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• W2015690692 abstract "The biosynthesis of taxol (paclitaxel) and related taxoids in Pacific yew (Taxus brevifolia) is thought to involve the cyclization of geranylgeranyl diphosphate to a taxadiene followed by extensive oxygenation of this diterpene olefin intermediate. A cell-free preparation from sapling yew stems catalyzed the conversion of [1-3H]geranylgeranyl diphosphate to a cyclic diterpene olefin that, when incubated with stem sections, was converted in good radiochemical yield to several highly functionalized taxanes, including 10-deacetyl baccatin III and taxol itself. Addition of the labeled olefin to a yew bark extract, followed by radiochemically guided fractionation, provided sufficient product to establish the structure as taxa-4(5),11(12)-diene by two-dimensional NMR spectroscopic methods. Therefore, the first dedicated step in taxol biosynthesis is the conversion of the universal diterpenoid precursor geranylgeranyl diphosphate to taxa-4(5),11 (12)-diene, rather than to the 4(20),11(12)-diene isomer previously suggested on the basis of the abundance of taxoids with double bonds in these positions. The very common occurrence of taxane derivatives bearing the 4(20)-ene-5-oxy functional grouping, and the lack of oxygenated derivatives bearing a 4(5)-double bond, suggest that hydroxylation at C-5 of taxadiene with allylic rearrangement of the double bond is an early step in the conversion of this olefin intermediate to taxol. The biosynthesis of taxol (paclitaxel) and related taxoids in Pacific yew (Taxus brevifolia) is thought to involve the cyclization of geranylgeranyl diphosphate to a taxadiene followed by extensive oxygenation of this diterpene olefin intermediate. A cell-free preparation from sapling yew stems catalyzed the conversion of [1-3H]geranylgeranyl diphosphate to a cyclic diterpene olefin that, when incubated with stem sections, was converted in good radiochemical yield to several highly functionalized taxanes, including 10-deacetyl baccatin III and taxol itself. Addition of the labeled olefin to a yew bark extract, followed by radiochemically guided fractionation, provided sufficient product to establish the structure as taxa-4(5),11(12)-diene by two-dimensional NMR spectroscopic methods. Therefore, the first dedicated step in taxol biosynthesis is the conversion of the universal diterpenoid precursor geranylgeranyl diphosphate to taxa-4(5),11 (12)-diene, rather than to the 4(20),11(12)-diene isomer previously suggested on the basis of the abundance of taxoids with double bonds in these positions. The very common occurrence of taxane derivatives bearing the 4(20)-ene-5-oxy functional grouping, and the lack of oxygenated derivatives bearing a 4(5)-double bond, suggest that hydroxylation at C-5 of taxadiene with allylic rearrangement of the double bond is an early step in the conversion of this olefin intermediate to taxol. The highly functionalized diterpenoid taxol (paclitaxel) 1Paclitaxel is the generic name for taxol, which is now a registered trademark of Bristol-Myers Squibb. Because of the far greater familiarity with the word taxol, we use it here in lieu of “paclitaxel.” 1 (1Wani M.C. Taylor H.L. Wall M.E. Coggon P. McPhail A.T. J. Am. Chem. Soc. 1971; 93: 2325-2327Crossref PubMed Scopus (3800) Google Scholar) is of interest because of its efficacy in the treatment of a range of cancers (2Holmes F.A. Kudelka A.P. Kavanagh J.J. Huber M.H. Ajani J.A. Valero V. Georg G.I. Chen T.T. Ojima I. Vyas D.M. Taxane Anticancer Agents: Basic Science and Current Status. American Chemical Society, Washington, D. C.1995: 31-57Google Scholar). A principal limitation to the wider therapeutic use of taxol was the limited supply of the drug isolated from the bark of Pacific yew (Taxus brevifolia Nutt.:Taxaceae); the yields are low (0.01-0.1% dry weight), the species is very slow growing and sparsely distributed, and the harvest is destructive (3Cragg G.M. Sander K.M. Cancer Cells. 1991; 3: 233-235PubMed Google Scholar, 4Kingston D.G.I. Pharmacol. Ther. 1991; 52: 1-34Crossref PubMed Scopus (320) Google Scholar). Alternate approaches for obtaining taxol include isolation from the renewable foliage and other tissues of plantation-grown Taxus species (5Witherup K.M. Look S.A. Stasko M.W. Ghiorzi T.J. Muschik G.M. Cragg G.M. J. Nat. Prod. 1990; 53: 1249-1255Crossref PubMed Scopus (234) Google Scholar, 6Kelsey R.G. Vance N.C. J. Nat. Prod. 1992; 55: 912-917Crossref Scopus (60) Google Scholar, 7Fett-Neto A.G. DiCosmo F. Planta Med. 1992; 58: 464-466Crossref PubMed Scopus (53) Google Scholar, 8Wheeler N.C. Hehnen M.T. J. Forest. 1993; 91: 15-18Google Scholar, 9Vidensek N. Lim P. Campbell A. Carlson C. J. Nat. Prod. 1990; 53: 1609-1610Crossref PubMed Scopus (177) Google Scholar, 10Wheeler N.C. Jech K. Masters S. Brobst S.W. Alvarado A.B. Hoover A.J. Snader K.M. J. Nat. Prod. 1992; 55: 432-440Crossref PubMed Scopus (141) Google Scholar), production in tissue culture systems (11Gibson D.M. Ketchum R.E.B. Vance N.C. Christen A.A. Plant Cell Rep. 1993; 12: 479-482Crossref PubMed Scopus (97) Google Scholar, 12Fett-Neto A.G. DiCosmo F. Reynolds W.F. Sakata K. Bio/Technology. 1992; 10: 1572-1575Crossref PubMed Scopus (137) Google Scholar), and semisynthesis of the drug and its analogs from baccatin III 2 and related late stage taxane diterpenoid (taxoid) metabolites that are more readily available (13Guénard D. Guéritte-Voegelein F. Potier P. Acc. Chem. Res. 1993; 26: 160-167Crossref Scopus (477) Google Scholar, 14Georg G.I. Ali S.M. Zygmut J. Jayasinghe L.R. Exp. Opin. Ther. Patents. 1994; 4: 109-120Crossref Scopus (55) Google Scholar, 15Nicolaou K.C. Dai W.-M. Guy R.K. Angew. Chem. Int. Ed. Engl. 1994; 33: 15-44Crossref Scopus (685) Google Scholar, 16Kingston D.G.I. George G.I. Chen T.T. Ojima I. Vyas D.M. Taxane Anticancer Agents: Basic Science and Current Status. American Chemical Society, Washington, D. C.1995: 203-216Google Scholar). Total synthesis of taxol, at present, is not commercially viable (17Borman S. Chem. Eng. News. 1994; 72: 32-34Crossref Scopus (23) Google Scholar), and efficient and economical supply of the drug must rely on biological production systems for the foreseeable future (18Cragg G.M. Schepartz S.A. Suffness M. Grever M.R. J. Nat. Prod. 1993; 56: 1657-1668Crossref PubMed Scopus (265) Google Scholar). Therefore, it is essential to understand the origin of taxol and related taxoids in yew species from the universal precursor of plant diterpenoids, geranylgeranyl diphosphate 3 (19West C.A. Porter J.W. Spurgeon S.L. Biosynthesis of Isoprenoid Compounds. Vol. 1. John Wiley & Sons, New York1981: 375-411Google Scholar). To this end, we have undertaken a systematic study of taxol biosynthesis at the enzyme level, with the goal of targeting the slow steps of the pathway for ultimate manipulation. The biosynthesis of taxol 1 (Scheme 1) is presumed to involve cyclization of geranylgeranyl diphosphate 3, via (1 S)-verticillene 4, to taxa-4(20),11(12)-diene 5 (20Harrison J.W. Scrowston R.M. Lythgoe B. J. Chem. Soc. C. 1966; : 1933-1945Crossref Scopus (60) Google Scholar, 21Begley M.J. Jackson C.B. Pattenden G. Tetrahedron. 1990; 46: 4907-4924Crossref Scopus (42) Google Scholar, 22Guéritte-Voegelein F. Guénard D. Potier P. J. Nat. Prod. 1987; 50: 9-18Crossref PubMed Scopus (108) Google Scholar) since taxoids bearing the 4(20) - and 11(12) -pair of double bonds are very common (23Kingston D.G.I. Molinero A.A. Rimoldi J.M. Prog. Chem. Org. Nat. Prod. 1993; 61: 1-206Google Scholar). Such a cyclization to establish the taxane skeleton could then be followed by oxidative elaboration of this diterpene olefin intermediate (22Guéritte-Voegelein F. Guénard D. Potier P. J. Nat. Prod. 1987; 50: 9-18Crossref PubMed Scopus (108) Google Scholar, 24Floss H.G. Mocek U. Suffness M. Taxol: Science and Applications. CRC Press, Boca Raton, FL1995Google Scholar). Previous investigations on taxol biosynthesis have involved feeding early precursors, such as labeled acetate, to various yew tissue preparations (25Strobel G.A. Stierle A. van Kuijk F.J.G.M. Plant Sci. 1992; 84: 65-74Crossref Scopus (55) Google Scholar, 26Zamir L.O. Nedea M.E. Garneau F.X. Tetrahedron Letts. 1992; 33: 5235-5236Crossref Scopus (52) Google Scholar), or have focused on late stage modifications to the taxane nucleus, such as the origin of appended acyl moieties (27Fleming P.E. Mocek U. Floss H.G. J. Am. Chem. Soc. 1993; 115: 805-807Crossref Scopus (61) Google Scholar, 28Fleming P.E. Knaggs A.R. He X.-G. Mocek U. Floss H.G. J. Am. Chem. Soc. 1994; 116: 4137-4138Crossref Scopus (39) Google Scholar). No experimental work on the enzymology of taxol biosynthesis has been reported, and the first dedicated step of the pathway and the identity of the presumed olefinic precursor of taxol have remained uncertain. In this communication, we describe the enzymatic cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12) -diene as the first committed reaction, and a very slow step, of taxol biosynthesis. Two-year-old T. brevifolia saplings in active growth were obtained from the Weyerhaeuser Research Center, Centralia, WA. [1-3H]Geranylgeranyl diphosphate (118 Ci/mol) was prepared as described previously (29LaFever R.E. Stofer Vogel B. Croteau R. Arch. Biochem. Biophys. 1994; 313: 139-149Crossref PubMed Scopus (70) Google Scholar). Bark extracts and taxoid standards (taxol, cephalomannine, and baccatin III, and their 10-deacetyl derivatives) were obtained from Hauser Chemical Research Inc., Boulder, CO. (+)-Taxusin was isolated from the wood of T. brevifolia (30Della Casa De Marcano D.P. Halsall G. J. Chem. Soc. Chem. Commun. 1969; : 1282-1283Crossref Google Scholar). Other diterpenoid standards were from our own collection. Methods previously developed for the isolation and assay of terpenoid cyclases from conifer stem tissue were employed (29LaFever R.E. Stofer Vogel B. Croteau R. Arch. Biochem. Biophys. 1994; 313: 139-149Crossref PubMed Scopus (70) Google Scholar, 31Lewinsohn E. Gijzen M. Savage T.J. Croteau R. Plant Physiol. 1991; 96: 38-43Crossref PubMed Scopus (111) Google Scholar). Yew sapling stem sections were frozen in liquid N2and pulverized in a hammer mill to separate the bark and adhering tissue from the woody core. The resulting “bark” powder was extracted with a buffer containing vinylpyrrolidone polymers and polystyrene resin to adsorb phenolics, and the 27,000 × g supernatant was partially purified by ion-exchange chromatography on DEAE-cellulose (31Lewinsohn E. Gijzen M. Savage T.J. Croteau R. Plant Physiol. 1991; 96: 38-43Crossref PubMed Scopus (111) Google Scholar). The preparation was then dialyzed to assay conditions (30 m M HEPES (pH 8.0), 5 m M dithiothreitol, 10% glycerol) and adjusted to ∼50 μg of protein/ml before incubation in the presence of 10 μM geranylgeranyl diphosphate and 1 m M MgCl2(29LaFever R.E. Stofer Vogel B. Croteau R. Arch. Biochem. Biophys. 1994; 313: 139-149Crossref PubMed Scopus (70) Google Scholar). Following incubation (1-3 h), the pentane-soluble products of the reaction mixture were extracted and the extract passed through a short column of silica gel to provide the hydrocarbon fraction. Quantification was by aliquot counting, and the products were analyzed by radio-GLC. 2The abbreviations used are:GLCgas-liquid chromatographyCCcolumn chromatographyDEPTdistortionless enhancement by polarization transferDQF-COSYdouble quantum filter homonuclear correlated spectroscopyHETCORheteronuclear correlationHMBCheteronuclear multiple bond correlationHMQCheteronuclear multiple quantum correlationHPLChigh pressure liquid chromatographyMSmass spectrometryTOCSYtotal correlation spectroscopy. To obtain sufficient amounts of the biosynthetic olefin for use in in vivo studies and for GLC-MS analysis, the enzyme preparation was scaled up by a factor of 10. gas-liquid chromatography column chromatography distortionless enhancement by polarization transfer double quantum filter homonuclear correlated spectroscopy heteronuclear correlation heteronuclear multiple bond correlation heteronuclear multiple quantum correlation high pressure liquid chromatography mass spectrometry total correlation spectroscopy. The biosynthetic olefin (5.36 μCi of 3H) that had been purified by CC (silica gel; pentane) and argentation TLC (silica gel; 10% AgNO3:pentane) was suspended by sonication in a buffer (1.5 ml) consisting of 5 m M KH2PO4(pH 5.5), 10 m M sucrose, 2 m M KCl, 2 m M phenylalanine, 1 m M dithiothreitol, 1 m M MgCl2, 1 m M sodium acetate, 1 m M sodium benzoate, 0.2 m M EDTA, 0.1 m M Na2H2P2O7, and 0.05% Tween 20. Following filter sterilization, the suspension was split and vacuum-infiltrated (six times) into two 1.75-g batches of yew stem discs (1-2 mm thick) prepared from sapling stem sections that had been surface-sterilized in 70% ethanol (1 s), followed by 3% sodium hypochlorite, 0.5% Tween 20 (1 min) and washing, and then sectioned using sterile technique. The tissue was incubated for 8 days at room temperature, in air, in the light with slow shaking in a glass vial and then thoroughly extracted with hexane (2 × 10 ml), which was passed through a silica gel column that was rinsed with diethyl ether:pentane (1:1) to remove residual substrate and labeled metabolites less polar than (+)-taxusin. The column was then rinsed with 10 ml of chloroform:acetonitrile:methanol (6:3:1), and this material was used to extract the tissue, as before. The process was repeated with three additional portions of chloroform:acetonitrile:methanol, at which point a methanol extraction afforded no additional extracted radioactivity. This taxoid fraction was decolorized, without loss of tritium, by elution through Davisil (Alltech C18on silica) with a gradient from 55% water, 25% methanol, 20% acetonitrile to 100% acetonitrile. Approximately 90% of the applied tracer was recovered in organic solvent-soluble products. Aliquots of the taxoid fraction were analyzed by HPLC on a 250 mm × 4.6-mm Econosil C8 column (5 μm, Alltech) by gradient elution (30 ml) from solvent A (55% water, 25% methanol, 20% acetonite) to 75% solvent A plus 25% solvent B (acetonitrile), while collecting 1-ml fractions for liquid scintillation counting. Retention times of the standards were as follows: 10-deacetylbaccatin III (3.08 min), baccatin III (5.24 min), 10-deacetylcephalomannine (20.48 min), 10-deacetyltaxol (22.99 min), cephalomannine (26.21 min), and taxol (27.82 min). The taxoid fraction was subsequently diluted with 2 mg each of 10-deacetylbaccatin III, cephalomannine, and taxol and subjected to preparative TLC on silica gel G with chloroform:methanol (20:1). The products (10-deacetylbaccatin III, RF 0.17; cephalomannine plus taxol, RF 0.47-0.51) were eluted with methanol, diluted with an additional 200 mg of authentic carrier, and crystallized from aqueous methanol to constant specific activity and m.p. Because cephalomannine and taxol were not separable, the eluted mixture was split in half; one-half was diluted with cephalomannine, the other with taxol. Repeated crystallization gave 10-deacetylbaccatin III, 150 nCi/mmol, m.p. 243−245°C decomposes cephalomannine, 14.0 nCi/mmol, m.p. 183−186°C decomposes taxol, 31.7 nCi/mmol, m.p. 215−218°C decomposes. All m.p. values are in agreement with the literature (23Kingston D.G.I. Molinero A.A. Rimoldi J.M. Prog. Chem. Org. Nat. Prod. 1993; 61: 1-206Google Scholar). An extract from 750 kg of T. brevifolia bark powder, that had been enriched in non-polar materials by partitioning from methanol into heptane, was batch processed through silica gel with hexane to afford a hydrocarbon fraction (3.1 g). This material was diluted with 0.5 μCi of the enzymatically prepared olefin and chromatographed repeatedly on a silica gel column with hexane, while monitoring fractions by liquid scintillation counting and GLC-MS, to yield a fraction (230 mg; >90% recovery of label) eluting between sandaracopimaradiene and abietatriene. This material, a very complex mixture of sesquiterpene and diterpene hydrocarbons, was next purified by argentation column chromatography (10% AgNO3-silica gel with a 0 to 10% gradient of Et2O in pentane) to afford 72 mg of an oil. Another passage through silica gel (with pentane) followed by reversed-phase column chromatography on Davisil (Alltech C18on silica, 2.5-20% CCl4in acetonitrile) and argentation TLC (10% AgNO3-silica gel with 5% ether in pentane) yielded about 1 mg of 85% taxadiene (RF 0.8-0.9, with 2% mixed sesquiterpene olefins and 11% abietatriene). NMR spectra were recorded at 499.8 MHz (1H, DQF-COSY, TOCSY) and at 125.7 MHz (13C(1H), DEPT, HETCOR) on a Varian VXR-500S instrument, and at 300.1 MHz (HMQC, HMBC) using a Bruker AMX-300 instrument. All experiments were run at ambient temperature (21°C) with a 5 μM solution in CDCl3. Chemical shifts are reported in δ (ppm) using tetramethylsilane as an internal standard. The DQF-COSY (32Piatini U. Soerenson O.W. Ernst R.R. J. Am. Chem. Soc. 1982; 104: 6800-6801Crossref Scopus (1888) Google Scholar) and TOCSY (33Griesinger C. Otting G. Wuetrich K. Ernst R.R. J. Am. Chem. Soc. 1988; 110: 7870-7872Crossref Scopus (1199) Google Scholar) experiments (mixing time 60 ms) were run in the phase sensitive mode. The 512 t1increments of 96 scans each were sampled in 2000 data points for each of the 512 t1increments. Zero-filling the F1 domain to 2000 and a Gaussian weighting function were applied in both F1and F2domains prior to double Fourier transformation. The HETCOR (34Bax A. Morris G.A. J. Magn. Reson. 1981; 42: 501-505Google Scholar) spectrum was obtained using 2000 data points in the F2domain and 256 t1increments (800 scans each), which were zero-filled to 1000 in the F1domain. A sine-bell squared weighting function phase shifted by π/2 was applied in both domains prior to double Fourier transformation. The HMQC spectrum was measured employing the pulse sequence of Bax et al. (35Bax A. Griffey R.H. Hawkins B.L. J. Magn. Reson. 1983; 55: 301-315Google Scholar). Delay Δ was set to 3.571 ms corresponding to the average one bond carbon-proton coupling constant, 145 Hz. In this experiment, 256 t1increments were sampled in 2000 data points using 464 scans for each of the t1increments. Data in the F1domain were zero-filled to 1000, and the sine-bell squared weighting function phase shifted by π/2 was applied in both F1and F2domains prior to double Fourier transformation. Analogous parameters were adopted for the HMBC spectrum that was obtained using the pulse sequence of Bax and Summers (36Bax A. Summers M.F. J. Am. Chem. Soc. 1986; 108: 2093-2094Crossref Scopus (3274) Google Scholar) and involved low-pass J-filtering to suppress correlations due to one-bond couplings. Delay Δ2was set to 62.5 ms corresponding to the average long range (through two or three bonds) carbon-proton coupling constant, 8 Hz. Radio-GLC was performed on a Gow-Mac 550P gas chromatograph coupled to a Packard 894 gas proportional counter (37Satterwhite D.M. Croteau R. J. Chromatogr. 1988; 452: 61-73Crossref PubMed Scopus (28) Google Scholar) using a 30 m × 0.53-mm diameter fused silica column coated with a 1.2-μm film of Superox FA (Alltech); 8 p.s.i. He, isothermal at 200°C with injector and detector at 220°C. GLC-MS analysis was performed on a Hewlett-Packard 5840A/5985B system using a 30 m × 0.25-mm diameter fused silica column with 0.25-μm film of Superox FA (Alltech) operated at 10 p.s.i. H2and programmed from 100°C (5-min hold) at 10°C/min to 220°C. Electron impact spectra were recorded at 70 eV. Pacific yew saplings were chosen as an experimental system because the taxoid content of immature tissue is relatively high (9Vidensek N. Lim P. Campbell A. Carlson C. J. Nat. Prod. 1990; 53: 1609-1610Crossref PubMed Scopus (177) Google Scholar), sapling stems contain a high proportion of phloem parenchyma cells in which taxol is thought to be produced (25Strobel G.A. Stierle A. van Kuijk F.J.G.M. Plant Sci. 1992; 84: 65-74Crossref Scopus (55) Google Scholar, 38Ellis, D., Zeldin, E., Russin, W., Brodhagen, M., Evert, R., McCown, B., (1993) Proceedings of the International Yew Research Conference, (Native Yew Conservation Council), Berkeley, CA, March 12-13, 1993.Google Scholar, 39Strobel G.A. Stierle A. Hess W.M. Plant Sci. 1993; 92: 1-12Crossref Scopus (28) Google Scholar), and saplings can be maintained in a greenhouse to minimize the effects of environmental variation (10Wheeler N.C. Jech K. Masters S. Brobst S.W. Alvarado A.B. Hoover A.J. Snader K.M. J. Nat. Prod. 1992; 55: 432-440Crossref PubMed Scopus (141) Google Scholar, 40Vance N.C. Kelsey R.G. Sabin T.E. Phytochemistry. 1994; 36: 1241-1244Crossref PubMed Scopus (47) Google Scholar). A soluble enzyme extract was prepared from T. brevifolia stem sections by methods previously developed for the isolation of other terpenoid cyclases from gymnosperm stem that minimize the deleterious effects of co-extracted phenolic materials (31Lewinsohn E. Gijzen M. Savage T.J. Croteau R. Plant Physiol. 1991; 96: 38-43Crossref PubMed Scopus (111) Google Scholar). Incubation of this preparation with 10 μM [1-3H]geranylgeranyl diphosphate, in the presence of 1 m M MgCl2, yielded radiolabeled, pentane-soluble products that were purified by column chromatography on silica gel to afford the hydrocarbon fraction (1 nmol; 5% conversion in 1 h). The production of the hydrocarbon material from geranylgeranyl diphosphate by the enzyme preparation was absolutely dependent on the presence of the divalent metal ion, as expected for a terpenoid cyclase (41Alonso W.R. Croteau R. Methods Plant Biochem. 1993; 9: 239-260Google Scholar), and negligible activity was observed in thermally inactivated control incubations. Radio-GLC analysis of the hydrocarbon fraction revealed the presence of a single major component that eluted between the tricyclic diterpene olefin standards sandaracopimaradiene and abietatriene (Fig. 1 a). To determine if this presumptive diterpene olefin could serve as a precursor of taxol, the purified biosynthetic product (5.36 μCi) was suspended in buffer and vacuum-infiltrated into two batches of T. brevifolia stem discs. Following incubation for 8 days, the labeled products were extracted and separated into a taxoid fraction containing metabolites more polar than (+)-taxusin (the tetraacetate of taxa-4(20), 11(12) -dien-5,9,10,13-tetraol). This fraction represented about 30% incorporation of radioactivity from the olefin and, when separated by reversed-phase radio-HPLC, revealed the presence of at least a dozen labeled compounds of polarity between 10-deacetylbaccatin III (10-deacetyl 2, Rt 3.08 min) and taxol 1 (Rt 27.82 min), including the common bark taxoids baccatin III 2, cephalomannine 6, and taxol, and their 10-deacetyl derivatives (40Vance N.C. Kelsey R.G. Sabin T.E. Phytochemistry. 1994; 36: 1241-1244Crossref PubMed Scopus (47) Google Scholar) (Scheme 1). Negligible incorporation of olefin label into this taxoid fraction was observed in control experiments with thermally inactivated tissue. To confirm the identity of the labeled taxoids, 10-deacetylbaccatin III, cephalomannine, and taxol (for which substantial amounts of authentic standards were available) were isolated by TLC, diluted with the corresponding unlabeled carrier, and crystallized to constant specific activity and melting point, thereby verifying the incorporation of the tritium-labeled olefin into 10-deacetylbaccatin III (4.1%), cephalomannine (0.5%), and taxol (1.1%). (The incorporation percentages should be regarded as minimum levels, as some tritium label (depending on the kinetic isotope effect) will be lost on oxidation at C-2 (Scheme 1) of the taxane skeleton of these advanced metabolites.) It is not uncommon for the level of 10-deacetylbaccatin III to exceed the levels of taxol and cephalomannine in T. brevifolia stem tissue (40Vance N.C. Kelsey R.G. Sabin T.E. Phytochemistry. 1994; 36: 1241-1244Crossref PubMed Scopus (47) Google Scholar). These results indicated that the diterpene olefin product of the cell-free system did serve as a precursor of taxol and other very closely related taxoids of yew stem, thus suggesting that the cyclization product of geranylgeranyl diphosphate was a taxadiene. To identify the biosynthetic diterpene olefin product, it was necessary to acquire sufficient material for spectroscopic analysis. To this end, the hydrocarbon fraction from an extract of 750 kg of dried T. brevifolia bark powder was diluted with 0.5 μCi of the enzymatically derived, tritium-labeled olefin, and the corresponding product (∼1 mg) was isolated by radiochemically guided chromatographic fractionation. The mass spectrum of the product gave principal ions at m/z 122 (100%), 107 (26%), 121 (25%), 123 (20%), and 105 (10%), and a parent ion at m/z 272 (1.5%) consistent with a cyclic diterpene olefin of molecular formula C20H32. Radio-GLC analysis of the isolated product demonstrated coincidental elution of radioactivity and the principal mass peak, as expected. Based on recovery, it was estimated that the level of this olefin in bark tissue was in the 5-10 μg/kg range. The structure of the diterpene olefin was determined to be taxa-4(5), 11(12) -diene 7 by analysis of one- and two-dimensional 1H and 13C NMR spectra. Proton connectivities were determined by DQF-COSY and TOCSY experiments, and the signals of all carbons with directly attached protons were assigned using HETCOR and HMQC spectra. Finally, the HMBC spectrum was used to assign quaternary carbons and to check the correctness of the connectivities established by the interpretation of the other spectra. The 13C DEPT experiment revealed a total of 20 carbon resonances (Table I), of which five corresponded to methyl carbons, seven to methylene carbons, three to methine carbons, and five to non-proton bearing carbons. One of the methine resonances and three non-proton bearing carbon resonances occurred in the downfield region: 120.0-140.0 ppm. All remaining carbon resonances were in the high field region: 20.0-45.0 ppm.Table I:1H and 13C NMR chemical shifts δ (ppm) for taxa-4(5),11(12)-diene measured in CDCl3solution at 293 K with tetramethylsilane as internal standard The 1H NMR spectrum (Table I) showed a one-proton multiplet (∑J = 11.1 Hz) at δ 5.28. All other signals were in the region from 0.65 to 2.63 ppm. In the TOCSY spectrum, the olefinic CH-5 (δ 5.28) proton multiplet exhibited cross-peaks to four different sets of protons corresponding to the methylene CH2-6 (δ 1.88, 2.01) and CH2-7 (δ 1.18, 1.67) protons, the methine CH-3 (δ 2.51) proton, and the methyl CH3-20 (δ 1.68) protons. By analyzing the HETCOR spectrum, the carbon signals at δ 24.0, 38.4, 39.7, and 121.1 were assigned to C-20, C-7, C-3, and C-5, respectively; they correlated with the corresponding proton signals (see above). The resonances of the C-3 and C-7 carbons showed a cross-peak with the proton singlet at δ 0.82 in the HMBC spectrum. Therefore, the proton signal was assigned to the methyl CH3-19 protons. Additionally, this proton resonance (δ 0.82) correlated with another two carbon signals at δ37.2 and 41.4, corresponding to the quaternary C-8 and methylene CH2-9 carbons, respectively. That the signal at δ37.2 belonged to a quaternary carbon, and the signal at δ 41.4 to a methylene carbon, was further supported by the DEPT data. Analysis of the HMBC spectrum also allowed the carbon signal at δ 138.5 to be assigned to the olefinic C-4 carbon, as it exhibited a cross-peak with the methyl CH3-20 proton signal (δ 1.68) due to a two-bond linkage. The methylene CH2-9 proton signals at δ 1.42 and 1.82 were identified based upon their correlation with the C-9 carbon signal at δ 41.4 in the HETCOR spectrum. Following the interpretation of the TOCSY and DQF-COSY spectra, the methylene CH2-2 and CH2-10 proton signals were assigned. The CH-3 proton signal (δ 2.51) displayed two cross-peaks at δ 1.57 and 1.66, corresponding to the methylene CH2-2 protons. One of the CH2-9 proton signals (δ 1.42) correlated with the signal at δ2.15, and the other signal (δ 1.82) showed a cross-peak at δ2.61. Therefore, both signals at δ2.15 and 2.61 were assigned to the methylene CH2-10 protons. Moreover, they exhibited a mutual cross-peak due to geminal coupling. In addition, both correlated with the carbon signal at δ24.5 in the HETCOR spectrum. Consequently, the latter was assigned to the C-10 carbon. The HETCOR analysis also allowed the carbon signal at δ28.4 to be assigned to the C-2 carbon. The gem-dimethyl groups (CH3-16, CH3-17) attached to the quaternary C-15 carbon of the taxadiene A-ring were easily identified in the HMBC spectrum. Both methyl proton resonances at δ 1.01 (CH3-16) and 1.33 (CH3-17) displayed cross-peaks with the same carbon signals at δ 44.2, 137.7, and 39.0, corresponding to the carbons C-1, C-11, and C-15, respectively. Furthermore, the CH3-16 proton resonance correlated with the carbon CH3-17 signal at δ26.3 (assigned using HETCOR analysis) and, due to the same three-bond coupling, the proton signal of the methyl CH3-17 correlated with the carbon CH3-16 resonance at δ30.7 (assignment based on HETCOR analysis). HMBC analysis also revealed the correlation of the carbon C-11 resonance with the proton singlet at δ 1.66. At the same time, this proton resonance exhibited cross-peaks with the carbon signals at δ 29.8 and 129.5. These facts allowed assignment of the proton resonance at δ 1.66 to the methyl CH3-18 protons; the carbon signals were assigned as the methylene CH2-13 carbon (δ 29.8) and the olefinic carbon C-12 (δ 129.5). Having established the carbon resonances of the methine CH-1 and methylene CH2-13, the HETCOR analysis permitted assignment of the proton signals of the groups at δ 1.74 (CH-1), and at δ 1.88 and 2.08 (CH2-13). Both the methylene CH2-13 proton resonances, as well as the methine CH-1 proton resonance, exhibited cross-peaks at δ 1.59 and 2.08 in the DQF-COSY spectrum. At the same time, these two proton signals (δ 1.59, 2.08) showed a mutual cross-peak. Therefore, they were assigned to the methylene CH2-14 protons. Only one of the CH2-14 proton resonances displayed a cross-peak with the carbon signal at δ 22.6 in the HETCOR spectrum. However, this signal (δ 22.6) corresponded to a methylene carbon based upon the DEPT data and was assigned to C-14. The remaining unassigned methylene carbon resonance at δ 23.2 was then assigned to the methylene CH2-6, even though no cross-peak between protons and carbon of the CH2-6 group could be identified in the HETCOR and HMQC spectra. The assignment of each carbon and hydrogen of the C20H32 olefin left little doubt that the product was taxa-4(5), 11(20) -diene 7. To confirm that the biosynthetic product generated in yew stem extracts and the olefin isolated from bark were the same, large scale enzyme incubations with geranylgeranyl diphosphate were carried out and the olefin fraction was analyzed by combined GLC-MS. The retention time and mass spectrum of the enzymatic product (>99% pure) were identical to those of taxa-4(5), 11(12) -diene isolated from the bark extract (Fig. 1, b and c). (1 S)-Verticillene 4, a possible enzyme-bound intermediate in the cyclization, was not detectable as a product of the cell-free extract. The cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12) -diene, as the first dedicated step in the biosynthesis of taxol and related metabolites, is consistent with earlier suggestions that the pathway involves preliminary formation of a parent taxane olefin followed by oxidative modification (20Harrison J.W. Scrowston R.M. Lythgoe B. J. Chem. Soc. C. 1966; : 1933-1945Crossref Scopus (60) Google Scholar, 21Begley M.J. Jackson C.B. Pattenden G. Tetrahedron. 1990; 46: 4907-4924Crossref Scopus (42) Google Scholar, 22Guéritte-Voegelein F. Guénard D. Potier P. J. Nat. Prod. 1987; 50: 9-18Crossref PubMed Scopus (108) Google Scholar). However, the identification of taxa-4(5), 11(12) -diene 7 as the olefinic intermediate, rather than taxa-4(20), 11(12) -diene 5 as originally proposed on the basis of metabolite co-occurrence (22Guéritte-Voegelein F. Guénard D. Potier P. J. Nat. Prod. 1987; 50: 9-18Crossref PubMed Scopus (108) Google Scholar), was unexpected. Consequently, the previously proposed cyclization to 5 (20Harrison J.W. Scrowston R.M. Lythgoe B. J. Chem. Soc. C. 1966; : 1933-1945Crossref Scopus (60) Google Scholar, 21Begley M.J. Jackson C.B. Pattenden G. Tetrahedron. 1990; 46: 4907-4924Crossref Scopus (42) Google Scholar) (Scheme 1) can be reformulated as involving ionization of the geranylgeranyl diphosphate ester with closure of the first ring and deprotonation to afford (1 S)-verticillene. Protonation at C-7 can then initiate transannular cyclization to generate the taxenyl cation, which upon deprotonation at C-5 yields the endocyclic double bond of the taxadiene product. The next step of the pathway to taxol is presumed to involve oxygenation of taxadiene (22Guéritte-Voegelein F. Guénard D. Potier P. J. Nat. Prod. 1987; 50: 9-18Crossref PubMed Scopus (108) Google Scholar, 24Floss H.G. Mocek U. Suffness M. Taxol: Science and Applications. CRC Press, Boca Raton, FL1995Google Scholar). The observations that no oxygenated taxoids bearing the 4(5) -double bond have yet been reported, whereas taxoids with the exo-methylene at the 4(20) -position and that also bear an oxygen function at C-5 are exceedingly common (23Kingston D.G.I. Molinero A.A. Rimoldi J.M. Prog. Chem. Org. Nat. Prod. 1993; 61: 1-206Google Scholar), therefore suggest that hydroxylation at C-5 of taxa-4(5),11(12) -diene, with migration of the double bond, must occur as an early, if not the first, oxygenation step of the pathway (Scheme 2). The transformation of taxa-4(5), 11(12) -diene 7 to taxa-4(20), 11(12) -diene-5-ol 8 would also set the stage for the later elaboration of the unusual oxetane moiety 11, which is considered (22Guéritte-Voegelein F. Guénard D. Potier P. J. Nat. Prod. 1987; 50: 9-18Crossref PubMed Scopus (108) Google Scholar, 24Floss H.G. Mocek U. Suffness M. Taxol: Science and Applications. CRC Press, Boca Raton, FL1995Google Scholar) to arise by conversion of the 5-hydroxy-4(20) -methylene 9 to the corresponding epoxide function 10 followed by ring expansion (Scheme 2). Although the enzyme that catalyzes the transformation of geranylgeranyl diphosphate to taxadiene has not yet been characterized in any detail, the low cyclization activity in T. brevifolia stem extracts relative to that of other diterpene cyclases of gymnosperm stem tissue (29LaFever R.E. Stofer Vogel B. Croteau R. Arch. Biochem. Biophys. 1994; 313: 139-149Crossref PubMed Scopus (70) Google Scholar), coupled to the very low levels of this key olefin intermediate in the bark, suggest that the cyclization step to generate the taxane skeleton is very slow (relative to subsequent oxygenations) and, thus, is an important target for manipulation. With the initial step of taxol biosynthesis in Pacific yew now defined, it would be of interest to determine if the taxol-producing fungus Taxomyces andreanae (42Stierle A. Strobel G. Stierle D. Science. 1993; 260: 214-216Crossref PubMed Scopus (1431) Google Scholar) employs a similar cyclization reaction. We thank David Bailey (Hauser Chemical Research Inc., Boulder, CO) for the bark extract and authentic taxoid standards, Nicholas Wheeler (Weyerhaeuser Research Center, Centralia, WA) for the T. brevifolia saplings, and Gerald Pattenden (University of Nottingham, Nottingham, United Kingdom) for the mass spectrum of verticillene." @default.
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• W2015690692 title "Cyclization of Geranylgeranyl Diphosphate to Taxa-4(5),11(12)-diene Is the Committed Step of Taxol Biosynthesis in Pacific Yew" @default.
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