 30–40% CH3CN/25 mM NH4HCO3. Product fractions were pooled, lyophilized, dissolved in 1.0 mL 25 mM NH4HCO3. UV analysis (ε258 24100 mol−1cm−1) and liquid scintillation counting were used to determine the solution concentration and specific activity (0.55 mM and 334 Ci/mol). Solution was used for protein cross-linking experiments without further purification.
Acid Stability Study of GPP
500 μL of 1.0 mM solution of GPP in 50:50 25 mM NH4HCO3/CH3CN containing 0.2 % TFA was allowed to sit at rt. 50 μL aliquots were removed at prescribed intervals (h) and analyzed by reverse-phase HPLC. A gradient of solvent A (25mM NH4HCO3) and solvent B (CH3CN) and detecting at 214 nm (flow rate, 0.7 mL/min) was employed. Elution was performed by a 40 min linear gradient of 20–80% B. Integration of the peak corresponding to GPP was performed using HPLC software. Hydrolysis half life was determined from a plot of remaining starting material (%) as a function of time.
Acid Stability Study of 3b
The same procedure as described for GPP was performed with a 0.30 mM solution of 3b in in 50:50 25 mM NH4HCO3/CH3CN containing 0.2 % TFA.
Purification of yPFTase and hPGGTase-I
yPFTase was purified as described by Mayer et al.41
and published in our earlier work.16
hPGGTase-I was purified using modifications of published procedures.12,42,43
A continuous fluorescence assay was employed to monitor yPFTase activity as described by Gaon et al.16
modified from the original published procedures.44,45
Enzyme Inhibition Experiments
The concentration of 3b was varied (0, 0.4, 0.6, 1.5, 3, 4.5, 6 μM) while the natural susbtrate, 1, and yPFTase were maintained at a fixed concentrations (10 μM and 11 nM). Enzymatic rates were obtained from linear regression analysis of the time-dependent fluorescence emission data using the fluorimeter software and the IC50 was calculated from a plot of the enzymatic rate versus concentration of 3b.
X-Ray Crystallography Studies
Crystals of the rPFTase:3b
complex was prepared by soaking 3b
into preformed crystals using methods previously described.32
X-ray diffraction data for the PFTase:3b
complex was collected on a Rigaku rotating anode generator equipped with osmic mirrors and a Raxis-IV image plate detector. With the detector set at 150 mm, data were collected in 323 contiguous 0.30° oscillation images each exposed for 8 minutes. The data extend to 2.2 Å resolution and have a Rmerge
of 4.6% with a 5.8-fold multiplicity. The structure was refined using CNX2002 (Accelrys Inc.) to an Rfactor
of 18.3% and an Rfree
Photolysis Reaction of Protein Prenyltransferases with [32P]3b
All reactions (100 μL) were performed in silinized quartz test tubes (10 × 75 mm) and contained 52 mM Tris-HCl (pH 7.5), 5.8 mM DTT, 12 mM MgCl2, 12 μM ZnCl2, 25 mM NH4HCO3, 10 μM [32P]3b (~10 fold above IC50 value) and 400 nM pure enzyme (yPFTase, hPGGTase-I) or 21.2 μg of partially purified (after ion-exchange FPLC) protein sample containing hPGGTase-I. For substrate protection experiments, FPP or GGPP was added to a final concentration of 100 μM. Reactions were photolyzed for 2 h at 4°C using a Rayonet Mini-Reactor fitted with 6– 350 nm bulbs and a spinning platform. Samples were spun to ensure an even exposure to the light. Loading buffer (100 μL) was then added to each sample and samples were heated to 100°C for 3 min followed by analysis with 12% Tris-Glycine SDS-polyacrylamide gel electrophoresis. Gels were stained with Sypro®-orange and subjected to 32P-phosphorimaging at rt.
Preparation of Hevea Brasiliensis Washed Rubber Particles
WRPs used in this study were prepared as previously described by R. Krishnakumar et al.46
and Cornish et al.47
Photolysis Reaction of Hevea Brasiliensis Washed Rubber Particles with [32P]3b
All reactions (~100 μL) were performed in silinized quartz test tubes (10 × 75 mm) and contained 0.1 M Tris-HCl (pH 7.5), 1.25 mM MgSO4, 5 mM DTT, 10 μM [32P]3b and one H. brasiliensis WRP (35–40 μL/particle, 4.51 μg protein/μL). For substrate protection experiments, FPP was added to a final concentration of 100 μM. Reactions were photolyzed for 6 h using the apparatus described above. Loading buffer (50 μL) was then added to each sample and samples were heated to 100°C for 3 min followed by gel analysis as described above.
Tryptic Digest of Labeled Proteins from Photolysis Reaction of Hevea Brasiliensis Washed Rubber Particles with [32P]3b
Proteins from previously described photolysis reaction (no SDS-PAGE analysis) were extracted with 5 mL of sequential extraction reagent (5.0 M urea, 2.0 M thiourea, 2.0% CHAPS, 2% SB-3–10, 40 mM Tris, and 0.2% Bio-Lyte 3 to 10 ampholyte). Samples were incubated in this buffer for 1 h at rt with constant rocking, and then centrifuged at max speed in a microcentrifuge for 15 min. The supernatant was then transferred to 50 mL conical tube and 4 volumes of cold acetone were added. Acetone precipitated protein pellets were washed twice with 80% acetone in water. Pellets were dried under N2 and prepared for fractionation using the ZOOM IEF fractionator according to manufacturer’s instructions. After IEF fractionation, samples from the different pH ranges were acetone precipitated as described above. Samples were then dissolved in 45 μL Laemmli sample buffer (0.06 M Tris-HCl pH 6.8, 0.01% bromophenol blue, 1.5% SDS, 10% glycerol) and analyzed by SDS-PAGE using 4–20% gradient gel. Gels were stained with Coomassie and subjected to 32P-phosphorimaging at rt. Radiolabeled protein bands were excised using pipette tip (3 spots from each band). For tryptic digestion of proteins, samples were reduced and alkylated using 10 mM dithiothreitol and 100 mM iodoacetamide in water, then digested with 100 ng of trypsin in 25mM NH4HCO3.
Mass Spectral Analysis of Labeled Proteins
Samples from tryptic digestion were spotted onto a MALDI target and eluted with 70% CH3CN, 0.2% formic acid containing 5 mg/mL MALDI matrix (α-Cyano-4-hydroxycinnamic acid). 0.5 μl was then spotted onto the MALDI target. Analysis was performed using an Applied Biosystems 4700 Proteomics Analyzer with TOF/TOF Optics. MALDI-MS data was acquired in reflector mode from a mass range of 700 – 4000 Daltons and 1250 laser shots were averaged for each mass spectrum. Each sample was internally calibrated using the trypsin autolysis products (m/z of 842.51 and 2211.10) as internal standards. The eight most intense ions from the MS spectrum were then subjected to MS/MS analysis. The mass range was 70 to precursor ion with a precursor window of −1 to 3 Daltons with an average 5000 laser shots for each spectrum. A peak list was created by GPS Explorer software (Applied Biosystems) from the raw data based on signal to noise filtering and included de-isotoping. The resulting file was then searched by MASCOT (Matrix Science) using user specified databases. A tolerance of 20 ppm was used if the sample was internally calibrated and 200 ppm tolerance if the default calibration was applied. Protein identification was validated by the following criteria: greater than 20 ppm mass accuracy on all MS ions and all ions in at least two MS/MS spectra, which were not modified, had to be accounted for.