Editing Paclitaxel (section)

==Production==
[[Image:PacificYew 8566.jpg|thumb|Undisturbed Pacific yew bark contains paclitaxel and related chemicals.]]

=== Bark processing ===
[[Image:Yew bark Taxol PD.jpg|thumbnail|The bark is peeled and processed to provide paclitaxel.]]

From 1967 to 1993, almost all paclitaxel produced was derived from bark of the Pacific yew, ''[[Taxus brevifolia]]'', the harvesting of which kills the tree in the process.<ref name="Gersmann 2011">{{cite news| vauthors = Gersmann H, Aldred J |title=Medicinal tree used in chemotherapy drug faces extinction|url=https://www.theguardian.com/environment/2011/nov/10/iucn-red-list-tree-chemotherapy|access-date=15 February 2017|work=The Guardian|date=10 November 2011|url-status=live|archive-url=https://web.archive.org/web/20170216143120/https://www.theguardian.com/environment/2011/nov/10/iucn-red-list-tree-chemotherapy|archive-date=16 February 2017}}</ref> The processes used were descendants of the original isolation method of [[Monroe Wall]] and [[Mansukh Wani]]; by 1987, the U.S. [[National Cancer Institute]] (NCI) had contracted Hauser Chemical Research of [[Boulder, Colorado]], to handle bark on the scale needed for [[Phases of clinical research|phase]] II and III trials.{{citation needed|date=June 2014}} While both the size of the wild population of the Pacific yew and the magnitude of the eventual demand for paclitaxel were uncertain, it was clear that an alternative, sustainable source of the [[natural product]] would be needed. Initial attempts to broaden its sourcing used needles from the tree, or material from other related ''[[Taxus]]'' species, including cultivated ones,{{citation needed|date=June 2014}} but these attempts were challenged by the relatively low and often highly variable yields obtained. Early in the 1990s, coincident with increased sensitivity to the ecology of the forests of the [[Pacific Northwest]], paclitaxel was extracted on a clinically useful scale from these sources.{{sfn|Goodman|Walsh|2001|pp=172–5}}

=== Semisynthesis ===
Concurrently, synthetic chemists in the U.S. and France had been interested in paclitaxel, beginning in the late 1970s.{{citation needed|date=June 2014}} As noted, by 1992 extensive efforts were underway to accomplish the [[total synthesis]] of paclitaxel, efforts motivated by the desire to generate new chemical understanding rather than to achieve practical commercial production. In contrast, the French group of [[Pierre Potier]] at the [[Centre national de la recherche scientifique]] (CNRS) addressed the matter of overall process yield, showing that it was feasible to isolate relatively large quantities of the compound [[10-deacetylbaccatin]] from the European yew, ''[[Taxus baccata]]'', which grew on the CNRS campus and whose needles were available in large quantity.{{citation needed|date=June 2014}}  By virtue of its structure, 10-deacetylbaccatin was seen as a viable starting material for a short [[semisynthesis]] to produce paclitaxel. By 1988, Poitier and collaborators had published a semisynthetic route from needles of the European yew to paclitaxel.{{sfn|Goodman|Walsh|2001|pp=100–1}}

The view of the NCI, however, was that even this route was not practical.{{citation needed|date=June 2014}} The group of [[Robert A. Holton]] had also pursued a practical semisynthetic production route; by late 1989, Holton's group had developed a semisynthetic route to paclitaxel with twice the yield of the Potier process.<ref>{{cite book | vauthors = Holton RA, Biediger RJ, Boatman PD | chapter = Semisynthesis of taxol and taxotere | veditors = Suffness M | title = Taxol: Science and Applications | date = 1999 | pages = 97–121 | location =  Boca Raton | publisher = CRC press | isbn = 978-0-13-873736-8 }}</ref>  The main innovation was "Ojima−Holton coupling", a ring-opening method independently discovered by Holton and Ojima.<ref name=Ojima2018>{{cite journal | vauthors = Ojima I, Wang X, Jing Y, Wang C | title = Quest for Efficacious Next-Generation Taxoid Anticancer Agents and Their Tumor-Targeted Delivery | journal = Journal of Natural Products | volume = 81 | issue = 3 | pages = 703–721 | date = March 2018 | pmid = 29468872 | doi = 10.1021/acs.jnatprod.7b01012 | pmc = 5869464 | doi-access = free | bibcode = 2018JNAtP..81..703O }}</ref> [[Florida State University]], where Holton worked, signed a deal with [[Bristol-Myers Squibb]] (BMS) to license their semisynthesis and future patents.{{citation needed|date=June 2014}} In 1992, Holton patented an improved process with an 80% yield, and BMS took the process in-house and started to manufacture paclitaxel in Ireland from 10-deacetylbaccatin isolated from the needles of the European yew.{{citation needed|date=June 2014}} In early 1993, BMS announced that it would cease reliance on Pacific yew bark by the end of 1995, effectively terminating ecological controversy over its use.{{citation needed|date=June 2014}} This announcement also made good their commitment to develop an alternative supply route, made to the NCI in their [[cooperative research and development agreement]] (CRADA) application of 1989.{{cn|date=September 2024}}

As of 2013, BMS was using the semisynthetic method utilizing needles from the European yew to produce paclitaxel.<ref>{{cite web|url=http://wgcriticalcare.com/injectable-pharmaceuticals/wp-content/uploads/2014/01/WGCC-Paclitaxel-PI-June-2013.pdf|title=Paclitaxel Injection, USP|website=Injectable Pharmaceuticals|language=en-US|access-date=22 April 2016|url-status=dead|archive-url=https://web.archive.org/web/20160918114404/http://wgcriticalcare.com/injectable-pharmaceuticals/wp-content/uploads/2014/01/WGCC-Paclitaxel-PI-June-2013.pdf|archive-date=18 September 2016}}</ref> Another company which worked with BMS until 2012,<ref>{{cite web|url=http://www.phytonbiotech.com/history/|title=History|access-date=22 April 2016|url-status=live|archive-url=https://web.archive.org/web/20160524143938/http://www.phytonbiotech.com/history/|archive-date=24 May 2016}}</ref> Phyton Biotech, Inc., uses plant cell fermentation (PCF) technology.<ref>{{cite web|url=http://www.phytonbiotech.com/paclitaxel/|title=Phyton BioTech Paclitaxel|access-date=22 April 2016|url-status=live|archive-url=https://web.archive.org/web/20160807223136/http://www.phytonbiotech.com/paclitaxel/|archive-date=7 August 2016}}</ref> By cultivating a specific ''Taxus'' [[cell line]] in fermentation tanks, they no longer need ongoing sourcing of material from actual yew tree plantations.<ref>{{cite book|chapter=Suspension Culture of Plant Cells under Heterotrophic Conditions | vauthors = Imseng N, Schillberg S, Schürch C, Schmid D, Schütte K, Gorr G, Eibl D, Eibl R | date = 2014 | veditors = Meyer HP, Schmidhalter D |title=Industrial Scale Suspension Culture of Living Cells|publisher=Wiley-Blackwell |isbn=978-3-527-33547-3 |pages=224–257 }}</ref> Paclitaxel is then captured directly from the suspension broth by a resin allowing concentration to highly enriched powder containing about 40% paclitaxel. The compound is then purified by one [[chromatographic]] step followed by [[crystallization]].<ref>Gilbert Gorr and Roland Franke. Commercial Pharmaceutical Production of Complex APIs via Plant Cell Fermentation (PCF) Technology. Presentation at CPhI 2015, 13 Oct..</ref> Compared to the semisynthesis method, PCF eliminates the need for many hazardous chemicals and saves a considerable amount of energy.<ref name="2004_EPA_award">{{cite web|url=https://www.epa.gov/greenchemistry/presidential-green-chemistry-challenge-2004-greener-synthetic-pathways-award|title=2004 Greener Synthetic Pathways Award: Bristol-Myers Squibb Company: Development of a Green Synthesis for Taxol Manufacture via Plant Cell Fermentation and Extraction|url-status=live|archive-url=https://web.archive.org/web/20061002105234/http://www.epa.gov/greenchemistry/pubs/pgcc/winners/gspa04.html|archive-date=2 October 2006}}</ref>

In 1993, paclitaxel was discovered as a natural product in ''Taxomyces andreanae'', a newly described [[endophytic]] [[fungus]] living in the yew tree.<ref>{{cite journal | vauthors = Stierle A, Strobel G, Stierle D | title = Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew | journal = Science | volume = 260 | issue = 5105 | pages = 214–216 | date = April 1993 | pmid = 8097061 | doi = 10.1126/science.8097061 | bibcode = 1993Sci...260..214S }}</ref> It has since been reported in a number of other endophytic fungi, including ''Nodulisporium sylviforme'',{{citation needed|date=November 2019}} ''Alternaria taxi'', ''Cladosporium cladosporioides'' MD2, ''[[Metarhizium anisopliae]]'', ''Aspergillus candidus'' MD3, ''Mucor rouxianus'', ''Chaetomella raphigera'', ''Phyllosticta tabernaemontanae'', ''[[Phomopsis]]'', ''Pestalotiopsis pauciseta'', ''[[Phyllosticta citricarpa]]'', ''[[Podocarpus]]'' sp., ''[[Fusarium solani]]'', ''Pestalotiopsis terminaliae'', ''Pestalotiopsis breviseta'', ''Botryodiplodia theobromae'', ''Gliocladium'' sp., ''Alternaria alternata'' var. ''monosporus'', ''[[Cladosporium cladosporioides]]'', ''Nigrospora'' sp. and ''[[Pestalotiopsis versicolor]]''. However, there has been contradictory evidence for its production by endophytes, with other studies finding independent production is unlikely.<ref>{{cite journal | vauthors = Staniek A, Woerdenbag HJ, Kayser O | title = Taxomyces andreanae: a presumed paclitaxel producer demystified? | journal = Planta Medica | volume = 75 | issue = 15 | pages = 1561–1566 | date = December 2009 | pmid = 19809969 | doi = 10.1055/s-0029-1186181 | bibcode = 2009PlMed..75.1561S | s2cid = 260283080 }}</ref><ref>{{cite journal|doi=10.1007/s13225-013-0228-7|title=Getting to the bottom of taxol biosynthesis by fungi|year=2013|vauthors = Heinig U, Scholz S, Jennewein S|journal=Fungal Diversity|volume=60|pages=161–170|s2cid=18642421|url=https://link.springer.com/content/pdf/10.1007%2Fs13225-013-0228-7.pdf |archive-url=https://ghostarchive.org/archive/20221009/https://link.springer.com/content/pdf/10.1007%2Fs13225-013-0228-7.pdf |archive-date=9 October 2022 |url-status=live|doi-access=free }}</ref>

===Biosynthesis===
[[File:Biosynthesis of Taxol.png|thumb|class=skin-invert-image|Biosynthesis of Taxol]]
Taxol is a tetracyclic [[diterpene]], and the biosynthesis of diterpenes starts with an FPP molecule being elongated by the addition of an IPP molecule in order to form geranylgeranyl diphosphate ([[GGPP]]).<ref name=":2">{{cite book| vauthors = Dewick PM |title=Medicinal Natural Products|date=6 February 2009|publisher=John Wiley & Sons, Ltd|isbn=978-0-470-74276-1|location=Chichester, UK |language=en |doi=10.1002/9780470742761 }}</ref> The biosynthesis of Taxol contains nineteen steps.<ref>{{cite journal | vauthors = Howat S, Park B, Oh IS, Jin YW, Lee EK, Loake GJ | title = Paclitaxel: biosynthesis, production and future prospects | journal = New Biotechnology | volume = 31 | issue = 3 | pages = 242–245 | date = May 2014 | pmid = 24614567 | doi = 10.1016/j.nbt.2014.02.010 }}</ref> These 19 steps can be considered in several steps, with the first step being the formation of the taxane skeleton, which then undergoes a series of oxygenations. Following the oxygenations, two acetylations and a benzoylation occur on the intermediate. The oxygenation of the taxane core is believed to occur on C5 and C10, C2 and C9, C13 followed by C7, and a C1 hydroxylation later on in the pathway. Later in the pathway, an oxidation at C9 forms a ketone functional group and an oxetane, forming the intermediate baccatin III. The final steps of the pathway include the formation of a C13-side chain which is attached to baccatin III.<ref>{{cite journal | vauthors = Croteau R, Ketchum RE, Long RM, Kaspera R, Wildung MR | title = Taxol biosynthesis and molecular genetics | journal = Phytochemistry Reviews | volume = 5 | issue = 1 | pages = 75–97 | date = February 2006 | pmid = 20622989 | pmc = 2901146 | doi = 10.1007/s11101-005-3748-2 | bibcode = 2006PChRv...5...75C }}</ref> The biosynthesis of Taxol is illustrated in more detail in the figure, with steps 1-7 all occurring in the enzyme [[taxadiene synthase]] (TS on the figure). Taxol's biosynthesis begins with E,E,E-GGPP losing pyrophosphate via an [[SN1]] mechanism (step 1 in the figure). The double-bond attacks the cation via electrophilic addition, yielding a tertiary cation and creating the first ring closure (step 2). Another electrophilic attack occurs, further cyclizing the structure by creating the first 6-membered ring and creating another tertiary cation (step 3). An intramolecular proton transfer occurs, attacking the verticillyl cation (step 4) and creating a double bond, yielding a tertiary cation. An electrophilic cyclization occurs in step 5, and an intramolecular proton transfer attacks the taxenyl cation (step 6). This forms the fused ring structure intermediate known as taxadiene. Taxadiene then undergoes a series of 10 oxidations via [[NADPH]], forming the intermediate taxadiene-5α-acetoxy-10β-ol (multiple steps later in the figure). A series of hydroxylations and esterficiations occur, forming the intermediate 10-deacetyl-baccatin III, which undergoes a further series of esterifications and a side-chain hydroxylation.<ref name=":2" /> This finally yields the product taxol.

===Total synthesis===
{{Main|Paclitaxel total synthesis}}

[[Image:Taxol number.svg|thumb|class=skin-invert-image|Paclitaxel, with rings labeled and accepted numbering scheme shown.]]
By 1992, at least thirty academic research teams globally were working to achieve a [[total synthesis]] of this [[natural product]], with the synthesis proceeding from simple natural products and other readily available starting materials.<ref name=Hall2003>{{cite journal | vauthors = Hall N | title = Creating complexity—the beauty and logic of synthesis | journal = Chemical Communications | issue = 6 | pages = 661–664 | date = March 2003 | pmid = 12703766 | doi = 10.1039/b212248k }}</ref> This total synthesis effort was motivated primarily by the desire to generate new chemical understanding, rather than with an expectation of the practical commercial production of paclitaxel. The first laboratories to complete the total synthesis from much less complex starting materials were the research groups of [[Robert A. Holton]], who had the [[Holton taxol total synthesis|first article to be accepted for publication]], and of [[K. C. Nicolaou]] who had the [[Nicolaou Taxol total synthesis|first article to appear in print]] (by a week, on 7 February 1994). Though the Holton submission preceded the Nicolaou by a month (21 December 1993 versus 24 January 1994),<ref>See N. Hall, ibid. See also the [[American Chemical Society]] publication [[Chemical and Engineering News]] (C&EN), 21 February 1994, page 32, and primary citations appearing at Holton and Nicolaou taxol total synthesis articles.</ref> the near coincidence of the publications arising from each of these massive, multiyear efforts—11–18 authors appearing on each of the February 1994 publications—has led the ending of the race to be termed a "tie"<ref name=Flam1994>{{cite journal | vauthors = Flam F | title = Race to synthesize taxol ends in a tie | journal = Science | volume = 263 | issue = 5149 | pages = 911 | date = February 1994 | pmid = 7906053 | doi = 10.1126/science.7906053 | author-link = Faye Flam | bibcode = 1994Sci...263..911F }}</ref> or a "photo finish",<ref name=Hall2003/> though each group has argued that their synthetic strategy and tactics were superior.<ref name=Flam1994/>

As of 2006, five additional research groups had reported total syntheses of paclitaxel: [[Wender Taxol total synthesis|Wender et al.]] in 1997, and [[Kuwajima Taxol total synthesis|Kuwajima et al.]] and [[Mukaiyama Taxol total synthesis|Mukaiyama et al.]] in 1998 with further [[linear synthesis|linear syntheses]], and [[Danishefsky Taxol total synthesis|Danishefsky et al.]] in 1996 and [[Takahashi Taxol total synthesis|Takahashi et al.]] in 2006 with further [[convergent synthesis|convergent syntheses]].{{update inline|date=March 2017}} As of that date, all strategies had aimed to prepare a 10-deacetylbaccatin-type core containing the ABCD ring system, followed generally by last stage addition of the "tail" to the 13-[[hydroxyl group]].<ref name=Hall2003/>

While the "political climate surrounding [paclitaxel] and [the Pacific yew] in the early 1990s ... helped bolster [a] link between total synthesis and the [paclitaxel] supply problem," and though total synthesis activities were a requisite to explore the [[structure-activity relationship]]s of paclitaxel via generation of analogs for testing, the total synthesis efforts were never seen "as a serious commercial route" to provide significant quantities of the natural product for medical testing or therapeutic use.{{sfn|Goodman|Walsh|2001|pp=179–182}}