terpenes are omnipresent natural products mainly found as constituents of plant oils , and have long held importance as flavours and fragrances , and as poisons and medicines .
isolation , structural elucidation and synthetic studies on this vast family of natural products were already being conducted over 100 years ago , providing constant challenges to chemists and thus resulting in a continuous development of the field .
taxol ( 1 ) , a successful anti - cancer drug , is one example of societal and scientific merit , being one of the most famous terpenes from a medicinal standpoint and one of the most densely functionalised and complex molecules from a structural standpoint .
its unique mechanism of action involving the stabilisation of microtubules has been fascinating biologists as well , and thus extensive research efforts have been carried out around the globe by chemists and biologists alike .
taxol ( 1 ) is a potent anticancer drug originally used as a treatment for ovarian and breast cancer , but its versatile effects now extend to treatments of lung , liver and other types of cancer .
initially isolated from the bark of the yew tree , substantial work in the 1980s and 1990s allowed for a semisynthetic route to taxol ( 1 ) that reduced the accompanying detrimental effects to the yew tree population , and currently , its commercial supply relies on cell - culture production ( taxol is currently manufactured with plant cell - culture technology through a collaboration of bristol - myers squibb and phyton biotech , inc . , a dfb pharmaceuticals company ) .
this diterpene is one of few natural products that continues to engender worldwide attention , and tremendous efforts have already culminated in seven total syntheses and one formal synthesis .
however , the inventory of man - made taxol ( 1 ) produced by total synthesis is still less than 30 mg , even when including the most recent approach by takahashi wherein an automated synthesis was employed for the majority of their synthetic steps . in contrast , the current industrial output of 1 through the use of biological machinery is of ton - scale ( 10 mg ) , clearly indicating the magnitudes of difference in chemical versus biological scale and efficiency .
closing this huge gap is not simply a matter of engineering , but rather a learning objective with which to guide chemical innovation , in the tactics , strategies , and methods of synthesis(for a discussion of the terms  strategy  versus  tactics  , see reference 21 ) .
up until the pioneering bioengineering work of stephanopoulos in 2010 , there was no access to large quantities of the less - oxidised taxane members such as taxadiene ( 7 ) .
although not necessarily as a means of supplanting its current ton - scale production , a reexamination of taxol ( 1 ) over 15 years after its first total syntheses could be of academic and medicinal merit .
in this article , we present the first scalable synthetic entry to the taxane family , setting the stage for rapid access to minimally oxidised taxane analogs and a scalable laboratory preparation of taxol ( 1 ) itself . inspired by nature s efficiency in creating vast numbers of complex terpene natural products , a research program was initiated in 2007 to mimic the essence of the two - phase biosynthesis of terpenes . in the first biogenetic phase ( the cyclase phase )
, linear hydrocarbon building blocks are brought together and cyclised efficiently , and in the second phase ( the oxidase phase ) , c = c and c 
the taxanes represent a large family of terpenes comprising over 350 natural products , for which this two - phase terpene synthesis strategy , if realised in a laboratory setting , could target not only taxol ( 1 ) but also related taxanes that differ in oxidation levels ( figure 1 ) . as such
the goal is to divergently access all  pre - taxol  compounds ( both natural and unnatural ) and to unveil new insights to chemical reactivity during an  oxidative ascent  of the taxane pyramid .
this article outlines the completion of our artificial  cyclase phase  for the taxane terpene family , as a prelude to a total synthesis of taxol ( 1 ) .
thus , the logic ( including strategic and tactical concerns ) and execution of a concise and scalable synthesis of minimally oxidised taxanes , including nature s putative cyclase phase endpoint , taxadiene ( 7 ) , are reported .
structurally , taxol ( 1 ) is a highly oxygenated diterpene adorned with acetyl and benzoyl groups , as well as a signature taxol side chain at the c13 oxygen atom ( see carbon numbering on 1 , figure 1a ) . for retrosynthetic discussion purposes , 1 is treated as if it were devoid of acyl groups , and this target is substituted with oxygenated hydrocarbon 2 . placing 2 at the apex of an  oxidase phase pyramid  , the number of c  o bonds decreases as one moves down the pyramid , with taxanes 3 , 4 and 5 corresponding to the oxidation pattern of 2-acetoxybrevifoliol , taxinines e and j , and yunnanxane , respectively ( figure 1b ) .
these highly oxidised taxanes all feature a c2-hydroxyl group and can be further simplified to taxa-4(5),11(12)-dien-2-one ( 6 ) , dubbed  taxadienone  , which represents a benchmark intermediate for a comprehensive access to the taxane family .
furthermore , if taxadiene ( 7 ) were to be desired , one could simply deoxygenate 6 to generate the least oxidised natural product in the taxane family ( figure 1c ) .
a full scholarly analysis of the taxane pyramid has been previously described , along with the merits in choosing a c2-oxidised taxane such as 6 .
strategic disconnections of 6 were built upon the landmark syntheses of 1 and the many studies geared toward the taxane framework . out of the many possible disconnections of the 6 - 8 - 6 tricyclic backbone , one was chosen such that : 1 ) the forward synthesis would be short , convergent , and scalable ; and 2 ) its asymmetric synthesis would only rely on one enantioselective reaction , after which the resulting stereochemical information could be propagated to set all other stereocentres diastereoselectively .
a vicinal difunctionalisation / diels  alder strategy to forge the taxane ab ring system ( see ring numbering in 2 , figure 1 ) seemed to adhere most to the above criteria .
the diels  alder strategy was previously utilised in the context of taxane synthesis by shea , jenkins , williams and others , and in fact , williams has reported the only total synthesis of ( )-taxadiene ( 7 ) , in 26 steps , back in 1995 .
furthermore , the decision to implement a vicinal difunctionalisation strategy was underpinned by recent developments in the asymmetric formation of all - carbon quaternary centres .
thus , a concise , enantioselective strategy was conceived , allowing for a gram - scale access to 6 in only seven steps , as well as a gram - scale synthesis of ( + ) -taxadiene ( 7 ) .
in the forward direction , known compounds 10 ( made from 8 in a modified one - pot procedure ; see supplementary information ) and 11 ( made from 9 using a known one - step procedure ) were combined to generate enone 12 by means of a lewis acid - modified organocopper 1,6-addition ( figure 2 ) .
this convergent synthesis of 12 is operationally simple and provides decagram quantities per reaction batch ( 86% ; over 50 grams of this material has been synthesised to date ) .
this enone was deemed to be a suitable substrate for alexakis enantioselective conjugate addition to establish the quaternary centre ( c8 ) . however , this strategy was contingent upon the feasibility of trapping the resulting aluminum enolate by tmscl .
although alexakis noted that isolation of such silyl enol ethers is difficult due to facile desilylation , a modified quenching procedure involving dilution with thf , addition of tmscl , followed by addition onto florisil in 1:10 et3n / hexanes , allowed the isolation of tms enol ether 14 in 89% yield and in 93% ee ( 1.0 g scale ; enantioselectivity was measured for desilylated ketone 15 on chiral hplc ; see supplementary information ) .
only 2 mol% of cutc and 4 mol% of chiral phosphoramidite ligand 13 were required , and this asymmetric reaction was routinely conducted on gram scale ( over 20 grams of this material has been synthesised to date ) .
tms enol ether 14 was indeed quite prone to desilylation , and formation of undesired ketone 15 was often the sole reaction pathway when attempting mukaiyama aldol reactions with commonly used lewis acids ( such as ticl4 , sncl4 , sn(otf)2 , sc(otf)3 , bf3oet2 , tmsotf , zncl2 , or mgbr2oet2 ) or other catalysts ( such as liclo4 , zr(obu)4 , bi(otf)3 , agotf , or sicl4 ) , even when performed under strictly anhydrous conditions .
surprisingly , water was found to be the enabling additive that allowed for an aldol reaction with acrolein .
gratifyingly , kobayashi conditions with gd(otf)3 in 1:10:4 h2o / etoh / phme generated the corresponding aldol product smoothly as a 2:1 mixture of diastereomers that are isomeric at c3 .
this aldol product was then oxidised in the same reaction pot with jones reagent to generate keto - enone 16 in 85% over two steps ( 3.2 g scale , 2:1 ratio of diastereomers at c3 ; this inseparable mixture of stereoisomers was carried onward for the ensuing step ) . a diels
 alder reaction to generate the 6 - 8 - 6 tricyclic skeleton has been previously demonstrated on similar systems , and the mixture 16 reacted predictably with bf3oet2 to furnish tricyclic compound 17 ( 2.3 g scale ) .
the desired diketone 17 was obtained in 47% yield , along with its diastereomer ( see supplementary information ) in 29% yield , with complete diastereoselectivity at c1 .
these cyclised diketones displayed very different polarities and were thus easily separated by silica gel chromatography at this stage .
only one more carbon remained to be installed to complete the taxane framework : this was achieved via enol triflate formation followed by negishi coupling to generate taxadienone ( 6 ) in 84% over two steps ( 2.4 g scale ) .
this material solidified quite readily , and a single - crystal x - ray analysis confirmed both the absolute and relative stereochemistry of the synthesised abc ring system .
this lowly oxidised core is the key intermediate for our future research efforts to elaborate the taxane pyramid . in summary ,
the total synthesis of ( + ) -taxadienone ( 6 ) was achieved in a total of 8 steps , with a longest linear sequence of 7 steps .
a few grams of taxadienone ( 6 ) could be synthesised by one chemist over the course of 7 days , with an overall yield of 18% from 8 or 20% from 9 . with the key taxane 6 secured in gram quantities , taxadiene ( 7 )
was then targeted , primarily as a means of spectroscopic comparison to previously reported data , but also to demonstrate the feasibility of a large - scale laboratory production of enantioenriched 7 .
furthermore , taxadiene ( 7 ) is produced in negligible amounts in nature ( less than 1 mg can be obtained from 750 kg of tree bark from t. brevifolia ) and therefore its optical rotation has never been recorded . to this end , a three - step deoxygenation sequence was performed in 52% yield overall on gram - scale , to generate ( + ) -7 , which was spectroscopically indistinguishable from that of natural 7 , previously synthesised ( )-7 , and bioengineered ( + ) -7 .
the optical rotation of [  ] ( chcl3 ) = + 165 ( c = 1.0 ) is reported herein for the first time and is comparable to that obtained for a bioengineered sample of ( + ) -7 ( see supplementary information ) .
in retrospect , the forward synthesis of ( + ) -taxadienone ( 6 ) appears to be rather simple and perhaps intuitive , however , in practice , this optimised approach required many rounds of strategic and tactical revision .
strategically , the retrosynthesis designed in figure 1c was one of many that were considered at the outset . a small snapshot of the many evaluated blueprints is illustrated in figure 4 .
for example , the known difficulties in forming the 6 - 8 - 6 tricyclic framework of taxanes led us to first consider a ring - closing metathesis strategy to close the central 8-membered ring ( disconnection a ) .
however , the realisation that the required substrate 18 would take many steps to build , and the fact that the stereocentres at c1 and c8 would have to be formed with two separate enantioselective reactions , dissuaded us from pursuing this route . an aldol route was then conceived , partly due to the facile formation of 19 via ketone 15 ( disconnection b ) . although installation of the stereocentres at c1 and c8 might still require two independent enantioselective transformations , the hope was that the reaction conditions used for the aldol cyclisation would concomitantly epimerise the c1 stereocentre into the desired configuration
however , despite a plethora of attempted experiments , the desired cyclisation from 19 did not proceed .
thereafter , strategies involving closure of the ab ring by a diels  alder reaction were envisioned .
one attempt involved the formation of ketone 20 by a sequence involving a coupling of the enolate of 2,6-dimethylcyclohexanone and a primary alkyl bromide , a shapiro reaction onto acrolein , oxidation , and diels  alder ( disconnection c ) , much akin to the transformation from 14 to 17 ( figure 2 ) .
however , ketone 20 already required many steps to construct , and the stereocentre at c8 was challenging to control , despite existing methods in asymmetric enolate alkylation .
lastly , enones 21 were considered as viable intermediates toward the formation of 6 ( disconnection d ) , due to our initial difficulties in forging the c2c3 bond by mukaiyama aldol reaction ( vide supra ) . although enones 21 were formed in short order and
already contained all but one carbon of the taxane framework , they did not undergo [ 4 + 2 ] cyclisation , likely due to the rigidity of the sp carbons at c3 and c8 .
furthermore , a methyl 1,4-addition at c8 was not possible , since reaction first occurred at the less hindered c14 , even when a large tert - butyldiphenylsilyl group was appended at c14 .
after many more strategic revisions and the success in forming the required c2c3 bond from tms enol ether 14 using gd(otf)3 , the final synthetic strategy depicted in figure 2 was realised . tactically , although quite efficient at present , most steps shown in figure 2 were initially difficult to scale and suffered from low and inconsistent yields .
even the first transformation from 8 to 10 ( figure 2 ) was not trivial , despite it being a known two - step transformation . modifying the reaction stoichiometry and reaction times was necessary to coax this process into giving good yields consistently on a decagram - scale , with eventual success as a one - pot operation .
the second transformation , a merging of the two similar - sized fragments 10 and 11 , can be conducted in good yields ( 86% ) on a decagram - scale , but initially , this reaction was plagued with inconsistent yields due to side product formation : the original reaction conditions of tert - butyllithium , bf3oet2 and cui resulted in 1,6-addition of tert - butyllithium ( whereas sec - butyllithium is not a very competent nucleophile for this reaction ) , iodination of 12 ( whereas the use of cubrsme2 circumvents this problem ) , and deconjugation of 12 to give a ,-substituted ketone ( this problem was rectified by optimising the work - up procedure ; see supplementary information ) .
the third , asymmetry - inducing step from 12 to 14 was straightforward when run on a small scale ( < 100 mg ) and delivered high enantioselectivity with 0.5 mol% cutc and 1 mol% ligand loading .
however , upon increasing the reaction scale to gram - scale , the reaction conversion suffered significantly . eventually , a higher catalyst loading ( 2 mol% cutc and 4 mol% ligand ) and precise temperature control allowed this reaction to give reliable yields and consistent enantioselectivity on gram - scale .
also worth mentioning is a modified quenching procedure that was developed to address the troublesome tms trapping of the aluminum enolate ( videsupra ) .
the fourth and the most difficult reaction was the aldol reaction of 14 and acrolein , which only returned desilylated ketone 15 under most reaction conditions .
a variety of lewis acid - mediated reactions ( vide supra ) , as well as anionic silicon - metal exchange reactions , never led to the desired product .
this aldol reaction only proceeded when lanthanide triflates such as yb(otf)3 or gd(otf)3 and very specific solvent systems were used .
the final challenge was a scalable diels  alder reaction from 16 to 17 , which has been known to proceed in moderate yields on similar substrates .
although efficient on a small scale , the yield decreased when the reaction was conducted on gram - scale , possibly due to formation of oligomers and polymers .
0.01 m ) and through slow addition of substrate 16 to the lewis acid solution ( see supplementary information ) . developing a scalable route to the taxane core involved the study and modification of fundamental aspects of the described chemistry , rather than a mere exercise in scale - up .
the conciseness of the synthetic route is a direct result of trying to achieve a scalable synthesis , and the reliability of the yields attests to the small variability of reactions ran on a larger scale . despite the efficiency of the described approach ,
there are two obvious limitations in the synthesis of taxadienone ( 6 ) : 1 ) the single functional group manipulation from cyclised diketone 17 to the corresponding enol triflate ( rendering the route 85% rather than 100% ideal ) ; and 2 ) the 2:1 diastereoselectivity in the aldol reaction of 14 and acrolein .
these issues are currently being addressed by : 1 ) developing a scalable , one - step enol triflation / methyl coupling reaction ; and 2 ) generating creative acrolein equivalents and/or reaction conditions with various additives to increase to diastereoselectivity of the aldol step . in summary ,
a scalable , enantioselective entry to the taxane family of natural products was achieved in only seven steps from commercially available starting material ( 1820% overall yield ) .
this triply convergent approach to taxa-4(5),11(12)-dien-2-one ( 6 ) allowed for a minimisation of concession steps , wherein six out of seven steps formed skeletal ( c  c ) bonds .
every one of these steps was performed on a gram - scale , attesting to the scalability and robustness of the sequence .
furthermore , ( + ) -taxadiene ( 7 ) was synthesised , enabling further structural confirmation , as well as the first optical rotation determination of this natural product .
the simple chemical route to ( + ) -7 nicely complements the recent pioneering studies of stephanopoulos and co - workers , whose bioengineering strategy also delivers gram - scale quantities of ( + ) -7 , albeit as a 9:1 mixture of olefin isomers ( taxa-4(5),11(12)-diene ( 7 ) and taxa-4(20),11(12)-diene ) ( see supplementary information ) .
studies are currently underway to utilise the existing functional group handles in 6 to oxidise various sites on the taxane skeleton , in order to create a pyramid - like library of unnatural and natural taxanes en route to taxol ( 1 ) .
 taxanes are a large family of terpenes comprising over 350 members , the most famous of which is taxol ( paclitaxel )  a billion - dollar anticancer drug . here , we describe the first practical and scalable synthetic entry to these natural products via a concise preparation of ( + ) -taxa-4(5),11(12)-dien-2-one , which possesses a suitable functional handle to access more oxidised members of its family . 
 this route enabled a gram - scale preparation of the  parent  taxane , taxadiene , representing the largest quantity of this naturally occurring terpene ever isolated or prepared in pure form . 
 the taxane family s characteristic 6 - 8 - 6 tricyclic system containing a bridgehead alkene is forged via a vicinal difunctionalisation / diels  alder strategy . 
 asymmetry is introduced by means of an enantioselective conjugate addition that forms an all - carbon quaternary centre , from which all other stereocentres are fixed via substrate control . 
 this study lays a critical foundation for a planned access to minimally oxidised taxane analogs and a scalable laboratory preparation of taxol itself . 
Introduction
Results
Discussion
Supplementary Material