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Enhanced yield of medicinal products from Tripterygium cell cultures Samija, Mijo Daniel 1992

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ENHANCED YIELD OF MEDICINAL PRODUCTSFROM TRIPTERYGIUM CELL CULTURESbyMIJO DANIEL SAMIJAB.Sc., The University of British Columbia, 1986A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES( DEPARTMENT OF CHEMISTRY )We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAFebruary 1992© Mijo D. SamijaIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of ^Ch Y'''1 Sir yThe University of British ColumbiaVancouver, CanadaDate^rtiarr 2/) frI'i2_(Signature)DE-6 (2/88)triptolidetripdiolide18(4- 3)-isodehydro-abietenolideABSTRACTThe growth of cell suspensions of the Asian medicinal plant Tripterygium wilfordiiwas manipulated in order to produce large amounts of pharmacologically active diterpene andtriterpene natural products. A method was developed where elicitation with a strain of the fungusBotrytis stimulated the production of oleanane and friedelane triterpene acids. In rapidly growingtwelve liter bioreactor cultures, triterpene yields were increased five to ten fold with this process,routinely providing more than 25 mg/L each of 22a-hydroxy-3-oxoolean-12-en-29-oic acid (B)and 30,22a-dihydroxyolean-12-en-29-oic acid (D) and more than 5 mg/L each of 22P-hydroxy-3-oxoolean-12-en-29-oic acid (A) and 30,220-dihydroxyolean-12-en-29-oic acid (C).Yield improvement for the triptolide family of diterpenes (triptolide and tripdiolide)was approached through the synthesis of potential intermediates of the natural biosyntheticpathways, the first step in a technique where synthetic elaborations would be completed by cellcultures. These synthetic intermediates were also sought to establish the details of triptolidebiosynthesis. Advances were made towards the synthesis of one potential intermediate,18(4- 3)-isodehydroabietenolide, starting from (L)-dehydroabietic acid.11TABLE OF CONTENTSAbstract^ iiList of Tables vList of Figures viAcknowledgments^ viiiINTRODUCTIONThe Pharmacology and Composition of Tripterygium^ 1(I) Medicinal History^ 1(II) Laboratory Research 2(III) Botanic al Information 4(IV) Analysis of Pharmacologically Active Components^ 5(V) Medical Uses of Tripterygium^ 23Methods of Yield Optimization in Plant Cell Cultures^ 26(I) Nutritional Studies On Cell Cultures of Tripterygium^ 28(II) Elicitation^ 33(i) Disease Resistance in Plants 33(ii) Phytoalexins^ 35(iii) Elicitors 35(iv) Mechanism of Elicitor Action^ 38(v) Elicitation of Triterpene Acid Biosynthesis ^ 39(III) Biotransformation of Synthetic Precursors , 43DISCUSSIONMetabolite(I)(11)(HI)(IV)(V)(VI)Production from Elicitation of Tripterygium^ 59Growth and Elicitation of TRP 4a Cultures 59Analysis of the Metabolites from TRP 4a Cultures ^ 62(i) Tripdiolide Determination 63(ii) Triterpene Determination^ 64Elicitation in the Production of Tripdiolide and Triterpenes^ 67Optimization of Triterpene Production in 12 Liter Bioreactors 71(i) The Growth Of Elicited Cultures ^ 71(ii) Analysis of Culture Extracts 73(iii) The Results of 72 Hour Elicitation 75(iv) A Summary of the 72 Hour Elicitations^ 93(v) Extended Elicitation ^ 97(vi) The Results of Extended Elicitation 99(vii) Conclusions on Elicited Triterpene Production^ 116(viii) Further Studies on Triterpene Elicitation  116(ix) Error Analysis^ 118The Natural Products Enhanced by Elicitation^  119Purification of Triterpenes A, B, C and D 120Synthesis of Precursors for Biotransformation to the Triptolides 122iiiEXPERIMENTAL(I)^Identification of TRP 4a Metabolites^ 137(i) Metabolite Production from Cell Cultures^ 137(ii) Tripdiolide Analysis 139(iii) Triterpene Analysis 139(iv) Isolation of Triterpene Acids A, B, C and D^ 141(II)^Synthetic Reactions Toward 18(4 - 3)-isodehydroabietenolide^ 143REFERENCES 151APPENDIX^ 157ivLIST OF TABLES1^Isolation and Testing of Natural Products from Tripterygium^ 172^Elicitation at 1.0% in Late Growth (Culture Series 252)  763^Elicitation at 1.0% and 0.1% in Mid Growth (Culture Series 254) ^ 794^Elicitation at 1.0% and 0.1% in Late Growth (Culture Series 255)  825^Elicitation at 1.0% and Extended Elicitation in Early Growth (Culture Series 256) ^ 856^Elicitation at 5.0% in Mid Growth (Culture Series 258)^  887^Elicitation at 1.0% and 5.0% in Early Growth (Culture Series 259) ^ 918 The Effect of Column Chromatography on the Triterpene Assay  969^The Effect of Inoculum Cycle on Elicited Triterpene Yields ^ 9710 Extended Elicitation from Early Growth (Culture Series 261)  100-10211 Extended Elicitation from Early Growth (Culture Series 262) ^ 106-10712 Extended Elicitation from Early Growth and from Day 0 (Culture Series 266) ^ 111-11313 Consistency in the Elicitation Results for the Triterpene Acids ^ 118vLIST OF FIGURES1 Sesquiterpenes of Tripterygium^ 6-72 Diterpenes of Tripterygium 8-93 Triterpenes of Tripterygium ^ 10-154 Other Natural Products of Tripterygium ^ 165 The Activation of Elicitation^ 366 Elicited Ursane Triterpenes 397 Partition of 1- 14C IPP in Cell Free Biotransformations ^ 418 Biosynthesis of Terpene Classes ^ 449 Biosynthesis of Abietane Diterpenes 4510 Synthesis of Racemic Butenolide by vanTamelen and Leiden (Biogenetic) ^ 4811 Synthesis of Racemic Butenolide by Garver and vanTamelen (Decalone) ^ 5012 Synthesis of Racemic Butenolide by Berchtold et al. (Naphthalene) ^ 5213 Synthesis of Chiral Butenolide by vanTamelen et al. (Dehydroabietic Acid) ^ 5314 Synthesis of Chiral Butenolide by Tokoroyama et al. (Dehydroabietic Acid) ^ 54-5515 Synthesis of Chiral Butenolide by Roberts (Dehydroabietic Acid) ^ 5716 Planned Butenolide Synthesis (Dehydroabietic Acid) ^ 5817 Growth Curves for PRD2Co Cultures of TRP 4a in 12 Liter Aerated Bioreactors  ^6118 Lactonization of Oleanane Triterpenes ^ 6719 Growth Curves and Culture Dry Weight of TRP 4a Series 266 ^ 7220 Growth Curves of TRP 4a Culture Series 252 ^ 7721 Triterpene Levels from Culture Series 252 7722 Growth Curves of TRP 4a Culture Series 254 ^ 8023 Triterpene Levels from Culture Series 254 80vi24 Growth Curves of TRP 4a Culture Series 255 ^ 8325 Triterpene Levels from Culture Series 255 8326 Growth Curves of TRP 4a Culture Series 256^ 8627 Triterpene Levels from Culture Series 256 8628 Growth Curves of TRP 4a Culture Series 258 ^ 8929 Triterpene Levels from Culture Series 258 8930 Growth Curves of TRP 4a Culture Series 259^ 9231 Triterpene Levels from Culture Series 259 9232 Triterpene Levels from Elicitation at Various Culture Ages ^ 9433 Optimization of Triterpene Production ^ 9434 Growth Curves of TRP 4a Culture Series 261 10335 Triterpene Levels from Culture Series 261 ^ 103-10436 Growth Curves of TRP 4a Culture Series 262 10837 Triterpene Levels from Culture Series 262^ 10938 Growth Curves of TRP 4a Culture Series 266 11439 Triterpene Levels from Culture Series 266 ^ 114-115viiACKNOWLEDGMENTSI am very grateful for the opportunity and the careful direction provided by ProfessorJ. P. Kutney in both synthetic and cell culture work. I extend my thanks also to Gary Hewitt forhis fundamental input into the planning and execution of the elicitation project and to DavidChen for providing such reliable day-to-day management of cell cultures. Elizabeth Bugantêwas an essential collaborator in the formative elicitation studies and Huifen Gu providedexcellent work and most of all unwavering motivation throughout the cell culture triterpeneanalyses. I will miss these two, visiting scholars from The Philippines and China respectively.Thank-you to the others who contributed to our understanding of the elicitation ofTripterygiurn, including Stephen Cheung and especially Leo Law. I express highest regards forMalcolm Roberts who provided indispensable advice and encouragement throughout mysynthetic studies. My worthy associates also included Kang Han, Francisco Kuri-Brena, YongHuang Chen, Krystyna Piotrowska and several others who were never too busy for helpfulsuggestions and support. The staff and faculty of this Chemistry department provided awonderful environment and excellent facilities that made my time here very enjoyable.Thank-you family and friends.viiiINTRODUCTIONTHE PHARMACOLOGY AND COMPOSITION OF TRIPTERYGIUMThis thesis was written to describe methods for increasing production of pharmacologicallyuseful compounds synthesized in the Asian medicinal plantTripterygium wilfordii.(I) MEDICINAL HISTORYIn China, the first written accounts of medicine (now 2000 years old) describeTripterygium as a source of extracts used to treat fever, chills, joint pain, swellings andskin rashest.In contrast, a recent article in the Chinese Medical Journal 2 names a commercialTripterygium extract as the current most effective treatment of rheumatoid arthritis (RA). Theoral ingestion of extract relieves the joint pain and inflammation that are symptoms of thisautoimmune disorder. During the course of this treatment, two beneficial physiological responsesare observed. The first is a rapid anti-inflammatory effect and the second a slowimmunosuppressive effect 2,3 . These responses are similar to those obtained with corticosteroids,the most widely prescribed drugs for RA treatment* , and are presently the most valuablepharmaceutical activities displayed by Tripterygium. Tripterygium extracts are also prescribed inthe treatment of other disorders related to the overactive or unregulated immune system: chronicnephritis (kidney) and hepatitis (liver), ankylosing spondilitis (joints) and systemic lupuserythematosis (skin and organs) 2,4 .*corticosteroids are not among the compounds isolated from Tripterygium (table 1, p.17 )1Tripterygium extracts exhibit a very effective male contraceptive activity that is reversedonce ingestion ceases. This application is still at the stage of clinical trials 1,5 and work isunderway to isolate the causative agent.The pharmaceutical use of Tripterygium occurs mainly in China, where it is dispensed bytwo types of practitioner: the medical doctor and the expert in traditional herbal medicine. Thefirst group has prepared a refined extract using university, industrial and clinical researchfacilities. The response of patients to this preparation is monitored closely to analyze the specificmode of action that the drug employs and to better understand the actual disease. The othergroup, the herbalists, use extracts that have been available for hundreds of years. The extractionprocedures were developed by observing the overall health of patients following treatment,without any specific understanding of the therapeutic mechanisms involved. Controlled aqueousor ethanolic extraction of Tripterygium provides tonics free of the toxic components contained inthe whole plant 1,6 . It is appropriate to note that the fevers, swellings and rashes for which herbaltreatment is a long-standing remedy are often symptoms of the immunological disorders forwhich refined Tripterygium extracts are now prescribed.(II) LABORATORY RESEARCHThe original studies on the chemical composition of Tripterygium began in the 1930's inChina7 . This research was carried into the U.S. by agricultural chemists 8 11 when it waslearned that Chinese farmers spread powdered Tripterygium root over their crops to kill chewinginsects and worms. The study produced a small catalog of compounds but was ended in the1950's, possibly due to the availability of more potent synthetic pesticides.In the 1970's, Prof. S.M. Kupchan and co-workers (Univ. Virginia) carried out a projectto isolate the medicinal agents of Tripterygium. His analysis of the components present in a2utilized bio-assays to select only the active principles. The extracts were chromatographed intoseveral pure compounds which demonstrated potent anti-leukemic activity 12,13 . This was a veryimportant advance in Tripterygium research, providing a group of possible pharmaceuticals forfurther study and developing the methods for compound screening that would be used bysubsequent groups.The ability of Tripterygium extracts to cause a reversible suppression of male fertilitywas first noted in China during the course of a clinical trial on rheumatoid arthritis treatment.This led to separate studies of the extracts viability as a male contraceptive'.As well as searching for the antifertility agent, several groups are now interested inisolating compounds responsible for the anti-inflammatory and immunosuppressive propertiesthat make Tripterygium useful against immune disorders2,14,15 . Each group is screening theextracts for a single active principle with a narrow range of effects. For example, to prepare auseful male contraceptive it would be desirable to separate out the immunosuppressive agents soas not to weaken the disease resistance of the consumer. Prof. J.P. Kutney's laboratory is workingon the isolation, identification and potential production of Tripterygiums medicinally useful anti-leukemic, anti-fertility, anti-inflammatory and immunosuppressive compounds. The methodsused and the results of this research will be reviewed in this paper. The standard source ofTripterygium extracts is the dried root of the plant which is native to China. In contrast, Prof.Kutney has worked exclusively with tissue cultures of Tripterygium which demonstrate rapidgrowth and an increased production of metabolites.3(III) BOTANICAL INFORMATIONTripterygium is a member of the family Celastraceae, which is a group of woody vinesand shrubs. It grows wild in the forested hills of Southeast China and Taiwan. The species ofTripterygium include T. wilfordii, T. hypoglaucum, T. regelii and T. forrestii. Within this thesis,Tripterygium will refer to Tripterygium wilfordii Hook. f. (Hooker filius), on which most studieshave been carried out. This plant is a perennial twining vine with pale green serrated leaves and ared pigment in the stems and roots. It bears small white flowers which develop into seeds havingthree longitudinal ridges, which is the source of the European name tri-pterygion (Greek; threesmall wings) and a Chinese name translating into "three wing nut". As a wild plant, the Chineserefer to Tripterygium as mang cao* (rank grass) while in the medicinal context it is referred toexclusively as lei gong teng (thunder god vine). The Celastraceae family contains other plantsnotable for the production of pharmacologically active compounds. Maytenus, for example,which produces the anti-cancer compound Maytansine 16,17 . The red quinone methide celastrol( ) is found, as the name suggests, in many Celastraceae and is a member of a group ofcytostatic triterpene quinones.Pharmaceutical extracts of Tripterygium are prepared from the root of mature plants. Theroots two outer layers (referred to as the bark or the cortex ) contain the toxic alkaloids that areused as an insecticide and the interior core (xylem ) contains the medicinal agentsl. The leaves ofTripterygium are exceedingly toxic to humans due to stored alkaloids 18 20 .* these are Pinyin phonetic transcriptions of the Chinese pronunciation4(IV) ANALYSIS OF PHARMACOLOGICALLY ACTIVE COMPONENTSThe screening of Tripterygium extracts for useful compounds began in China where Chouand Mei isolated sugar and pigment components (1936)21 . In searching for an insecticide, theAmerican chemist Haller identified the main pigment component, celastrol, and isolatedinsecticidal alkaloids (1941) 8 ' 10. This study was continued by Beroza, who identified several ofthe alkaloid structures (1953)22,23 . In a search for medicinally useful compounds, Kupchan et al.discovered a group of anti-leukemic diterpenes (1972) 12 . These compounds were produced on alarger scale by plant tissue cultures in work carried out by Kutney's group 24,25 (1981), whoseresearch is now focussed on improving the production method and identifying anti-inflammatory, immunosuppressive, and anti-fertility factors. The isolation of these compoundsrequires several repetitions of a sequence involving chromatography of biologically activeextracts followed by an assay for active fractions. The following figures (figs. 1 - 4) provide acompendium of the natural products which have been isolated to date* . The source of thesecompounds was primarily Tripterygium wilfordii, with a few isolated from T. regelii, T.hypoglaucum and T. forrestii. Following the structures, Table 1 correlates these compounds withthe discovering groups and lists pharmacological activities that have been observed.* A numbering scheme for terpene skeletons is provided in Appendix I.5R6OR1^OR5R2 R2—O ^-07-1513^'OH^7-A3dihydroagarofuran skeleton triptofordins triptogelinsR1 R2 R3 R4 R5 R6 Triptofordin1 Cn – – – Bz – A2 Bz – OH – Bz – B3 Bz – OAc =0 Ac – C-14 Bz – OAc OH Ac – C-25 Bz =0 OAc – Cn OAc D-16 Bz OH OAc – Cn OAc D-27 Bz OAc OH OAc Cn OAc F-18 Bz OBz OH OAc Ac OAc F-29 Bz OAc OAc OAc Bz OAc F-310 Bz OH OH OAc Cn OAc F-411 Bz =0 OAc OAc Bz OAc E12 Bz =0 OAc OAc Ac OAc13 Bz =0 OAc OAc Cn OAcTriptogelin14 Bz OBz OAc OBz OBz – A-115 Bz OBz OAc OH OBz – A-216 Bz OBz OAc OH – – A-317 Bz OBz OAc =0 – – A-418 Bz OBz OAc – – – B-1Figure 1 (a) SESOUITERPENES OF  TRIPTERYGIUM (Triptofordins and Triptogelins)Acetyl (Ac), benzoyl (Bz), (trans-)cinnamyl (Cn) (0FO)unsubstituted (–).Ft6OOAcOBz 0^CH'CHO:-07—OAc OH27^( trans- cinnamyl ) triptofordinine A-128^( cis- cinnamyl )^triptofordinine A-2OBz^OBz29^regilidineR2 R319 — Bz — wilforzine20 — Bz Ac wilforine21 — Ac Ac wilformine22 — Fr Ac wilforgine23 — Nc Ac wilfornine24 OH — Ac wilforidine25 OH Bz Ac wilfordine26 OH Fr Ac wilfortrineFigure 1 (b) SESOUITERPENE ALKALOIDS OF  TRYPTERYGIUMAcetyl (Ac), benzoyl (Bz), nicotinyl (Nc), 3-furoyl (Fr),(‘0_ . t.--.\ ) (R^)Ri \—r■r^Runsubstituted (—).7H31 tripdiolideH30 triptolide,s .9H32 triptonideH33 triptolidenol18(4 - 3)abeo-abietaneskeletonOHOH34 R= H^triptophenolide35 R= CH3 triptophenolide methyl etherH36 triptonolideOHH HOHOC H337 neotriptophenolide^ 38 isoneotriptophenolideFigure 2 DITERPENES OF TRIPTERYGIUM ABEO-ABIETANE BUTENOLIDES AND ABIETANES8OHOCH341 neotriptonoterpeneHeco2H 45 (I)-dehydroabietic acidHOOCH342 triptonodiolHO2Cabietane skeletonOCH340 triptonoterpene (b)HOOH39 triptonoterpene (a)Figure 2 DITERPENES (cont.)oleanane skeleton46 13 - amyrinHO47 313,29-dihydroxyolean-12-ene48 313,11 a-dihydroxyolean-12-ene^49 oleanolic acidFigure 3 TRITERPENES OF TRIPTERYGIUM (a) OLEANANES (i)10po2H''OH50 3f3-epikatonic acid'OH51 22p-hydroxy-3-oxoolean-12-en-29-oic acid^52 22a-hydroxy-3-oxoolean-12-en-29-oic acidHO^ C^ HO^ D53 3p, 22p-dihydroxyolean-12-en-29-oic acid^54 3p,22a-dihydroxyolean-12-en-29-oic acidFigure 3 TRITERPENES OF TRIPTERYGIUM (a) OLEANANES (ii)1155 wilforlide BHO56 wilforlide A57 triptodihydroxy acid methyl esterFigure 3 TRITERPENES OF TRIPTERYGIUM (a) OLEANANES (iii)12ursane skeleton58 a- amyrinCO2H''OH59 triptotriterpenic acid Cg02CH 3^ CO2C H3'OH 'OH60 regelin^ 61 regelinolFigure 3 TRITERPENES OF TRIPTERYGIUM (b) URSANES13friedelane skeleton62 polpunonic acid 63 salaspermic acid64 3,24 - dioxofriedelan-29-oic acid^65Figure 3 TRITERPENES OF TRIPTERYGIUM (c) FRIEDELANES14Quinone Methides66 tingenone68 celastrol67 2213-hydroxytingenone69^0-sitosterolFigure 3 TRITERPENES OF TRIPTERYGIUM (d) OTHER15R70 celacinnine (trans - cinnamyl)71 celabenzine (benzoyl)72 celafurine (3-furoyl)HN ..,• •■,.„,.., ... „ /....•NR[ (3,—i-0R'{ c>_ciol74 neotriptonolide73 wilforonideSpermidine AlkaloidsButenolidesFigure 4 OTHER NATURAL PRODUCTS OF TRIPTERYGIUM 16Table 1^Isolation and Testing of Natural Products from TripterygiumCompound ReferenceSourceBiological Activity *mice (m) human cell culture (c)wholeplantcellculture1A19 + 02B19 + 03C-119 + 04C-219 + 0SD-120, 26 + 06D-220, 26 + o7F-118 + 08F-218 + 09F-318 + 010F-418 + 011E20, 26 + 012 26 + 0 possibly insecticidal13 26 + 0 possibly insecticidalTable 1(a)^Sesquiterpene TriptofordinsCompound ReferenceSourceBiological Activitymice (m)^human cell culture (c)wholeplantcellculture14 27 + 0A-115 27 + 0A-216 27 + 0A-317 27 + 0A-418 27 + 0B-1Table 1(b)^Sesquiterpene Triptogelins* few of the compounds have been subject to a wide range of tests; while human patients have been treated withplant extracts containing these components, purified compounds have yet to be used (except triptolide 30 )17Table 1 (cont.)Compound ReferenceSourceBiological Activitymice (m) human cell culture (c)wholeplantcellculture19 22, 23, 28, 29 + 0 insecticidalwilforzine20 22, 23, 30 + o insecticidalwilforine21 28, 31, 32 + 0 insecticidal, immunosuppressive (m)wilformine(euonine)22 22, 23, 28 + 0 insecticidalwilforgine23 31 + 0 insecticidal, immunosuppressive (m)wilfornine24 33 + o insecticidalwilforidine25 22, 23, 33, 34, + 0 insecticidalwilfordine 3526 22, 23, 32, 33, + 0 insecticidal, antileukemic (m),wilfortrine 35, 36 immunosuppressive (m)27 37 + 0triptofordinine A-128 37 + 0triptofordinine A-229 38 + 0regilidineTable 1(c)^Sesquiterpene Alkaloids18Table 1^(cont.)Compound ReferenceSourceBiological Activitymice (m) human cell culture (c)wholeplantcellculture30triptolide12, 13, 24, 25,39, 40, 41, 42,43, 44, 45, 46+ .4. antileukemic (m), antitumor (c), cytostatic (c),anti-inflammatory (m), immunosuppressive (m)31tripdiolide6, 12, 13, 24,25, 42+ + antileukemic (m), antitumor (c)32triptonide12 + 033triptolidenol42, 47 + 034triptophenolide48 + 035triptophenolide methylether 48 + 036triptonolide49 + 037neotriptophenolide48 + 038isoneotriptophenolide50 + 0•39triptonoterpene (a)47 + 040triptonoterpene (b)51 + 041neotriptonoterpene51 + 042triptonodiol51 + 043- - -25 0 +44 24, 25 0 +45dehydroabietic acid24 0 +Table 1(d)^Diterpenes19Table 1^(cont.)Compound ReferenceSourceBiological Activitymice (m) human cell culture (c)wholeplantcellculture(i)4613 - amyrin25,52 0 +47313,29-dihydroxyolean-12-ene25, 53 0 +48313,1 la-dihydroxyolean-12-ene25, 54 0 +49oleanolic acid24, 25 0 +50313-epikatonic acid55 + 051A25 0 +52B25, 26 0 +53C25 0 +54D16, 25, 55, 56,57+ + antileukemic (m), anti-inflammatory (m)55wilforlide B55, 58 + + partially converted to 52 in aqueous solution56wilforlide A55, 58 + + partially converted to 54 in aqueous solution57triptodihydroxy acidmethyl ester36, 37 + 0Table 1(e)^Triterpene Oleananes(i) Ursanes(ii) Friedelanes(iii)Quinone Methides(iv) Sterols(v)20Table 1 (cont.)Compound Reference-^SourceBiological Activity mice (m)^human cell culture (c)wholeplantcellculture(ii)58a - amyrin25, 52 0 ±59triptotriterpenic acid C56, 59, 60 + 060regelin56, 61 + 061regelinol56, 61 + 0(iii)62polpunonic acid24, 25, 37 + +63salaspermic acid55, 62 + +643,24-dioxofriedelan-29-oic acid55 ± 065- - -25 0 -I-(iv)66tingenone24, 25, 63,64,65+ ± cytostatic (c)672213-hydroxytingenone24, 25, 64, 65 0 + cytostatic (c)68celastrol(tripterine)1, 7, 9, 24,55 +± immunosuppressive (c)cytostatic (c)(v)6913-sitosterol24, 25 ± +Table 1(e) (cont.)^Triterpene Oleananes(i) Ursanes(ii) Friedelanes(iii)Quinone Methides(iv) Sterols(v)21Table 1^(cont.)Compound ReferenceSourceBiological Activitymice (m)^human cell culture (c)wholeplantcellculture(i)70celacinnine66 + 071celabenzine66 + 072celafurine66 + 0(ii)73wiforonide50 + 074neotriptonolide51 + 0Table 1(f)^Spermidine Alkaloids(i) Butenolidesbi)22(V) MEDICAL USES OF TRIPTERYGIUMThe pharmacological activities of these compounds and those of compounds yetundiscovered combine to produce the medicinal properties of whole plant extracts. In thepreparation of a medicinal extract from the plant, a simple separation of compounds is availablesince the distribution of natural products is not uniform. The triptofordins and alkaloids are foundmainly in leaves, stem and root bark, while the diterpenes and triterpenes occur in the root corel ,3, 4, 6, 18. Since the leaves and root bark demonstrate a high level of toxicity, pharmaceuticalpreparations primarily utilize the root core. The lower toxicity of root core preparations allowthem to be administered in higher dosages than whole root extracts. The basic Tripterygiumextract is prepared by boiling 15 to 25 grams of root core in water or by soaking 2 to 4 grams inethanoll , 3 . The resulting solutions provide one daily dose for rheumatoid arthritis treatment. Acommercially refined preparation called GTW is produced by extracting the root core into hotwater and then into chloroform. The extracted mixture is refined by column chromatography toproduce GTW which has an unknown composition save that it contains glycosidic compounds*,diterpenes and triterpenes. The extract of 1.5 to 2.3 kilograms of root core provides the 60 to 90milligrams GTW prescribed as a daily dosages.The extracts of Tripterygium root core display anti-inflammatory / immunosuppressiveproperties due to the following physiological mechanisms: capillary permeability and theproduction of humoral inflammatory mediators are decreased and cell-based inflammation andimmune responses are suppressed. These effects are due to an inhibition of the generation andactivation of T-cells (the main regulatory agents of the immune system). Antibody production isalso blocked, possibly another result of a lowered T-cell activity 4, 67 .* GTW from: "glycosides of Tripterygium wilfordii"23GTW was prescribed at 60-90 mg/day to treat rheumatoid arthritis (equal toapproximately 1.0-1.5 mg/kg/day). The single dose toxicity (LD50) * in mice was 160 mg/kg,while in dogs 10 mg/kg/day caused chronic lethargy and weight loss'. This demonstrated aneffective dosage approximately six times lower than the harmful dosage.A smaller dosage of 20 mg of GTW per day was sufficient to produce infertility in humanmales. After two months at this level, the sperm number was reduced by 200 times and spermmotility was eliminated. Reversible damage to sperm generating cells accompanied the infertilityobserved in animals. Immunosuppressive side effects were not seen in human subjects at thislevel of GTW while higher dosages had produced an increase in secondary infections. Malefertility was totally recovered after two months without the drugl , 4, 15, 68 .Triptolide (j) is the only pure compound that has been tested on human subjects and itwas shown to be effective in treating rheumatoid arthritis (RA). This property was revealed whenthe majority of the anti-RA activity of Tripterygium was extracted into ethyl acetate and thetriptolide isolated from this extract demonstrated a high level of activity. Treatment with puretriptolide impaired heart action in several patients, suggesting that some other componentspresent in GTW enhance the activity of triptolide to keep the dosage below a harmful leve1 40 .Triptolide has a strong anti-leukemic activity in mice. An injection with triptolide (at 0.1mg/kg intravenous 12, 13 or at 0.25 mg/kg intraperitonea144 ) greatly extended survival time aftermice were injected with leukemic cells. The LD50 for triptolide (intravenous injection of mice)has been determined to be 0.8 mg/kg for a single dose and 0.16 mg/kg/day over seven days withconcurrent degeneration of heart tissue and bone marrow'. This shows that the toxic dosage isvery close to the therapeutic dosage and thus triptolide has not been applied as an anti-leukemicpharmaceutical for human patients.* LD50 ; the dosage lethal to 50% of test animals24.... ....... Is 'HtriptonideThe specific pharmacology of triptolide involves a strong inhibition of T-cell activity(cell-mediated immunity) that can be observed with mouse spleen cells in vitro at 50 ng/ml(0.14 P4), a cytostatic activity observed in human cell cultures at less than 3.0 µg/ml (8.3 gM)and a cytotoxic activity against human carcinoma cell cultures at 1.7 ng/ml (0.005 gM)13, 41 . Aninhibition of antibody production occurs in mice at 1.0 mg/kg (intraperitoneal) 39 which couldresult from the impairment of T-cell activity combined with cytostatic effects.A mechanism for the action of triptolide has been demonstrated by Kupchan andSchubert 13 : triptolide may be able to modify the activity of cell growth regulating enzymesthrough the alkylation of thiol groups in cysteinyl residues. The alkylation process was modelledby opening the electrophilic 9,11-epoxide of triptolide and tripdiolide with propanethiol (pH 7.4)to produce the thio-ether at carbon 9. This opening can not occur without the donation of ahydrogen bond from the hydroxyl at carbon 14, as in the case of triptonide. The significance ofepoxide opening is supported in that triptonide lacks anti-leukemic activity.As well as the 9,11-epoxy-14P—hydroxy system, triptolide also contains an electrophilica-methylene-y-lactone. As a class these lactones have demonstrated anti-tumor activity 13 . Theepoxide and lactone systems must both remain intact for triptolide analogs to exhibitanti-leukemic or anti-tumor activity 69, 70 .25METHODS OF YIELD OPTIMIZATION IN PLANT CELL CULTURESWhen a potential pharmacological application is found for a natural product, medicaltesting is begun and a large quantity of pure compound must be isolated. Several milligrams issufficient for in vitro analysis and one hundred milligrams will supply a study on a population ofmice, but gram quantities are often required for testing on human subjects as at least a dozenpatients must be treated for a period of several months.Tripterygium is harvested from the forests of southern China. Word of the plant appearedin North American publications after farmers, gathering the roots for insecticide, had causedmajor landslides8 . Individual natural products are very dilute in the plant and so a large amountof material is required. In the example of tripdiolide, an isolation from dried root core of only0.001% (10 mg/kg) is reported 12 along with 0.0005% from dried stem and leaves42 .The level of natural products is affected by plant growth conditions such that the poorestenvironment can result in the highest yields. One experiences this effect with cultured food cropsthat display a milder flavour than the wild counterpart. Adverse conditions that are stressful tothe plant increase the level of metabolites thought to act in defensive roles.While the metabolite levels of cultivated crops can be manipulated through the growthconditions, the laboratory is a superior environment for this method. Kupchan's initial exposureof the anti-leukemic compounds of Tripterygium 12 initiated our group's interest in using plantcell cultures as a system for enhancing tripdiolide production and screening for further activecompounds. Tripterygium cultures produced tripdiolide in 36-times the natural level onceproduction was optimized (Kutney et al. 71 ). Tripdiolide, assayed by TLC-fluorimetry, was foundmainly in the nutrient medium after cells had been filtered out. This tripdiolide amounted to0.036% of the whole culture dry weight, a level reached in 25-35 days compared to a crop time26of at least a year for whole plants. Seventy-eight liters of cell suspension (approximately 860 gdry weight) provided the isolation of 200 mg of pure tripdiolide (and 179 mg of pure triptolide)following several column chromatographies (a 0.023% yield of tripdiolide25). Two other groupshave developed suspension cultures of Tripterygium72,73 but the maximum yields of tripdiolidewere relatively low ( below 0.01 % ).While most of the commercially available natural products are currently isolated fromplants, the drawbacks of this production method include a long growth time, difficulties inobtaining ton quantities of plant material and a general low yield which can be further decreasedby climatic conditions.Cell cultures offer the advantage of controlled growth conditions and allow mediaoptimization for increased metabolite production as well as a ready selection of high yielding celllines. The lack of extensive tissue formation in cell suspensions also provides a facilitated accessto cells for enzyme isolation and the study of biosynthetic pathways.Three basic methods for manipulating Tripterygium cultures to produce higher productyields are currently used by our laboratory. The primary method is alteration of nutrient andhormone levels to stimulate a metabolite production cycle. The second, a technique calledelicitation, is a method used to stimulate accumulation of antimicrobial compounds by activationof the infection resistance process. The third method is the biotransformation of syntheticprecursors. In a successful biotransformation one introduces a large quantity of a compound(similar or identical to a natural biosynthetic intermediate) into cell cultures and the plant enzymesystems convert it to a more elaborate product. The following text provides a description of thesethree methods.27(I) NUTRITIONAL STUDIES ON CELL CULTURES OF TRIPTERYGIUMThe natural products of Tripterygium listed in figure 1 are classified as secondarymetabolites (save (3-sitosterol, 69 ). Plant biochemistry can be divided into two systems. Thecentral one is primary metabolism which produces energy as well as the protein, carbohydrateand lipid compounds required in structural and homeostatic roles. Primary metabolic processesare mainly identical among various plant types. Of a lesser importance are the secondarymetabolic processes since they produce compounds that are not essential for plant growth. A lackof demand has allowed the evolution of secondary pathways along various lines in different planttypes. Assorted functions include the production of pigments, insect antifeedants, cytostatic orantimitotic compounds (which control infectious pathogens), toxins and insect pheromones.While these roles have been determined for some compounds, many others have no knownbiological activity. A diversity of secondary products allows one to identify genetic relationshipsbetween plant types according to the catalog of compounds produced. This is the technique ofchemotaxonomy. Tripterygium is part of the family Celastraceae and many of its secondaryproducts are observed in other family members such as Euonymus, Celastrus and Maytenus.Examination of figure 1 reveals that most of the secondary metabolites from Tripterygium areterpenoid compounds. The process of terpene biosynthesis will be discussed later in thisintroduction.In plant cell cultures as well as whole plants, primary and secondary metabolic activitiesare maximized at opposite stages of the growth cycle. Initially, a plant uses energy and chemicalprecursors for rapid growth. Then if a disease occurs, growth is inhibited while resources arediverted to secondary metabolism which provides compounds to resist further infection. When aplant has grown to reproductive readiness, growth slows and secondary metabolism is activatedto produce compounds that may discourage foraging insects or animals. The inverse relationshipbetween the growth cycle and secondary metabolism is carried over by plant cells grown in28culture, making metabolite production a two stage process. Cells are first grown up to a highmass and then stimulated to convert resources to the synthesis of secondary compounds.As described in Kutney et al.24 , the first tripdiolide from cultures occurred in a level onlyslightly higher than that in the whole plant. A program to optimize tripdiolide production wassubsequently carried out71 . Tripdiolide was determined both directly by quantitative TLC-fluorimetry and by inference from the cytotoxicity against KB cells (human carcinoma).To begin the plant cell cultures, small leaf cuttings from Tripterygium were incubated ona solid nutrient agar. Some of these explants developed a growth of callus tissue, a clump ofrapidly dividing cells that lacks obvious structure and often occurs in plants at the site of injury.Callus lines with detectable tripdiolide were used to start suspension cultures by a transfer tostirred liquid medium. The suspensions were maintained by successive subculture at the end ofeach growth cycle with cells dividing rapidly until the point of nutrient depletion. This methodprovided a somewhat heterogeneous population of cells called a clonal variant which couldreproduce for an indefinite period through mitotic division . One clonal variant of the manyproduced was selected for its stable growth and the ability to produce tripdiolide. This line wasthe TRP-4a variant and has been maintained in callus and suspension culture for the past eightyears. Over this time the TRP-4a variant has altered from a stage where cells aggregated in largeclumps with some root formation and has become a smooth suspension of small cell clumps withthe appearance of a fine sand and a pale green to tan coloration.The nutrient mixtures used to grow Tripterygium are based on either of two standardmedia, PRL-4 or MS(2% sucrose). These formulations supply all growth requirements for plantcells raised in darkness; sucrose is the carbon/energy source and simple salts supply nitrogen,phosphorous and sulphur for the synthesis of amino and nucleic acids. Tripterygium cultures aregrown in the dark at 25°C in half-liter stock cultures swirled in erlenmeyers (shake flasks) or inten liter production cultures grown in aerated bioreactors. Sterile conditions are maintainedthroughout the growth process.29The following additives, compounds that act as plant hormones to alter the cell state, werescreened with respect to their effect on growth and the production of tripdiolide.Additive * ActivityI indole-3-acetic acid auxin(accelerates cell enlargement)D 2,4-dichlorophenoxyacetic acid auxin (synthetic)(accelerates cell enlargement)NA 1-naphthaleneacetic acid auxin (synthetic)(accelerates cell enlargement)K kinetin cytokinin(induces cell division)Co fresh coconut milkundefined mixture containingcytokinins, facilitates cell growth inculture mediumLeaf explants were found to initiate callus growth most readily on a solidified PRI2Coagar. Callus and suspension cultures were then maintained with PRD2Co. These media are thebasic PRL-4 formula with the listed additions. I and D have similar biological activity; I is anatural product which is degraded rapidly in solution while D is a stable synthetic substitute.Coconut milk has many growth enhancing components which support cells in culture, thoughrecent results (unpublished; Biological Services, UBC) have demonstrated good growth on acompletely defined medium after a gradual reduction in coconut milk over many subcultures.PRD2Co supported a rapid and stable growth (requiring 14-18 days for stock suspensions) buttripdiolide was not produced by these cultures. Other media were screened with respect totripdiolide production and it was observed that maximal tripdiolide was produced followinginoculation of MSNA0.5K0.5 medium (MS medium plus additions) with a stock culture that had* The level added to media (mg/L) is denoted bya subscript (eg D0.5). Co is used at 10% v/v .30been raised to the end of growth phase in PRD2Co. From the time of this inoculation, tripdiolidereached a peak level after 25-35 days, which coincided with the end of the growth phase. Torecover the tripdiolide, cells and medium were separated and extracted with ethyl acetate.Growth was slower in MSNA0.51C0.5 and yielded a lower biomass, while tripdiolide in thefiltered culture medium reached levels up to 4.0 mg/L (0.036% of culture dry weight), more thanthirty times the yield from plants. The MSNA0.51(0.5 medium shares many basic constituentswith the PRD2Co formulation save the replacement of growth enhancing coconut milk with apure cytokinin, kinetin. D and NA have equivalent activity as auxins. MSNA0.51C0.5 has a greateroverall concentration of nutrients, including a doubling in the available nitrogen as well asincreased calcium, decreased thiamine, the presence of glycine and small variations in themicronutrient levels (Mn2+, Zn2+, Cu2+, Co2+, etc).Increases in the individual components of MSNA0.5K0 .5 have demonstrated that noparticular additive is responsible for the increased tripdiolide production 71 . It is a very goodgrowth medium, yet the transition from PRD2Co to MSNA0.51(0.5 results in a decreased biomassyield. The synthesis of other diterpene and triterpene secondary metabolites is activated alongwith that of tripdiolide24, 25 with a visible darkening of cultures owing to the appearance oforange quinone methides and dark compounds which may be polyphenols.The production of previously undetected secondary metabolites and a slower, lowbiomass growth are consistent with the hypothesis that a profound shift in metabolism occurswithin the cultures on exposure to MSNA0.51C0.5 : a transition from growth phase to a productionphase, not caused by a specific additive so much as by a sudden change in the environment.In the past several years that MSNA0.5K0.5 has been used to produce tripdiolide, the biomassyield has been observed to vary from culture to culture. Following extraction, poor yields oftripdiolide were obtained from cultures exhibiting the low and high extremes of cell growthwhile the intermediate growth cultures produced maximal tripdiolide. This appears consistent31with a model in which stress on the culture is required to increase the population of cells in aproduction phase, to an upper limit above which the cells are not able to function.MSNA0.51(0.5 medium stimulates the more mature "production" phase in Tripterygiumcells in a fashion that cannot be directly linked to a particular culture component or physiologicalmechanism. Elicitation is an alternate method to increase the level of secondary metabolites. Thetechnique appears to specifically activate disease resistance responses and in theory will produceonly those metabolites involved in fighting infectious pathogens.What is known of themechanism of elicitation is reviewed in the following section.32(II) ELICITATIONSome plant-derived secondary metabolites have antimicrobial properties which aredisplayed by the isolated compounds as well as from within the intact plants. With many plants,exposure to a pathogenic organism results in the rapid accumulation of these compounds and thislends resistance to spreading of the infection 74 . This type of metabolite induction is termedelicitation. Elicitation has been used to stimulate plant cell cultures such that the yield ofantimicrobial metabolites was increased. The techniques used for this purpose will be reviewedfollowing a brief summary of plant pathogen interactions.(i) Disease Resistance in PlantsThe laboratory applications of elicitation have been modelled mainly on thephysiological response of plants following exposure to a pathogenic fungus. Similar responsesare seen when the pathogen is bacterial, and to a lesser extent, when it is viral. During asuccessful infection process, a fungus penetrates the wall of a plant cell, digesting the wall andthen the cellular contents. The plants defences are challenged in this order :1. waxy cuticle or bark2. cell wall3. constitutive antimicrobial compounds4. defences induced by the pathogenInfection through the cuticle, a physical barrier of hydrocarbon wax, is usually fortuitousbut the fungus has specific resources for penetrating the underlying plant cell wall. This cell wallis a laminar array of polysaccharides containing a small amount of protein and lipid. Theoutermost layer, cellulose, is composed of (1 - 4) linked 13-D-glucose subunits. Beneath this,there is the pectin layer made up of galactose, rhamnose, arabinose and galacturonic acid33polymers and copolymers. The patterns of linkage and branching in these repeating sequencesare common to many various plant types. It has been found that every type of plant cell wallpolysaccharide can be digested into soluble oligomers by specific hydrolytic enzymes isolatedfrom plant pathogens 75 . During an infection the plant cell wall acts to delay exposure of thevulnerable cell membrane, meanwhile the plant uses further resources to kill the fungus or inhibitits multiplication.A plant's defenses can include constitutive antimicrobial compounds stored either in thecell wall or in intracellular compartments 74 . Maintaining this defence requires the continuousexpenditure of energy and metabolites. The more economical defence is induced only onexposure to a pathogenic organism. Common components of a plants inducible defence are :i) Phytoalexins^low molecular weight secondary metabolites withantimicrobial activity produced in response to animposed stressii) Qtll wall^- polymer deposition (callose or lignans)reinforcementiii) enzymes attacking^- cell wall hydrolasesthe pathogeniv) necrogenesis^- self destruction of plant cells following fungalexposureWhen plant tissue is exposed to a fungal pathogen, rapid alterations may be observed inthe plants respiration and hormonal balance76 . A typical result is cell wall thickening. In pineseedlings, a layer of callose ( (3-(l -. 3)-glucan polymer) was produced precisely at the boundaryof exposure to a fungus and this was detected only sixty minutes after the inoculation 77. Suchexposure may also stimulate a plant to rapidly accumulate fungal-specific cytotoxic or cytostaticsecondary metabolites.34(ii) PhytoalexinsExamples of secondary metabolites produced by various plants following fungalexposure are presented by Eilert et al. 78 and Brodelius79 . If compounds generated in this mannerexhibit antifungal activity and are present in healthy plants in only trace amounts, they areclassified as phytoalexinsThe phytoalexin families include isoflavonoids, terpenes, polyacetylenes, stilbenoids andalkaloids. A good demonstration of the role of phytoalexins in plant defences is the response ofsoybean seedlings to fungal invasion 74 . The antifungal isoflavonoid glyceollin I is absent incontrol seedlings. Following a root-dip of the seedlings in fungal inoculum, this compound wasproduced rapidly and specifically at the site of infection. After 8 hours, an intracellularconcentration of 0.6 mM was reached, and this was found to produce 90 % inhibition of fungalgrowth in vitro. The fungal infection was restricted to the exposed tissues; both the treated andcontrol plants could be grown to maturity. When the seedlings where pre-treated with aninhibitor of isoflavonoid biosynthesis, inoculation resulted in widespread fungal invasion whichstopped seedling growth. The mechanism of antifungal action for the glyceollin family involvesdirect disruption of fungal cell membranes 80 .(iii) ElicitorsAgents which cause the activation of plant defences (eg. phytoalexin production) aretermed elicitors. While the term is normally used to describe activation by pathogenic organisms,other elicitors include physical stress, chemical agents and electromagnetic or ionizing radiation.A review by Darvill and Albersheim81 is an extensive introduction to the elicitation ofphytoalexin production. In one study, Eilert et al. 78 discovered a 300 fold stimulation ofsanguinarine production from poppy cell cultures using a fungal elicitor (Botrytis). Thisantifungal alkaloid reached 2.9 % of the cells dry weight. Several mechanisms which are thoughtto be active in the elicitation of plant defences are outlined in the following scheme.35Plant cellself- I ysisFungal hydrolasesdigest plant cell wallPlant hydrolasesdigest fungal cell wall1 IunknownagentsreleasedsolubleplantoligosaccharidessolublefungaloligosaccharidesIc_54pathogenicfungusplantcellIMUTUALRECOGNITIONELICITATION OF DEFENCESIN NEIGHBOURINGPLANT CELLSFigure 5^The Activation of Elicitation36This figure reveals that the effects of an eliciting fungus are brought on by thetransmission of by chemical signals in the form of soluble oligosaccharides. This allowselicitation of neighbouring plant cells that have not been infected and therefore possess greatermetabolic capabilities. It also means that a particular plant will respond to various types of fungiwith the same set of defences (provided the fungi have similar cell walls). The two sources ofsoluble oligosaccharides have been demonstrated in the following experiments:(a) pathogenic fungi synthesize a complement of hydrolytic enzymes able to degrade theplant cell wall75 and the soluble oligosaccharide products have been shown to be elicitors. Whenpectin hydrolysates from cultured carrot cells82 were re-introduced to parent cultures, theproduction of the phytoalexin 6-methoxymellein was initiated within 24 hours (also observedwhen only the hydrolysis enzyme, pectinase, was added to cultures). By this activationmechanism, plants are responding directly to damage caused by a fungus.(b) alternately, some plants cells store hydrolases in their outer wall that are specific forfungal wall polysaccharides. Contact with a fungus brings the release of soluble fungaloligosaccharides which elicit the plants defences. To test this process, Darvill and Albersheim 81,83 prepared an acid hydrolysate of polysaccharides from a fungal pathogen. The resultingoligosaccharide mixture stimulated phytoalexin production in soybean. Fractionation of thishydrolysate yielded the most active component, a seven unit glucan (polyglucose) with 1341 — 6)connectivity and 0-(1 3) branching:glu^glu^glu^glu^gluglu^glu37Many fungi contain chitin (polyglucosamine) in their outer cell wall.. The hydrolyticenzyme chitinase, which degrades the polymer to oligomers, can be extracted from some plantspecies. The soluble digestion product of chitin (sold commercially as chitosan) oftendemonstrates elicitor activity with plant cell cultures 84 .Following fungal elicitation, some plants have demonstrated a rapid autolysis describedas the necrogenic response74 . This occurs in plant cells directly exposed to fungus and appears tocause the elicitation of more distant cells. Since mechanical cell disruption does not bringelicitation it is likely that necrogenesis is a specific physiological process for amplifying anelicitor signal.(iv) Mechanism of Elicitor ActionProtein receptors capable of binding oligosaccharides have been found on the surface ofplant cells. Involvement of these receptors in the transduction of elicitor signals wasdemonstrated by Ebel and Grisebach with soybean seedlings74 . A P-glucan elicitor was purifiedfrom a fungal hydrolysate and labelled with tritium. Cells from the seedlings had high affinitysurface binding sites for this oligosacccharide and the radioactivity was exchangeable in thepresence of the unlabelled glucan. Analogous glucans with different chain length or branchingpattern had a decreased ability to compete for binding sites and this binding affinity in vitro wasproportionate with elicitor response in whole plants. Surface receptors for the glucan elicitorisolated by Darvill and Albersheim ((b) on previous page) were also demonstrated 81,83 . In thesestudies, the extent of elicitation was dependant on the ratio of elicitor amount to cell biomassrather than on the elicitor concentration, thus plant cell receptors appear to bind elicitor tightly.The mechanism for transduction of the elicitor signal within the interior of plants cellshas not been identified. Calcium addition to the cell growth medium increased the sensitivitytowards elicitation74 , thus oligosaccharide receptors may control calcium influx. The target forelicitor stimulation has been identified as gene activation by several groups74,82,85 who38134demonstrated that phytoalexin production is preceded by the synthesis of mRNA. As well,elicited increases of enzyme activity in the phytoalexin synthesis pathways was blocked byinhibitors of protein synthesis. In overview, elicitor stimulation leads to increased genetranscription producing a high enzyme population in the phytoalexin synthesis pathways.(v) Elicitation of Triterpene Acid BiosynthesisExperiments in our laboratory (to be detailed in this thesis) have demonstrated elicitationof triterpene acids from Tripterygium . A report in Phytochemistry (1989) by Threlfall andcolleagues also describes the elicitation of cell cultures to produce triterpene phytoalexins86 . Thetropical shrub Tabernaemontana divaricata was cultured and elicited with a Candida albicanspreparation (a yeast-like fungus parasitic on plants). Since Threlfall et al.'s findings wereanalogous in several ways to results from Tripterygium, I will give their work a brief review.The elicitation from Candida caused an inhibition of cell culture growth followed by theproduction of ursane (triterpene) phytoalexins to a maximum of 1.4 % of culture dry weight :R1 R2 R3 R4i )^CO2H H CH3 CH3ii)^CO2H OH CH3 CH3iii )^CO2H H CH2OH CH3iv )^CH3 OH CH2OH CO2HFigure 6^Elicited Ursane Triterpenes39Thirty-six hours after the elicitation, the capacity for synthesis of triterpenes hadincreased five-fold, assayed in vitro by the activity of squalene synthetase (an enzyme requiredfor triterpene and phytosterol production).Threlfall also used an elicited population of enzymes to carry out phytoalexinbiosynthesis in vitro using disrupted cells 86. A plant cell suspension culture was exposed toelicitor and after a short period a cell free enzyme extract was prepared. These enzymes, togetherwith added cofactors, were able to convert radiolabelled isopentenyl pyrophosphate (IPP) into anursane triterpene phytoalexin. In parallel controls using non-elicited cells, the label was onlyincorporated into phytosterols, which lack phytoalexin activity. Radiolabelled phytosterols werenot detected in the elicited transformation, revealing that elicitation inhibits phytosterolproduction while activating phytoalexin production. Phytosterols are used for cell membraneformation. Inhibition of their synthesis could be responsible for the retarding effect of elicitationon cell growth. A summary of the effects of elicitation on metabolite production is presented onthe following page.40* IPPGPP indole alkaloidsFPPsesquiterpeneslvsqualeneGGPP ►^ diterpenes^* squalene-^ * triterpeneamyrins2,3-oxide phytoalexinsphytosterolsFigure 7 Partition of 1- 14C-IPP in Cell Free BiotransformationsUsing Elicited Cultures Inhibited (^), Enhanced ( 11 ), Isolated with radiolabel (*)IPP - isopentenyl pyrophosphateGPP - geranyl pyrophosphateGGPP - geranylgeranyl pyrophosphateFPP - farnesyl pyrophosphateIn this model, elicitation creates a high activity of squalene synthetase, depleting the poolof FPP and thus decreasing the rate of sesquiterpene and diterpene synthesis. Indole alkaloidscontaining terpene components were not produced by the elicited cultures although theyaccumulate in non-elicited preparations. This may result from a depletion of GPP brought aboutby an increase in the metabolite flux towards squalene. At the branch-point following squaleneoxidation, inhibition of phytosterol synthesis directs squalene-2,3-oxide exclusively towardspentacyclic triterpenes.41In our laboratory, the triterpene acids were selected as targets for elicitation due to theiranti-inflammatory activity demonstrated in small animals (Table 1, page 20). This was part ofour larger interest in isolating the anti-inflammatory, immunosuppressive, anti-fertility andanti-leukemic agents which give Tripterygium medicinal value. Threlfall et al. have provided aclear example of increases in non-essential triterpenes brought about through fungal elicitation.While it has not yet been proven that high triterpene content in whole plants is effective ininhibiting fungal infection, it seems very likely that the specific triterpenes synthesized afterfungal exposure are part of the natural resistance process and thus function as true phytoalexins.Anti-inflammatory activities from oleanane triterpene alcohols and hydroxy-acids havebeen revealed by Professor Shoji Shibata's group in a number of papers 87 -91 . These compoundsare structurally similar to the triterpene acids from Tripterygium, and were isolated in a study oftraditional medicines parallel to the work on-going in our research group. The role of triterpenesas accessible plant phytoalexins and as compounds with medicinal potential is becoming wellrecognized. Further research will likely lead to their development into commercialpharmaceuticals.42(III) BIOTRANSFORMATION OF SYNTHETIC PRECURSORSWhen plant cells are used to produce a compound, one limitation on yield is thatsecondary metabolite levels are regulated through enzymatic control. This is especially relevantfor a toxic compound which must be compartmentalized within the cell. It is generally necessaryto the cell that regulation of synthesis occurs early in a pathway to avoid the accumulation ofintermediates. For example, in the mammalian synthesis of cholesterol regulation occurs at thesix carbon stage where the transformation of hydroxy-methyl-glutaryl-CoA to mevalonic acid,thefirst dedicated terpene intermediate, is inhibited by the end product, a cholesterol ester. This typeof regulation hinders the production level from bioreactor cultures, where it would be nodrawback for cell death to occur from excess product accumulation. Regulation can be bypassedif one obtains quantities of a precursor that enters the synthesis after the regulation step, in whichcase high levels of biotransformation can be accomplished. Another attractive process is the useof soluble enzyme systems from cell-free plant extracts to carry out a biotransformation. Thesesystems require only a few added cofactors, such as oxidizing or reducing agents to drive non-spontaneous processes. The major advantage here is simplified product recovery. The workcarried out by Professor Kutney provides several examples of these methods92 . In one case, cellcultures of Catharanthus Roseus provided enzyme extracts to couple indole precursors into a bis-indole product in an efficient synthesis of vinblastine. Also, a similar enzyme preparationeffected a ring closure reaction in the production of the polycyclic phenol etoposide. Bothproducts are widely used, valuable cancer therapy agents currently isolated from plants.To utilize plant enzymes as synthetic tools, the natural biosynthetic intermediates of aproduction pathway must be determined. After this, an accessible intermediate can be targetedfor laboratory synthesis. Terpenes share a biosynthetic pathway which forms isopentenylpyrophoshphate (IPP) from acetic acid (as a thioester coenzyme adduct). All the various classesof terpenes are then generated from 1PP93 (figure 8).43Farnesyl PP1 7-- IPP(15+5) sesquiterpenesGeranyl PPI 7-- IPP(10+5)diterpenesOPPGeranylgeranyl PPOPPmonoterpenestriterpenes(Squalene)1O^00-P —O-P-0°OH^OHIsopentenyl Pyrophosphate ( IPP )IOPPDimethyl allyl PPetc.Figure 8^Biosynthesis of Terpene ClassesThe phosphate esters of terpenols are either incorporated directly into other molecules orcyclized to yield the terpene classes of figures 1 -4 . The sesquiterpene agarofurans are producedthrough the cyclization of farnesyl pyrophosphate and the diterpene abietanes from4411abietanes(oxidation)(OPP_^labdanesOPPgeranylgeranyl PPOPPpimaranesgeranylgeranyl pyrophosphate. The oleanane, ursane, friedelane and quinone methide classes oftriterpene are synthesized from squalene which is produced through a dimerization of farnesylpyrophosphate. The biosynthesis proposed for the abietane diterpene dehydroabietic acid (whichis produced by Tripterygium) follows this path:co2Hdehydroabietic acidFigure 9^Biosynthesis of Abietane Diterpenes45In the plant cell, these processes occur on an enzyme surface where only one opticalisomer is formed. The enzymes are thought to catalyze terpene synthesis by supplying electrondonating systems which stabilize carbocations or act as bases in an elimination process.To elucidate the synthesis of tripdiolide, one may work forward from an earlyintermediate such as geranylgeraniol or backward from the end products using late intermediateanalogs. In the first method, one introduces radiolabelled geranylgeraniol for a short incubationwith plant cells then extracts the products, most likely containing subsequent intermediates inlabelled form. With this approach, Misawa et a1:72 determined that the addition of farnesol to cellculture medium caused a small increase in the level of tripdiolide produced.The other method of analysis, working backward from the product, requires aretrosynthetic analysis guided by any known intermediates. One synthesizes the labelled form ofa possible precursor which closely resembles the end product and then tests for its incorporationinto the end product. Attempts by our group demonstrated that no tritiated dehydroabietic acid isincorporated into tripdiolide, therefore it is likely that tripdiolide biosynthesis branches from anearlier abietane-type intermediate.The late intermediate approach was chosen by our group since a very usefulbiotransformation, the epoxide incorporation, is likely to occur in a final stage of tripdiolideproduction. The triptolides have two main elaborations on the abietane skeleton, the butenolide atring A and the hydroxy-triepoxy system of ring C. These functionalities are isolated on thecarbon skeleton, making it likely that they are formed sequentially rather than concurrently. Thebutenolide is likely formed first due to its greater stability in solution. Support for this hypothesisis provided by the abeo-abietane compounds isolated from Tripterygium which display thecompleted butenolide at ring A but have only phenolic systems at ring C; conversely, epoxidecompounds lacking the butenolide have not been isolated.In the synthesis of potential precursors to tripdiolide, it was envisioned that a butenolidecompound could be readily prepared from dehydroabietic acid which is in turn an available46component of tree resins. A program was begun to synthesize butenolides with suitableunsaturation at ring C for biological epoxide addition. In this respect, the presence ofdehydroabietic acid in culture medium3 suggests that the plant metabolism can recognize anaromatic ring.Several other groups have developed synthetic strategies to the butenolide portion of thetriptolides. These processes fall into two main classes, the synthesis of racemic butenolide fromsmall achiral starting materials and the synthesis of optically pure butenolide from(1)-dehydroabietic acid. While the racemic product could not be used pharmacologically, it couldbe used in biotransformations to (1)-triptolides, though half of the compound would remainuntransformed.Synthetic planning to the butenolidesmust take into account a facile tautomerizationto the A/B-cis isomer. The trans ring junction isstable in aqueous solution at neutral pH whilein alkaline conditions a complete conversion tothe cis junction takes place (Berchtold's group 70 ,using methoxide in methanol for 48 hours atroom temperature). Alkaline or acidicequilibration though the extended enolate musttherefore be avoided.75 18(4 - 3) - isodehydroabietenolideA synthesis of racemic butenolide by vanTamelen and Leiden 94 is one of the most facilepathways yet designed (figure 10). It results in 15% yield after 12 steps with only 4 purificationsrequired.This is a biogenetic type synthesis inspired by the biosynthetic cyclization ofgeranylgeraniol. The key ring-forming step is the cyclization of a diterpene-like phenolderivative ( A) which forms both A and B rings with the correct trans junction.47SnCI4CH2Cl2 , 0°1.5h^ 010„Et0 HOOCH3A+BrOCH3 NaH THF, 0°- r.t.99%■(1) Ba(OH)2Et20/H20, 90°17h, 92%(2) LiAIH 4 , Et 20, 0°+Et0.....nr,0 0LiH, DMF75 .90%(1) LiBr/PBr3collidine/Et20, -40°- 0°(2) ZnBr2 , Et20, 0°70%MeS02Cl/Et 3NCH2Cl2 , 0°(1) mCPBACH2Cl2, r.t.(2) n-BuLi/i-Pr2NH, -78°51% from AFigure 10^Synthesis of Racemic Butenolide by vanTamelen and Leiden (Biogenetic)48This and most other syntheses of butenolides lead up to a C 14-phenol product whichfacilitates the synthetic incorporation of the epoxide system. The absence of a phenolic hydroxyl(that is usually protected as an ether) would cause no apparent difficulties in these sequences.Another racemic synthesis by Garver and vanTamelen95 (figure 11) utilizes a trans-decalone for an A/B-ring nucleus. First ring C is added with a Diels-Alder reaction then ring A ismodified to accept a one carbon unit at C3 to prepare for the appropriate butenolide, a processoutlined in figure 11. The C3 addition occurs through a 2,3-sigmatropic rearrangement of acarbene intermediate generated by loss of methanol from a dimethylformamide mixed acetal.A valuable tool for ring A transformations is introduced here at two stages of butenolidesynthesis. This is the oxidation of an olefin to the rearranged allylic alcohol through epoxidationfollowed by ring-opening with base.49HO"SOCl2/pyrEt20, 0°, 2h83%OCH 3N-1-0CH 3/ Hxylene, reflux4A sieves (CH3OH)3d, 80%OCH3(1)KOAc, DMSO75°, 24h,(2)NaOCH3, CH3OH25°, 2h70%1M HCI (aq), THF25°, 10 min80%(1)mCPBA, CH2Cl225°, 30h, 100%(2)Li N(Si(CH3)3)2THF, 0°• 25°, 2h(1)mCPBA, CH2Cl225°, 6h, 100%(2)LDA, THF25°, 24h, 91%Figure 11^Synthesis of Racemic Butenolide by Garver and vanTamelen (Decalone)50The butenolide was also synthesized under the direction of Berchtold (Lai et al. 7°; figure12) using a tetrahydronaphthalenone nucleus for rings B and C, then appending ring A throughalkaline condensations. In this process, the formation of a large quantity of the more stableAB-cis isomer was unavoidable during the conversion of a 04,5 olefin intermediate to thedesired A3,4 olefin. The proper trans fusion was recovered by oxidation to a dienone and thencatalytic hydrogenation (predominantly exo to the B-ring boat conformation).vanTamelen et al.96 carried out a chiral synthesis of the butenolide using(1)-dehydroabietic acid as a starting material (figure 13). After the aromatic C-ring was convertedto the phenol ester at C14, Curtius degradation was used to decarboxylate at C4 followed by Copeelimination to the exocyclic olefin. Oxidative cleavage to the C4 ketone allowed alkylation at C3using formaldehyde. The sequence used to complete the butenolide was a lengthy processdemanding several protections (12% yield from the formaldehyde adduct). Benzyl methyllithiumether was added at C4 and the product selectively oxidized to the correct hydroxy-acid. Therelease of a newer synthesis (figure 10) from the same group allows a more efficient butenolidecompletion if methyllithium is added to the C4 ketone.51(1) NaBH4 , EtOH25°, 2h(2) 2M HCI93%(1) neutral aluminaEtOAc, 25°, 2d(2) p-TsOH, benzenereflux , 2h80-95%mCPBACH2Cl2 , 25°Pd/C, H2 (g)EtOAc, 1.5h63% trans(+ 27% cis)OCH3(1) Et3 N, CH2Cl2 , 25°(2) CH3 SO2 CI / trimethylpyridineDMF, 10°- 25°92%OCH 3Figure 12^Synthesis of Racemic B utenolide by Berchtold et al. (Naphthalenone)52(1) SOCl2 , Bz/DMF, 50° (acyl chloride)(2) NaN3 , H20/acetone, 0° (acyl azide)(3) toluene , 100° (isocyanate)(4) LiAIH4 , THF, reflux (1° amine)(5) HCO2H/H2C0(aq) , reflux (2° amine)(6) (i) mCPBA, CHCI3 , - 20°(ii) Et3N, -20° to reflux40% overall0s04 / Na104AcOH/dioxane/H2020° , 30%HOCH3Li(i) i-Pr2 N Li (ii) H2CO (g)THF, -78°50% ...-^oxidize to aldehyde and continue as infigure 10 with final NaC102 oxidationROFigure 13^Synthesis of Chiral Butenolide by vanTamelen et al. (Dehydroabietic Acid)Tokoroyama et al. developed an alternate synthesis of chiral butenolide 97, 98 (figure 14)from the (1)-dehydroabietic acid derived olefin of figure 13. Attempts at direct carbon addition bysubstitution of a C3 alcohol had been unsuccessful, thus the butenolide was appended throughClaisen rearrangement of a sulphonium ylid produced at the exocyclic methylene. With thecarbon skeleton in place, two methods of oxidation were developed which produced either thebutenolide precursor of triptolide or the C2-hydroxylated butenolide precursor to tripdiolide.53Figure 13, At 02Hdehydroabietic acid45 Se02EtOH / H2OA , 63%SOCl2 Et20 , 25°, 71%HO"PhSH / NaOEtDMSO , 25°PhSCH2CI / t-BuOKDME , -10° , 83%N-chlorosuccinimideAgNO3CH3CN / H2O60%(continued)Figure 14^Synthesis of Chiral Butenolide by Tokoroyama et al. (Dehydroabietic Acid)54(i) LDA , THE , -78°(ii) Mo05 • pyr • HMPA40%02 / rose bengalUV light , 25°acetone / methanol41%AGOSOCl2 , Et2025° , 71%0CH 3ONaOH , EtOH / H2OA,55%N-bromosuccinimideH2O / DMSO25° , 40%NaC102 , dioxane / H2O95%(A)(1) PCC , CH2Cl2(2) CH2N2 , Et20 , 0°(B)(i) t-BuOK , THE , 0°(ii) AcCIFigure 14^(continued)55In our group, interest in butenolide synthesis was directed towards biotransformation ofreadily prepared precursors rather than on a total chemical synthesis of the triptolides. A higheryielding pathway than those previously described would be necessary if the synthesis were to beadapted to commercial production. Also, in the initial studies on the biosynthetic pathway, itwould be necessary to substitute a non-exchangeable atom of the precursor for its radioactiveisotope. To this end, synthetic plans were envisioned to incorporate a single carbon atom into thebutenolide skeleton at some late stage of the synthesis. The reagent used would have to beavailable in radioactive form, such as a 14C isotope; its addition at a late stage would decreaselosses of radioactivity in subsequent reactions.Malcolm Roberts, working with Professor Kutney, developed one such route thatincorporated 14CH3I into the butenolide ring. (A method that had been used previously byGarver and vanTamelen95 to append ring C of the triptolides). In this synthesis 25 (figure 15), anexocyclic olefin was prepared by oxidative decarboxylation of (1)-dehydroabietic acid in one stepusing lead tetraacetate. Ozonolysis to the C4 ketone was achieved in fair yield with by-productsfrom benzylic oxidation. A masked carboxylic acid was added to C3 through alkalinecondensation with carbon disulphide followed by trapping in-situ as the ketene dithioacetal. Thehindered, mild base used for the condensation did not cause isomerization to the A/13-cis form.The subsequent addition of a methylene unit to C4 was a step that could be used to incorporateradioactive carbon. Methyl iodide (available enriched in 14C) and dimethylsulphide were used toprepare a sulphur ylid that added to the C4 carbonyl to form an exocyclic epoxide. At this stage,hydrolysis of the dithioacetal group led directly to the butenolide.56(ii) CH3 I , 25° , 99%CS2 , THF , 25°HCI , CH3OH / CH3CN42% (purified)(i) 03 , - 78°CH3OH / CH2Cl2(ii) Me2S , 66%Pb(OAc)4 , pyrbenzene , reflux--:. H 50%CO2Hdehydroabietic acid45H 3C\S=CH 2 , THF , - 20°ts,H3L0Figure 15^Synthesis of Chiral Butenolide by Roberts (Dehydroabietic Acid)57Prior to the development of the ketene dithioacetal approach, our group had studied theformation of a butenolide through addition of cyanide to C3 of ring A. This addition, followed byelaboration of a C4 hydroxymethyl group would allow hydrolysis of the nitrile product withspontaneous lactonization. This approach is attractive due to the addition of carbon in theappropriate oxidation level for hydrolysis to the butenolide and due to the commercialavailability of 14 C-cyanide. The work that I have carried out in the synthesis of triptolideprecursors utilizes this strategy.KCN NC(1) mCPBA(2) Base(1) KOH , e(2) H3OGINCFigure 16^Planned Butenolide Synthesis (Dehydroabietic Acid)The central objective of this research is to develop large scale production methods for thetriptolides, which demonstrate antileukemic and antifertility properties, as well as for theoleanane triterpenes which have an anti-inflammatory activity. One approach taken has been thesearch for economical precursors that will allow production of the triptolides throughbiotransformation with plant cell cultures. Another approach has been to study elicitationconditions that will activate the biosynthetic pathways to the triptolides or the triterpenes. Theresults from these projects are described in the discussion which follows.58DISCUSSIONMETABOLITE PRODUCTION FROM ELICITATION OF TRIPTERYGIUMThere is currently a demand for pure samples of the pharmacologically active compoundstripdiolide and triptolide which are used in clinical research studies. If testing supports therelease of the triptolides as pharmaceuticals, the demand will be increased and a productionindustry will be established. At present, the richest source of tripdiolide is the TRP 4a cell cultureof Tripterygium wilfordii developed in this laboratory. These cells produce 4 mg of tripdiolideper liter of culture medium in 30 days 71 , 20-36 times more than can be isolated from plants.While the growth time is accelerated from years for the whole plant to weeks in culture,commercial cell culture is expensive and we have continued research on methods of improvingtripdiolide production.Promising results from the literature indicated that the technique of fungal elicitationwould be well suited to these goals. The method was attempted using TRP 4a cultures and itproved successful in greatly enhancing the yield of some metabolites as well as reducing theirproduction time. The initial studies of fungal elicitation described in this paper were aimed atseveral other products along with the triptolides. Foremost of these were a group of oleananetriterpenes, A,B,C and D (51,52,53 and a), first isolated from TRP 4a by Malcolm Roberts 25 .Of these, triterpene D had been isolated from whole plants and demonstrated anti-inflammatoryactivity (see table 1). The linking of this activity to triterpene D was of great relevance as theprimary medical use of Tripterygium has been the treatment of rheumatoid arthritis. Theelicitation of TRP 4a cultures was therefore directed towards improving the production ofoleanane triterpenes, tripdiolide and triptolide.(I) GROWTH AND ELICITATION OF TRP 4A CULTURESStock cultures of TRP 4a were maintained in liquid PRD2Co medium as shake flaskcultures. The 500 ml volumes were grown in the dark and subcultured into fresh medium at 8 %v/v at the end of their growth phase (14-18 days). Small-scale elicitation experiments were also59carried out with cultures prepared in this manner. Large scale elicitations were run in 12 Lbioreactors constructed from glass cylinders with stainless steel fittings. In the bioreactors, themedium, inoculum level and growth conditions were similar to those in shake flasks although airwas bubbled up through the culture while in the shake flasks it entered solely by diffusion. Tosuspend the large cultures, a metal cone with open ends (immersed with large end downwards)was used to channel the rising air. This created a strong current in the medium. The "air-lift"circulation prevented settling of the cells with a minimum of shearing forces. Heated air filters onthe inlet and outlet were used to maintain sterile conditions. Periodic sampling was carried out toassay growth (by refractive index) and culture purity (microscopically). This was carried outthrough a tube extending to the bottom of the bioreactor.Since the first publication of cell culture methods for Tripterygium in 1981 24 , stocks ofTRP 4a have been continuously maintained in liquid culture by the Biological Services section ofour chemistry department. The head of this section, Gary Hewitt, has noted that the growth ofTRP 4a cultures in PRD2Co medium (determined by the rate of dry weight accumulation) isinversely proportional to the refractive index (RI) of the medium (unpublished).The RI (25°C) offreshly inoculated PRD2Co is 1.3371 compared to 1.3329 for water. The main contribution toincreased RI is sucrose, the carbon and energy source for TRP 4a growth. At the end of growth,when sucrose is depleted, the RI reaches a minimum of 1.3333 (± 0.0002). Cell debris is filteredout before measuring the RI and an increased amount of dead cells in older cultures does notnoticeably affect the RI. As determination of the culture dry weight requires removal of a largesample (to insure that a representative ratio of cells to medium has been obtained), it waspreferable to estimate growth through the RI which requires only a few drops of the medium.The growth patterns of the bioreactor cultures used for the elicitation experiments to bedescribed in this paper are presented in figure 17 (for data, see Appendix II). The open squaresare RI data taken from all of the untreated PRD2Co cultures while the crosses mark RI in culturesafter the addition of elicitor.60••a0••1.3375 ^a PRDCo1.3370 • PRDCo + BotrytisO 4.OaO 0 0a 0 0O DI• a a00^0 .11.1• ••■•0 •• • 1300 •■a• ■D •0 1313365 71.3360 7RI13355 7(25°C)1.335013345 71.3340133351.3330 ^0 5• I^•^•^•10 15Culture Age (days)20^25Figure 17 Growth Curves for PRD2Co Cultures of TRP in 12 Liter Aerated Bioreactors RI 25°c of filtered broth fromexperiments 252, 254, 255, 256, 258, 259, 261, 262 and 266(24 cultures). RI values recorded after the addition of Botrytisare marked with a (+).This figure demonstrates that growth began at a slow rate and accelerated after the first ten dayswith the more rapid rate continuing until RI 1.3333. Cultures were harvested at this point toavoid a degradation of metabolites that has been observed after the depletion of sucrose 71 . Thevarious contributors to sucrose consumption: cell division, cell growth and metaboliteproduction, could not be distinguished using the RI curve. Three points on the growth curve werechosen as suitable times for the elicitation of cultures: early growth (RI 1.3360), mid growth (RI1.3350) and late growth (RI 1.3333). It was thought that elicitation in the slow growth phase(before RI 1.3360) would be delay the onset of rapid growth.The chosen method for elicitation was the addition of a fungal preparation directly into agrowing TRP 4a culture. For this purpose, fungal cultures (cells and liquid medium) weredisrupted using a tissue homogenizer and sterilized by heat treatment. Elicitors prepared in thisfashion contained all of the heat-stable fungal products present before exposure to plant cells.Live fungi were not used as they would proliferate rapidly and kill the TRP 4a cells beforesecondary metabolites could accumulate. It was not known at the outset whether an elicitorwould be a carbohydrate component of the fungal cell wall or a soluble factor.(II) ANALYSIS OF THE METABOLITES FROM TRP 4A CULTURESThe quantification of metabolites from TRP 4a cultures involved three steps: extraction,purification and analysis. Ethyl acetate was used as the extraction solvent. While pharmaceuticalextracts were prepared using hot water or ethano1 1 3, these solvents were not considered in thepresent study. Malcolm Roberts 25 had previously extracted TRP 4a cultures (MSNA0.51(0.5) intomethanol and then partitioned the extract between ethyl acetate and water. While anti-inflammatory and anti-fertility activities were found in the ethyl acetate extract, no significantactivity was observed in the water soluble material (also observed by Kupchan et al. 12 ). Thetriptolides and triterpenes under investigation in the present study had been fully extracted into62ethyl acetate as they were absent in follow-up extractions of TRP 4a using methanol. A steadydegradation of tripdiolide dissolved in methanol was be detected after one weeks storage at4.0°C. This did not occur in ethyl acetate solutions.To prepare for extraction, cultures were harvested and filtered to separate the cells fromthe liquid medium. If not extracted immediately, the cells were frozen. The medium was freeze-dried, dissolved in a small amount of water and extracted repeatedly with ethyl acetate. Freezingcaused the cells to break open and they released 50 % of their mass as liquid after thawing andfiltration. This liquid was extracted directly into ethyl acetate. The remaining cell solid wassuspended in ethyl acetate, treated with the tissue homogenizer, then filtered. In each of thesemethods, extraction was repeated until the evaporation of solvent revealed no further mass.These crude ethyl acetate extracts were then purified or analyzed directly, depending on thecomposition of the extract and the metabolite being determined.(i) Tripdiolide DeterminationAll of the crude ethyl acetate extracts were analyzed for tripdiolide without purification.This involved a fluorimetric method published by this group 99 . Samples were not purified astripdiolide requires very careful chromatographic handling to prevent decomposition. Theextracts from shake-flask cultures were often only 50 mg (with tripdiolide expected in quantitiesless than 5 mg) and thus the analysis would be disturbed by small losses on silica gel. Foranalysis, the extracts were dissolved in a fixed amount of ethyl acetate and spotted quantitativelyto a silica gel plate using a microliter syringe. The extract spots were alternated with spotscontaining increasing amounts of pure tripdiolide. The silica plate was developed inmethanol/chloroform and then sprayed with a cerium reagent which complexed with thetripdiolide to cause fluorescence under long wave UV light. A horizontal strip at the Rf oftripdiolide was bordered with black tape and the fluorescence was determined using a scanningfluorometer. The resulting peak areas were measured and the tripdiolide from extracts calculatedwith the standard curve generated from the pure samples present on each silica gel plate. It was63determined that this fluorescence assay was not specific for tripdiolide, thus a good degree ofresolution had to be obtained with the plates prior to the determination to avoid detecting othercompounds.The fluorimetric determination was somewhat laborious as it required the development ofmany chromatography plates (each sample was determined twice and each plate incorporated astandard curve of tripdiolide). Gas chromatographic analysis (GC) was attempted, but noconditions were found in which tripdiolide did not decompose to numerous products. Tripdiolidehas a melting point of 225°C, but is unstable at this temperature. Due to the greater stability ofoleanane triterpenes, it was considered likely that they could be analyzed by GC.(ii) Triterpene DeterminationThe oleanane triterpene acids A, B, C and D (5152,52 and 54 have high molecularweights (A, B = 470 C, D = 472)and high melting points (260°-300°C), thus they are notsuitably volatile for a direct GC analysis. HPLC (high pressure liquid chromatography) wouldnot be immediately applicable either, as the acid functionality absorbs only weakly and in theshort wavelength UV region. Previous attempts by this group had shown that the colored quinonemethide compounds ((26-63.) created a strong interference with UV detection due to extensivepeak tailing. For these reasons, either GC or HPLC would require derivitization of the triterpeneacids: GC for increased volatility and HPLC to install a strong UV chromophore. GC analysiswas chosen due to the high peak resolution obtainable with our capillary GC system.The most logical target for derivitization of the triterpenes was the polar carboxylic acidfunctionality. Treatment of the triterpene acids with diazomethane in methanol providedcomplete conversion to methyl esters (mp 120°-230°C) under mild conditions. The triterpeneesters would not elute from GC columns coated with polar stationary phases (cyanopropylsilicone or polyethylene glycol columns), even at their maximal operating temperatures (250°C). The shift to non-polar stationary phases, dimethyl silicone or phenylmethyl silicone,64provided peaks for the triterpene esters. This was in part due to their higher operatingtemperatures of up to 300°C. The most rapid resolution of triterpene methyl esters was obtainedusing a 12 m dimethyl silicone capillary column at 280°C (isothermal). These conditions elutedthe desired peaks in 28-35 minutes. This long analysis time might have been reduced by furtherderivitization of the triterpene esters, for example by a subsequent acetylation. This was notinvestigated as the chromatogram of methylated plant extracts was extremely crowded from 5-22minutes. The late elution of triterpene methyl esters placed their peaks in the first region of thechromatogram that was relatively undisturbed by the peaks of other compounds.The triterpene acids A, B, C and D (51,5?., .aa and 4) (a gift from my co-workerMalcolm Roberts) were treated with diazomethane and the methyl ester products wererecrystallized from methanol/water. Standard mixed solutions of the triterpene esters in ethylacetate were used to prepare a calibration curve. Calibrations for oleanolic and polpunonic acid(49, b.2)were prepared in the same fashion. An internal standard (methyl cholate) was added toall solutions prior to injection.During the analysis of plant extracts, methyl cholate was not a suitable internal standard.The peak from this compound eluted earlier than the triterpene esters and was often eclipsed bycomponents of the extract. If samples were analyzed with a consistent technique, the internalstandard was not required. Accurate repetition of the injection volume provided a repeatability of± 5% in the triterpene ester integrations between duplicate analyses. To compensate for changesin the column conditions, re-calibration with the triterpene ester standards was repeated beforeeach set of determinations.The maximum resolution of triterpene ester peaks was achieved by injection of dilutesamples. The triterpene esters under analysis constituted a very small proportion of the totalextract mass (0-10 %), thus concentrated samples exceeded the column capacity and causedgreatly decreased resolution. The extracts were diluted successively with accurate volumes ofethyl acetate until the triterpene peaks created less than 50 % of a full scale deflection in the65chromatogram (attenuation set at zero). With this method, the integrated area of triterpene peakswas kept well within the calibration range provided by the pure triterpene standards.Injection of pure triterpene A or C, oleanolic or polpunonic acid methyl esters resulted ina single peak (comprising 97% of the run integration). The injection of pure triterpene B or Dmethyl esters resulted in the elution of two peaks. Through a process of co-injection, theadditional peaks were identified as wilforlide B (55, the lactone isomer of triterpene B) andwilforlide A (5_6, the lactone isomer of triterpene D). Co-injection of the triterpene standardswith their lactone analogs increased the integration of the early eluting peaks without altering thepeak shape or retention time. Analysis of the triterpene B and D ester standards by TLC showedconclusively that lactones were not present prior to the GC analysis, demonstrating thatlactonization must have occurred during the GC chromatography.Lactonization of the ester and free acid forms of triterpenes B and D was observed tooccur partially on silica gel and almost completely under acid catalysis (H2SO4 in ethanol) or inthe mass spectrometer. This supports a conclusion that thermally induced lactonization hadoccurred during GC analysis. Lactonization was confined to the hot injector region (300°C) anddid not occur along the length of the column (280°C) as the lactone and ester peaks werecompletely resolved. Prolonged conversion would have resulted in the elution of a single broadpeak halfway between the normal retention times of the lactone and the methyl ester. Theanalyses of triterpene B and D esters in the plant extracts were reported using the combined areasof the lactone and ester peaks. The lactonization of the oleanane triterpenes (acid or ester forms)is outlined in figure 18 using the example of triterpene D.66HOHOTriterpene D (54 )HOWilforlide A (53)HOHOTriterpene C (53 )HOROH7/..--- ROHFigure 18^Lactonization of Oleanane TriterpenesR=H or CH3. The conformation of triterpene D acidwas determined by X-ray crystallography25 .(III) ELICITATION IN THE PRODUCTION OF TRIPDIOLIDE AND TRI1ERPENESThe PRD2Co culture medium provided the most rapid growth of TRP 4a cultures. Iffungal elicitation could be used to initiate tripdiolide synthesis in this medium, it would besuperior to the slower MSNA0.51(40.5 culture medium. Several types of fungal elicitor wereprepared by the Biological Services section using liquid cultures of fungi in PRL-4 medium67(PRD2Co less D and Co). The fungi included a strain of Botrytis, Sclerotinia sclerotiorum,Rhodotorula rubra, Trichoderma viride and two unidentified types isolated by the biologicalservices: POD 129 F-1 and POD 129 F-2. When the fungal cultures had grown to maturity(forming an aggregate of fungal mycelia rather than a loose suspension of cells) they were firsttreated with a tissue homogenizer until the mycelia had fragmented and were then autoclaved.These sterile fungal elicitor preparations were added to 500 ml TRP 4a cultures in amounts of0.1% and 1.0% by volume. The TRP 4a cultures were elicited at the beginning of their rapidgrowth phase (RI 1.3355) to insure maximum cell vigor.The first elicitation experiment was planned to screen the various fungi for the ability toinitiate tripdiolide production. Elicited cultures and non-elicited controls were harvested 18, 24,36, 48 and 72 hours after the time of fungus additions and extracted immediately with ethylacetate. The TLC fluorimetric analysis of these extracts showed maximal tripdiolideaccumulation in the 18-24 hour cell extracts of cultures elicited with the fungi Botrytis,Trichoderma and Sclerotinia at 0.1%. The levels of tripdiolide determined for these cultureswere from 9 to 14 mg per liter of culture. The same fungi at 1.0% or 72 hours as well as the otherfungi under all conditions produced tripdiolide levels in the cells close to those from controlcultures, 2 to 3 mg per liter of culture. The tripdiolide levels from the medium of all elicitedcultures were close to control levels of 5_ 1 mg per liter of culture. These results suggested thattripdiolide was produced rapidly after elicitation and was then degraded beyond 24 hours. Also,while tripdiolide produced in the MSNA0.5K0.5 cultures was found mainly in the medium (after25-35 days), the more rapid production in elicited PRD2Co cultures did not allow time for arelease from the cells. The unusual presence of tripdiolide in control PRD2Co cultures wasattributed to interference from other compounds in the fluorimetric determination. The assay wasnot specific for tripdiolide and relied on a thin layer chromatography for resolution.Elicitation had a notable effect on the appearance of cells. The cultures elicited withBotrytis and Sclerotinia (the fungi which induced a high tripdiolide assay) produced cells with a68dark gray/orange colour. The other elicited cultures were moderately dark green and the controlcultures were a very pale green. One factor in this darkening was the accumulation of triterpenequinone methides. A member of this group of red/orange compounds, tingenone (66), wasidentified in the elicited extracts by TLC comparison with an authentic sample.The 18 hour elicitation was scaled up from 500 ml shake flasks to a 12 liter bioreactor toallow isolation of the products. The large culture developed similarly to the small cultures andthe elicited cells were dark in comparison with normal PRD2Co cells. The elicited culture extractdemonstrated a high tripdiolide level through fluorimetric analysis, 10 mg tripdiolide per liter ofculture. Closer study of the cell extract by TLC in several solvent systems (chloroform/methanol,ethyl acetate/hexanes, chloroform ether) indicated that tripdiolide was not actually present,contrary to the positive fluorimetric assay result. After a purification of the extract usingpreviously successful column chromatographies24 produced no tripdiolide, it was concluded thatthe fluorimetric determination had not been accurate. Extracts from the small scale elicitorscreening experiment were re-examined, and it was found that cultures with a high tripdiolideassay also contained higher levels of many other compounds. The TLC conditions used in thefluorimetric assay provided an Rf of 0.3 for pure tripdiolide while a large proportion of theextracted compounds travelled to a higher position. These compounds had left a trail of residualmaterial back to the origin. This was especially true of the quinone methides (figure 1), where atail of orange was visible behind pure standards after TLC. The high tripdiolide readings forelicited extracts had been caused by increased amounts of other compounds whose trailingresidues were picked up by the sensitive fluorescence detector. This interference also accountedfor the low tripdiolide levels determined for control PRD2Co cultures that had not previouslybeen observed to produce any tripdiolide.The appearance of high levels of tripdiolide in the fluorimetric assay had demonstratedthat the elicitation technique was successful in increasing the production of some other secondarymetabolites besides the triptolides. Our initial efforts at identifying these compounds involved69screening the elicited sample extracts for oleanane triterpenes, the other family ofpharmacologically active compounds under investigation in this group. The crude extracts weretreated with diazomethane and directly analyzed by gas chromatography. The oleananetriterpenes A, B, C and D^52, 52 and 5,4) were present in low levels in control cell extracts(< 5 mg/L) and were nearly undetectable in all extracts of the growth medium. On the otherhand, some of the elicited cell extracts showed definite increases in triterpene yield. The 24 hourand 72 hour elicited cultures were tested by GC and the increases in the 72 hour levels werehigher than those in the 24 hour levels. Also, the cultures treated with 1.0% elicitor had highertriterpene levels than those treated with 0.1% elicitor. Under the optimal conditions of 1.0%elicitor for 72 hours, the cultures treated with Botrytis and Sclerotinia produced the largesttriterpene yields (20-40 mg of each triterpene per liter of culture). Treatment with Rhodotorula orthe local isolates POD 129 F-1 and POD 129 F-2 resulted in moderate triterpene yields (10-20mg per liter). Treatment with Trichoderma did not cause a significant increase in triterpene levelsabove those in the control cultures.The discovery that fungal exposure produced increases in the related oleanane triterpenesA-D (figure 3, page 11) made elicitation a valuable addition to our metabolite productiontechniques since triterpene D has demonstrated anti-inflammatory activity (Table 1, page 20).The primary medicinal property of Tripterygium extracts is the reversal of inflammation causedby rheumatoid arthritis; a readily available triterpene compound capable of reproducing thiseffect would be a very useful pharmaceutical.To confirm the elicited increase in triterpenes, large scale bioreactor experiments weredesigned which would provide enough extract to chromatographically purify the triterpenes. Thefungus Botrytis, which had demonstrated positive results in both the GC and fluorimetric assays,was chosen as the elicitor for these experiments.70(IV) OPTIMIZATION OF TRITERPENE PRODUCTION IN 12 LITER BIOREACTORS(i) The Growth Of Elicited CulturesThe growth of TRP 4a cells in 12 L bioreactors was described previously (see figure 17).The apparatus used allowed the operation of three bioreactors simultaneously , which providedfor the growth of one control and two elicited cultures. In some cases, a bioreactor becamecontaminated with outside microorganisms. When this occurred, the affected culture wasdiscarded and the experiment continued with the remaining cultures. TLC analysis did not showincreased triterpene levels from these infections.The growth curve for 12 L bioreactor cultures, figure 17, shows that elicitation slightlydiminished the rate of RI decrease. In contrast, elicited cultures displayed a large drop in cellfresh weight in comparison with non-elicited cultures grown for an equal time (data to bepresented in the following section). This indicates that the growth curve for elicited culturesincluded sucrose consumption that was not being directed towards the production of cellbiomass. Increased secondary metabolite production could account for some of this alteration inthe normal growth pattern.Cell dry weights were sampled during the growth of the culture series 266. Figure 19(data in appendix HI) shows the growth curves determined by RI (open circles) and by cell dryweight (closed circles). The error bars of ± 10% included in the dry weight data reflect thedifficulty in obtaining a homogeneous suspension of cells in a 25.0 ml culture sample. The nineculture samples analyzed revealed a cell water content of 92.9% (standard deviation ±1.5),indicating a close correlation of fresh weight to dry weight in both elicited and control cells. Thecultures monitored in the upper two plots had been elicited with Botrytis before the sampling,while the third culture was elicited at the mid-point of sampling. The growth curves show thatalthough elicited cultures accumulated cell mass more slowly than the control culture, a drop inrefractive index still indicated increases in the cell dry weight. At the end of growth (RI 1.3333),the dry weight levelled off (lower plot) while the RI continued to fall at a slow rate.71(266-1 (O d )15Dry Weight(g/L)10 —.._._5010 5 20 2513370R 11.3360--0-1.33501.33401.3330 I^I10 15Culture Age (days)15Dry Weight(g/L)10 —4,--..50 5120 25266-2 ( 8 d)I^I10 15Culture Age (days)01.3370R 11.3360—0--1.33501.33401.3330(266-3 (18 d)15Dry Weight(g/L)10 ----0,--.501.3370I^I^10^5^10 15 20^25Culture Age (days)Figure 19^Growth Curves and Culture Dry Weight of TRP 4a Series 266Refractive index at 25.0°C. Dry weights from freeze-dried cells in25.0 ml culture samples. The culture number is followed by the ageat which Botrytis was added (1.0 % v/v).R 1.3360I_0—1.33501.33401.333072As well as decreasing the biomass yield of PRD2Co cultures, elicitation decreased theproportion of viable cells. This was observed in a microscopic examination of samples from theculture series 266. Membrane integrity was assayed by dye exclusion (Biological Services, usingEvans' blue stain).The three cultures were examined on the 18th day of growth, culture 266-1(elicitor from day 0) displayed 31% lysed cells, culture 266-2 (elicitor from day 8) had 34%lysed cells and culture 266-3 (no elicitor) had only 12% lysed cells (from counts of 200-300 cellsin random fields). When live cultures were viewed without staining, the non-elicited cells wereperfectly round while the elicited cells showed an irregular border and appeared shriveled. Also,when any of the culture series were harvested, the non-elicited cultures provided cells with aloose, granular texture while while cells from the elicited cultures packed into a firm, dense cake.The viability of the cells, the appearance and the texture could well be related to a singlecomponent of the elicitation response (such as increased cell permeability) though this couldonly be determined through a more detailed study of the elicitation mechanism.(ii) Analysis of Culture ExtractsEthyl acetate extraction of twelve liter cultures provided from two to five grams ofmaterial. For triterpene analysis, one half of this extract (in methanol) was treated withdiazomethane. The esterification caused a lightening of the elicited extracts from orange toyellow, indicating an alteration in the chromophore (rings A and B) of the triterpene quinonemethides. The extracts of the cells and the culture medium were kept separate. The mediumextract mass was about 20% of the cell extract mass and the medium extract contained a muchsmaller number of compounds. This allowed an accurate GC analysis of the crude mediumextract while the crude cell extract required purification to remove some compounds whicheluted near the triterpene esters, disturbing the baseline of the chromatogram. Purification wasaccomplished by column chromatography on silica gel (with a gradient elution using increasingethyl acetate in toluene). The triterpene region of the GC chromatograms from cell extracts ofcontrol cultures contained very few compounds, thus control extracts were also analyzed without73purification. As the elicited culture extracts were purified and not the control culture extracts, anyloss of material on the silica gel would have diminished the reported magnitude of elicitedtriterpene yields. This increases the confidence in any elicited increases.The reported triterpene yields are the total of the cell and medium results. Typically themedium produced much lower triterpene yields. These yields were reported in units of triterpeneweight (mg) per unit volume of culture (L). The compounds oleanolic and polpunonic acid(compounds 49 and h2) were determined along with the triterpene acids A, B, C and D. Whilethese two substances were of little interest in themselves, they provided a broader scope to ouranalysis of the elicitation effect, particularly with respect to determination of the friedelanetriterpene family which includes polpunonic acid.74(iii) The Results of 72 Hour ElicitationWhen the various fungal elicitors were screened, 500 ml cultures of TRP 4a were elicitedin the early growth stage (RI 1.3355). A large increase in triterpenes was obtained using Botrytisat 1.0% v/v for 72 hours. The same conditions were incorporated into the first 12 L bioreactorexperiment, culture series 252, save that this series was elicited in late growth (RI 1.3332). Thiswas done in light of the results from tripdiolide production using TRP 4a in MSNA0.51C0.5,where maximum metabolite yield occurred at a late stage in growth71 . Also, it was thought thatthe greater mass of cells in an older culture would provide a more rapid triterpene synthesis.The conditions and the results of this experiment are presented in table 2. Culture 252-1was the 1.0% Botrytis, 72 hour elicited culture and 252-2 the PRD2Co control. The pattern ofgrowth for the two cultures is presented in figure 20 and the triterpene yields are plotted in figure21.There was a significant increase in the production of triterpenes A, B, C and D due toelicitation with Botrytis. Moreover, the elicited yields compared favourably with those from theslower growing MSNA0.5K0.5 cultures (30-40 days growth time) from which the triterpeneswere first isolated. The MSNA0.51(0.5 production levels were 1.2, 4.8, 2.7 and 9.3 mg/L for A, B,C and D acids25 . This translates to methyl ester yields of 1.2, 4.9, 2.8 and 9.6 mg/L forcomparison with the yields of the present discussion. (Triterpene acid yields are 0.97% of theester yields. )75Culture 252-1 252-2Initial CultureVolume (L)11 11Inoculum Level(v/v)[ Age (d) / RI ]9.1 %[19 / 1.3332]9.1 %[19 / 1.3332]Age atElicitation (d)[ RI ]16[1.3332]-Elicitor Level(v/v)1.0% not addedElicitation Period/ Age at Harvest(d)3 / 19 0 / 19FinalRI of CultureMedium1.3334 1.3333Fresh Weight ofCells(g)3303 2890EtOAc Extractweight (mg/L)448 321Triterpene A(mg/L)0.76 0.25Triterpene B(ma)4.49 0.97Triterpene C(ma)0.80 0.06Triterpene D(mg/L)3.83 0.22Oleanolic Acid(mg/L)- -Polpunonic Acid(mg/L)- -Table 2^Elicitation at 1.0% in Late Growth (Culture Series 252)Refined fractions for triterpene analysis were prepared by silica gel columnchromatography of the esterified culture extracts (252-1 only). Oleanolic andpolpunonic acids were not determined for this culture series.76■ elicited^ control- ABCD -1.33701.3360RI1.3350133401.3330 • • •^ . .0^5^10^15^20^25Culture Age (days)Figure 20 Growth Curves of TRP 4a Culture Series 252 Refractive index at 25.0°C:Legend symbols are followed by the age and level (v/v) at which Botrytis was added tocultures.5TriterpeneLevel^-(mg/L)^43210Figure 21^Triterpene Levels from Culture Series 252 Oleanane triterpenes A-D.Culture 252-1 is elicited with 1.0 % Botrytis at RI25 . 1.3332 (late growth) for 72 hours.Culture 252-2 (control) has no additions.The next experiment in 12 L bioreactors, series 254, was used to examine 72 hourelicitation in the middle of growth phase (RI 1.3354) rather than at the end. Culture 254-2 wassimilar to 252-1, with Botrytis added at 1.0%. Culture 254-1 was elicited with 0.1% Botrytis andculture 254-1 was the non-elicited control (table 3, figures 22 and 23).Both elicitation conditions provided an increase in triterpenes A and B above the controllevels, while triterpene C was only increased with 1.0% elicitation. Triterpene D appeared atcontrol levels under both elicitor concentrations. Due to increased levels of the less availabletriterpenes A and C, the 1.0% elicitation was more useful than the 0.1% elicitation and the mid-growth elicitation of series 254 was more useful than the late growth elicitation of series 252.The low level of triterpene D in elicited 254 cultures was anomalous as it was more often themajor triterpene product (as observed in the elicitor screening experiment and cultures 252, 259and 261 through 266). The triterpenes B and D are subject to losses through lactonization onsilica gel; this may have caused the appearance of lower levels.78Culture 254-1 254-2 254-3Initial CultureVolume (L)12 12 12Inoculum Level(v/v)[ Age (d) / RI ]8.3 %[18 / 1.3331]8.3 %[18 / 1.333118.3 %[18 / 1.33311Age atElicitation (d)[ RI ]14[1.3355]14[1.33353]-Elicitor Level(v/v)0.1 % 1.0 % not addedElicitation Period/ Age at Harvest(d)3 / 17 3 / 17 0 / 17FinalRI of CultureMedium1.3350 1.3349 1.3350Fresh Weight ofCells(g)2400 2220 2590EtOAc Extractweight (mg/L)478 439 471Triterpene A(mg/L)1.10 1.73 0.55Triterpene B(ma)2.94 2.33 1.26Triterpene C(ma)0.59 1.83 0.88Triterpene D(mg/L)1.18 1.24 1.36Oleanolic Acid(mg/I-)- - -Polpunonic Acid(ma)- - -Table 3^Elicitation at 1.0% and 0.1% in Mid Growth (Culture Series 254)Refined fractions for triterpene analysis were prepared by silica gel columnchromatography of the esterified culture extracts (254-1 and 254-2 only).Oleanolic and polpunonic acids were not determined for this culture series.79If.- ABCD -^- ABCD -0.1 %^ 1.0 %■ elicited1:1 control1.33701.3360RI1.33501.33404,0,44•:414rh"".•♦ 254-1 (14/0.1%) -254-2 (14/1.0%)— — D — 254-31.3330 • • •^ . .0^5^10^15^20^25Culture Age (days)Figure 22 Growth Curves of TRP 4a Culture Series 254 Refractive index at 25.0°C.Legend symbols are followed by the age and level (v/v) at which Botrytis was added tocultures.5TriterpeneLevel 4(mg/L)3210Figure 23 Triterpene Levels from Culture Series 254 Oleanane triterpenes A-D.Culture 254-1 is elicited with 0.1 % Botrytis atRI25. 1.3355 (mid growth) for 72 hourswhile culture 254-2 is elicited with 1.0 % Botrytis atRI25. 1.3353 (mid growth) for 72hours. Culture 254-3 (control) has no additions.80Culture series 255 repeated the 1.0%, 72 hour, late growth elicitation (RI 1.3335) ofculture series 252 and included a 0.1% elicited culture (table 4, figures 24 and 25). The triterpeneyields from both elicitations were the highest yet obtained. Extracts from this series wereanalyzed for oleanolic and polpunonic acid methyl esters and these compounds were revealed tobe major products of the elicitation response. The result of an increase from 0.1% to 1.0%elicitor was a large decrease in triterpenes D, 0 and P, a slight decrease in triterpene B and smallincreases in the levels of A and C. Cultures of series 254 and 255 indicated a possible advantagein triterpene A and C production from using 1.0% elicitation. As these were the minor triterpeneproducts, 1.0% elicitation was accepted as the most useful course for further experiments .The 1.0% elicited culture 255-3 was treated identically with the culture 252-1 and yet255-3 provided a far superior triterpene yield. While there were no obvious causes for thedifference, the slower growth of the 255 series prior to Botrytis addition (19 days to RI 1.3335compared with 16 days to RI 1.3332) indicates that the TRP 4a inoculum had varying growthpotential in the two culture series.81Culture 255-1 255-2 255-3Initial CultureVolume (L)12 12 12Inoculum Level(v/v)[ Age (d) / RI ]8.3 %[17 / 1.333318.3 %[17 / 1.3333]8.3 %117 / 1.33331Age atElicitation (d)[ RI ]- 19[1.3334]19[1.3335]Elicitor Level(v/v)not added 0.1 % 1.0 %Elicitation Period/ Age at Harvest(d)0 / 22 3 / 22 3 / 22FinalRI of CultureMedium1.3335 1.3334 1.3335Fresh Weight ofCells(g)4005 3212 3330EtOAc Extractweight (mg/L)530 564 569Triterpene A(mg/L)0.85 4.88 5.90Triterpene B(mg/L)4.69 18.2 17.8Triterpene C(mg/L)0.00 6.91 7.56Triterpene D(mg/L)0.61 9.83 5.25Oleanolic Acid(mg/L)0.88 12.8 6.34Polpunonic Acid(mg/L)0.41 10.8 6.53Table 4^Elicitation at 1.0% and 0.1% in Late Growth (Culture Series 255)Refined fractions for triterpene analysis were prepared by silica gel columnchromatography of the esterified culture extracts (255-2 and 255-3 only).4..,..4k.....-..,•-.---- ..^ •... u`. .. lt.%.,.. %.....":••,4.44.4.....„..ot%--D— 255-1♦ 255-2 (19/0.1%)255-3 (19/1.0%)4 1".. -....:*• •2510020KRTriterpeneLevel(mg/L)■ elicited^ control133701.3360RI1.33501.33401.3330^• •••■••••■•••• I ....I"0 5^10^15^20Culture Age (days)Figure 24 Growth Curves of TRP 4a Culture Series 255 Refractive index at 25.0°C.Legend symbols are followed by the age and level (v/v) at which Botrytis was added tocultures.- ABCDOP - - - ABCDOP -0.1 %^ 1.0 %Figure 25 Triterpene Levels from Culture Series 255 Oleanane triterpenes A-D,oleanolic acid (0), polpunonic acid (P). Culture 255-2 is elicited with 0.1 % Botrytis atRI25. 1.3334 (late growth) for 72 hours while culture 255-3 is elicited with 1.0 % Botrytisat R125 . 1.3335 (late growth) for 72 hours. Culture 255-1 (control) has no additions.83Previous cultures had been elicited (for 72 hours at 1.0%) in late growth (252-1, 255-3)and mid growth (254-2) and so experiment 256 was designed to test the effects of elicitoraddition in the early growth phase. At this stage, the cells had established a maximum rate ofgrowth and the supply of nutrients was still high while the mass of cells was quite low. Culture256-3 was elicited in early growth (RI 1.3360) with 1.0% Botrytis for 72 hours (table 5, figures26 and 27).The elicited triterpene levels from culture 256-3 were far higher than the control levelsand were comparable with the highest results thus far, those from the late growth elicited culture255-3. For these two cultures to have produced equal amounts of the triterpenes, the cells of theearly elicited culture needed to synthesize triterpenes at a greater rate than the more numerouscells of the late elicited culture, revealing an increased elicitor response from the younger culture.The high triterpene yields from early elicitation was an important result in light of the overallgrowth time of 13 days for culture 256-3 compared to 22 days for the late elicited culture 255-3.When the 1.0%, early growth elicitation was extended from 72 hours to 12 days, culture256-1, the triterpene levels were doubled. This indicated that the TRP 4a cells could producetriterpenes continuously while in the presence of elicitor. This contrasted with the resultsobtained by Threlfall's group86 where elicited plant cell cultures (Tabernaemontana divaricata)stopped accumulating triterpenes 48 hours after addition of the fungus. In the Tabernaemontanacultures, elicited pentacyclic triterpenes were not degraded after prolonged culture growth. Theincrease in triterpenes with Tripterygium elicited over 12 days suggested that either a degradationdid not occur or that synthesis exceeded degradation.84Culture 256-1 256-2 256-3Initial CultureVolume (L)12 12 12Inoculum Level(v/v)[ Age (d) / RI ]8.3 %[17 / 1.3335]8.3 %[17 / 1.3335]8.3 %[17 / 1.33351Age atElicitation (d)[ RI ]10[1.3360]- 10[1.3360]Elicitor Level(v/v)1.0 % not added 1.0 %Elicitation Period/ Age at Harvest(d)12 / 22 0 / 13 3 / 13FinalRI of CultureMedium1.3334 1.3353 1.3354Fresh Weight ofCells(g)2480 1818 1490EtOAc Extractweight (mg/L)831 411_444Triterpene A(mg/1-)21.4 1.09 6.18Triterpene B(mWL)42.6 5.38 23.6Triterpene C(ma)32.4 1.48 6.74Triterpene D(mg/L)17.8 1.04 7.90Oleanolic Acid(mg/1-)24.4 1.98 7.27Polpunonic Acid(mg/1-)39.3 2.29 10.1Table 5^Elicitation at 1.0% and Extended Elicitation in Early Growth (Culture Series 256) Refined fractions for triterpene analysis were prepared bysilica gel column chromatography of the esterified culture extracts (256-1 and256-3 only).85■ elicited^ control1.33701.3360RI1.33501.33404%•Notit.41•41I........----x---^256-1 (10/1.0%)--D —  256-2♦^ 256-3 (10/1.0%) 1.3330 • •0" • •5 10^15Culture Age (days)20 25Figure 26 Growth Curves of TRP 4a Culture Series 256 Refractive index at 25.0°C.Legend symbols are followed by the age and level (v/v) at which Botrytis was added tocultures.50TriterpeneLevel(mg/L)^403020100- ABCDOP - - - ABCDOP3d^ 12dFigure 27 Triterpene Levels from Culture Series 256  Oleanane triterpenes A-D,oleanolic acid (0), polpunonic acid (P). Culture 256-3 is elicited with 1.0 % Botrytis atR125. 1.3360 (early growth) for 72 hours while culture 256-1 is elicited with 1.0 %Botrytis at RIB. 1.3360 (early growth) for 12 days. Culture 256-2 (control) has noadditions. Control triterpene levels were included beside the 12 day elicited trial forcomparison, although the control was harvested 9 days earlier with the 3 day trial.86The results so far had shown an advantage to the use of 1.0% elicitor rather than 0.1%elicitor for the production of triterpenes A and C. The favourable triterpene increases from earlygrowth elicitation in the 256 series could also be interpreted as an effect of higher elicitor levels.The degree of response to elicitation has previously been related to the elicitor/cell mass ratio,rather than simply to the elicitor concentration (introduction p. 38, refs 81 and 83). Thissuggested a tight binding of the elicitor to the cells. In light of this observation, 1.0% elicitation(v/v) would have provided a greater relative concentration of elicitor to early growth culturesthan to late growth cultures due to the differences in cell mass. To further explore the effects ofincreased elicitor concentration, series 258 was carried out using 5.0% elicitor addition. Culture258-1 was elicited with 5.0% Botrytis for 72 hours in the mid growth stage (RI 1.3351)and 258-2was elicited equally in the mid-late growth stage (RI 1.3340). (An early elicited culture was lostto contamination.)The results are presented in table 6 and figures 28 and 29.The growth of series 258 cultures was quite slow prior to elicitation. The 5.0% elicitationyielded triterpenes in the low range of previous 1.0% elicited results (levels similar to those ofseries 252 and 254) while the levels were well below those from the most successful 1.0%elicitations of series 255 and 256. With these comparisons, 5.0% elicitation did not appearadvantageous to the lower elicitor levels. Due to the variances in triterpene production seen in thesuccessive culture series, a set of fermentors with parallel 1.0% and 5.0% elicitation was deemednecessary to confirm the low triterpene yield from an increased elicitor level.Of the two 5.0% elicitations, the earlier elicitation was more successful in generatingtriterpene production despite a much larger quantity of cells in the later elicited culture (freshweight at harvest was 3102 g vs. 1918 g from the earlier elicited culture). This increased rate ofelicited triterpene synthesis from younger cells was also noted in the comparison of 256-3 to255-3, and so appears to be a dependable effect.87Culture 258-1 258-2Initial CultureVolume (L)12 12Inoculum Level(v/v)[ Age (d) / RI ]8.3 %[18 / 1.333318.3 %[18 / 1.33331Age atElicitation (d)[ RI ]15[1.3351]21[1.33401Elicitor Level(v/v)5.0 % 5.0 %Elicitation Period/ Age at Harvest(d)3 / 18 3 / 24FinalRI of CultureMedium1.3348 1.3336Fresh Weight ofCells(g)1918 3102EtOAc Extractweight (mg/L)613 623Triterpene A(mg/I-)1.05 0.48Triterpene B(mg/I-)3.12 1.83Triterpene C(mg/I-)1.78 0.77Triterpene D(mg/I-)1.83 0.93Oleanolic Acid(mg/I-)0.93 0.81Polpunonic Acid(mg/I-)4.88 1.83Table 6^Elicitation at 5.0% in Mid Growth (Culture Series 258)Refined fractions for triterpene analysis were prepared by silica gel columnchromatography of the esterified culture extracts (258-1 and 258-2)........................♦^ 258-1 (15/5.0%)258-2 (21/5.0%)' 'x133701.3360RI1.33501.33401.3330 • •0^5^10^15Culture Age (days)••,...•20 25Figure 28 Growth Curves of TRP 4a Culture Series 258 Refractive index at 25.0°C .Legend symbols are followed by the age and level (v/v) at which Botrytis was added tocultures.5.0TriterpeneLevel^4.0(mg/L)3.02.01.00.0• 15dMI 21 d- AB C DO P -Figure 29 Triterpene Levels from Culture Series 258 Oleanane triterpenes A-D,oleanolic acid (0), polpunonic acid (P). Culture 258-1 is elicited with 5.0 % Botrytis atR125• 1.3351 (mid growth) for 72 hours while culture 258-2 is elicited with 5.0 % Botrytisat R1 25 . 1.3340 (mid/late growth) for 72 hours.Culture series 259 was planned to re-examine the effects of early growth elicitation using1.0% and 5.0% elicitor additions. The 1.0% early growth elicitation, 259-2, was run under thesame conditions as 256-3 (elicited 72 hours from RI 1.3360) and the 5.0% elicitation, 259-3, wastreated the same way. Culture 259-1 was a non-elicited control. The results are presented in table7 and figures 30 and 31.The triterpene levels from the 1.0% elicited culture, 259-2, were moderately higher thancontrol levels, save triterpenes C and 0, which were not increased. The elicited triterpenes A, B,D and P were accumulated to only one third of the levels reached in culture 256-3 although thetwo cultures were grown and elicited under identical conditions. This indicated once more thatadditional conditions such as the state of the inoculum had a great effect on the responsiveness ofcultures to elicitation.The 5.0% elicited culture, 259-3, did not yield significantly more triterpenes than thecontrol culture. Elicitation was not without any effect, as the harvest mass from 259-3 was 1600g compared to 1991 g from the 1.0% elicited culture and 2169 g from the control culture. Theseobservations suggested that an increase to 5.0% elicitation was detrimental to both cell growthand triterpene production.90Culture 259-1 259-2 259-3Initial CultureVolume (L)12 12 12Inoculum Level(v/v)[ Age (d) / RI ]8.3 %[16 / 1.3332]8.3 %[16 / 1.3332]8.3 %[16 / 1.3332]Age atElicitation (d)[ RI ]- 10[1.3360]10[1.3360]Elicitor Level(v/v)not added 1.0 % 5.0 %Elicitation Period/ Age at Harvest(d)0 / 13 3 / 13 3 / 13FinalRI of CultureMedium1.3355 1.3356 1.3358Fresh Weight ofCells(g)2169 1991 1600EtOAc Extractweight (mg/L)375 400 306Triterpene A(ng/L)0.70 1.79 0.83Triterpene B(ng,/1-.)2.09 7.30 3.17Triterpene C(mg/L)0.88 0.69 0.55Triterpene D(ma)1.80 4.73 1.86Oleanolic Acid(ng/L)1.54 1.46 1.40Polpunonic Acid(mg/I-)0.30 1.48 0.41Table 7^Elicitation at 1.0% and 5.0% in Early Growth (Culture Series 259)Refined fractions for triterpene analysis were prepared by silica gel columnchromatography of the esterified culture extracts (259-2 and 259-3 only).133401.3330^• •" 1 .."1.". 1 ••••■•.0 5^10^15^20Culture Age (days)Figure 30 Growth Curves of TRP 4a Culture Series 259 Refractive index at 25.0°C.Legend symbols are followed by the age and level (v/v) at which Botrytis was added tocultures.1.33701.3360RI1.3350— —D — 259 - 1259-2 (10/1.0%)+^ 259-3 (10/5.0%)258.0TriterpeneLevel^6.0(mg/L)4.02.00.0 ■ elicitedO control- ABCDOP - - - ABCDOP -1.0 %^ 5.0%Figure 31^Triterpene Levels from Culture Series 259 Oleanane triterpenes A-D,oleanolic acid (0), polpunonic acid (P). Culture 259-2 is elicited with 1.0 % Botrytis atRI25. 1.3360 (early growth) for 72 hours while culture 259-3 is elicited with 5.0 %Botrytis atRI25. 1.3360 (early growth) for 72 hours. Culture 259-1 (control) has noadditions.92(iv) A Summary of the 72 Hour Elicitations(a)^The effect on triterpene yield following elicitation with 1.0% Botrytis for 72 hours atvarious stages of culture growth is presented in figure 32. An excellent increase in triterpeneswas possible from both early and late elicited cultures, although high yields were not entirelydependable. Early elicitation was clearly advantageous when one considered the time requiredfor triterpene production and the mass of cell material that required extraction. When thetriterpene levels presented in figure 32 were divided by the culture time before harvest and theharvested cell weight, an optimized production of triterpenes through early growth elicitationwas revealed, figure 33.When the elicitation was carried out using 0.1%, 1.0% and 5.0% Botrytis, the highesttriterpene yields resulted from the 1.0% addition. The 0.1% elicitation resulted in slightly lowerlevels of the triterpenes A and C, while 5.0% elicitation did not raise the triterpene levelssignificantly above control levels and caused an inhibition in culture growth (with respect to thecell mass).Elicitation with Botrytis produced other effects on cultures 252-259 besides increasedtriterpene production. The harvest weight of elicited cultures was less than that of parallelcontrols by a degree proportional to the level of Botrytis addition and to the duration of exposure.The most visible result of elicitation was an alteration of the colour of the growing cultures.Elicitation caused a darkening from the normal pale green/tan of control (PRD2Co ) cultures to adarker green and further to a dark green/gray/orange in proportion to the resulting triterpene yieldrather than to the amount of elicitor added. The relation of a dark colour to production of the red-orange triterpene quinone methides was confirmed after the isolation of tingenone (66) andcelastrol (613) from the elicited extracts (Huifen Gu, unpublished). Culture darkening provided areliable estimation of triterpene synthesis prior to harvest and analysis.93- ABCD -ABCD - ABCD - ABCD - ABCD256-3^259-2early252-1^255-3late256-3^259-2early252-1^255-3lateFigure 32 Triterpene Levels fromElicitation at Various Culture Ages Oleanane triterpenes A-D. Elicitation with1.0 % Botrytis for 72 hours. Controls weregrown without Botrytis.1.5TriterpeneLevel(mg/L/kg/d)1.00.50.0-ABCD - ABCD - ABCD -ABCD -ABCD -1030TriterpeneLevel(mg/L)20254-2mid254-2midelici edculture256-3259-2254-2252-1255-3Ri25o atelicitation1.33601.33601.33531.33321.3335controlculture256-2259-1254-3252-2255-1■ elicited .^ control .■ elicited^ controlFigure 33 Optimization of Triterpene Production^Oleanane triterpenes A-D.Elicitation with 1.0 % Botrytis for 72 hours as described in figure 32. Levels are quoted intriterpenes produced (mg/L) per fresh weight of cells (kg) per day of growth to harvest.94(b) Culture stress factors (such as temperature) are capable of stimulating secondarymetabolite production 81 . The elicitatiOn of TRP 4a with Botrytis caused stress on the cultureswith regard to the decreased harvest mass and increased cell death. A study by Brodelius et al. 84has demonstrated that a direct relationship can exist between culture stress and fungal elicitation .In cultured tobacco cells, the fungal elicitor chitosan increased secondary metabolite productionin proportion to its concentration until an optimal level, above which the production was almostcompletely blocked. Studies measuring the electrical conductivity of the culture mediumrevealed that surpassing the optimal chitosan concentration produced a sudden increase inconductivity caused by cell membrane permeabilization (the release of ions to the medium).Chitosan is a cationic polyglucosamine that can be extracted from the outer cell walls of fungi. Itmay increase plant cell permeability by binding to anionic groups at the membrane surface tocause membrane disruption or by binding to specific elicitor receptors to open gated ionchannels. If increased cell membrane permeability is linked to the elicitation of TRP 4a, it isreasonable to expect that high elicitor levels (5 %) would disturb the osmotic balance of theculture. Thus a single mechanism, osmotic stress, could cause increased metabolite production,decreased cell growth and increased cell lysis. The substance mediating the elicitation of TRP 4aby Botrytis has not been identified; chitosan or another type of polysaccharide may well be theactive principle.(c) One uncertainty in the analysis of triterpenes B and D originated in the columnchromatography of extracts from the elicited cultures. Low triterpene D levels from elicitedcultures 254-258 may have been caused by conversion of the methyl ester into the lactonewilforlide A (a). If this reaction occurred due to exposure to silica gel during chromatography,the lactone would have eluted well in advance of the triterpene ester fractions that were analyzed.95Triterpene levels from the crude esterified extract and from the purified triterpene fractions ofculture 256-3 are presented below to demonstrate the results of purification. The decreasedtriterpene levels after purification resulted from the removal of overlapping peaks in the GCanalysis and from any losses during column chromatography.Triterpene Level (mg/L)Triterpene A B C D 0 PCrude extract 11.0 26.0 11.3 15.9 9.25 10.5Purified extract 6.18 23.6 6.74 7.90 7.27 10.1Table 8^The Effect of Column Chromatography on the Triterpene AssayTriterpene determination from culture 256-3.The increases in triterpene production through elicitation surpassed any effect of lossesduring chromatography as the control extracts were not purified. A greater uncertainty wasprovided by the lack of reproducible elicitation results from successive cultures treated underidentical growth conditions. The growth rate of cultures prior to elicitation had often varied, thusour attention was directed towards the influence of the stock cultures used as inoculum. The TRP4a stock cultures were grown in 500 ml volumes and required about 17 days to mature. The linewas maintained in four groups, subcultured at intervals to provide mature inoculum at four dayintervals. A correlation of elicited triterpene production to the inoculum cycle used for thesuccessive cultures is presented in table 9. Culture series 255 and 256 provided an increasedelicited triterpene production over other series, and this indicated that cultures from inoculumcycle B were the most responsive to elicitation. While the four inocula were derived from thesame source, developmental differences must have occurred to cause varied levels of secondarymetabolism.96Culture Series Inoculum Cycle Elicitation Results252 A +254 A +255 B +++256 B +++258 C +259 A2 ++Table 9^The Effect of Inoculum Cycle on Elicited Triterpene Yields  (+) indicates anincrease in triterpene yields above control levels. (Culture age and elicitor levels are notconsidered in this comparison.)(v) Extended ElicitationAn experiment was developed to monitor triterpene production at elicitation periodslonger than 72 hours, since series 256 had demonstrated a doubling of triterpene levels from the3 day to the 12 day elicitation. The time course of triterpene production was examined usingcultures elicited in the early growth phase (RI 1.3360). Small samples were withdrawn fromthese cultures at intervals for triterpene assay. Purification of the triterpenes from these smallsamples was not practical, yet the previous large scale isolations had confirmed the high levels oftriterpenes determined for the elicited cultures by GC analysis.The time course experiments, 261, 262 and 266, were cultured with inoculum B toremove the effect of varying inoculum cycles on the triterpene production. The B sub-group ofTRP 4a had previously demonstrated a maximum responsiveness to elicitation. The size ofsamples removed from the cultures was set at 200 ml to provide sufficient triterpene levels foranalysis and to insure that the cell density was representative of the whole culture. Largersamples could not be taken as the air-lift bioreactors would not operate below a volume of 10 L.The culture samples (containing 30-70 g cells, fresh weight) were frozen and then extracted the97following day; ethyl acetate extraction of the solid and liquid material was rapid and efficient dueto the small sample size.As GC analysis was carried out on the crude esterified extracts, some inflation of thetriterpene determinations was expected. While the assay was not highly accurate, the change inlevels with time provided a good assessment of increases or decreases in the triterpene content.A great advantage of determination from crude extracts was the removal of purificationlosses that occurred during column chromatography. Also, as the purification of triterpene esterfractions did not occur, determinations for triterpene B and D lactones 05 and a) were includedin the levels reported for B and D methyl esters. TLC analysis of the crude extracts had shownthe lactones present in about 10% of the triterpene acid levels. Since treatment with KOH/ethanolcould readily convert lactones to free hydroxy acids, the combined determination measured thetotal possible yield for the triterpene B and D acids.98(vi) The Results of Extended ElicitationCulture series 261 incorporated 1.0% Botrytis addition in early growth (day 10, RI1.3360); the same parameters were used for the most successful elicitation experiment 256-1.Cultures 261-1 and 261-2 were both elicited with these conditions while 261-3 was not elicited,providing a control culture. The results are presented in table 10 and in figures 34, 35.Throughout the growth, control triterpene levels remained below 3 mg per liter of culture. Theelicited triterpene levels rose much higher. Triterpenes A, B and D increased over the first sixdays (261-1) and nine days (261-2) of elicitation and then began to decrease while triterpenes C,O and P increased over the entire elicitation period. A decrease in some triterpene levels afterextended culture growth required a decrease in the rate of synthesis as well as a degradationprocess. This demonstrated that the cultures had become less sensitive to elicitation or the elicitorwas metabolized by the culture or it was neutralized by irreversible binding. In the controlculture, relatively stable triterpene levels indicated that above a low level of accumulationsynthesis was balanced by degradation. An alternate interpretation was that triterpene synthesiswas briefly elicited by the stress of transfer of the mature stock cultures (with depleted sucroseand depleted salts) into the concentrated PRD2Co of the bioreactors at day 0 and the initiallyformed triterpenes remained throughout the culture period.99Culture 261-1 261-2 261-3Initial Culture 12 12 12Volume (L)Inoculum Level(v/v) 8.3% 8.3% 8.3%[ Age (d) / RI ] [18 / 1.3332] [18 / 1.3332] [18 / 1.3332]Age atElicitation (d) 10 10 -[ RI ] [1.3358] [1.3358]Elicitor Level(v/v)1.0 % 1.0 % not addedCulture^261-1.Elicitation Period/ Age^(d)3 / 13 6 / 16 9 / 19 12 / 22RI of CultureMedium1.3352 - 1.3340 1.3335Volume Sampled(m1)192 204 198 200EtOAc Extractweight (mg/L)578 760 1460 1220Triterpene A(mg/1-)3.76 7.63 6.03 4.81Triterpene B(mg/1-)15.7 25.6 24.4 21.8Triterpene C(mg/1-)0.856 3.34 4.78 4.89Triterpene D(mg/1-)7.08 18.6 14.6 13.3Oleanolic Acid(mg/L)5.24 6.75 9.58 11.2Polpunonic Acid(mg/1-)2.36 6.71 12.2 15.1Table 10^Extended Elicitation from Early Growth (Culture Series 261)Triterpene analysis performed on crude esterified extracts of culture samples.Cultures originated with TRP 4a B inoculum. (cont. next two pages)100Culture^261-2Elicitation Period/ Age^(d)3 / 13 6 / 16 9 / 19 12 / 22RI of CultureMedium1.3352 - 1.3339 1.3335Volume Sampled(ml)204 203 199 200EtOAc Extractweight (mg/L)691 956 1260 1080Triterpene A(mg/I-)3.40 6.08 6.55 6.33Triterpene B(mg/I-)14.7 20.3 28.4 26.6Triterpene C(mg/I-)0.730 2.68 4.24 5.64Triterpene D(mg/I-)6.61 12.2 20.4 20.6Oleanolic Acid(mg/L)5.11 6.13 8.09 10.4Polpunonic Acid(mg/1-)2.07 5.13 10.1 14.8Table 10^Extended Elicitation from Early Growth (Culture Series 261) (cont.)101Culture^261-3Elicitation Period/ Age^(d)0 / 13 0 / 16 0 / 19 0 / 22RI of CultureMedium1.3351 - 1.3338 1.3335Volume Sampled(ml)202 208 186 200EtOAc Extractweight (mg/L)411 255 828 539Triterpene A(mg/I-)0.39 0.35 0.780 0.23Triterpene B(Ingil-)1.61 1.12 2.27 1.65Triterpene C(mg/1-)0.00 0.00 0.00 0.28Triterpene D(mg/l-)0.00 0.00 2.44 0.28Oleanolic Acid(mg/L)0.54 0.31 0.56 0.44Polpunonic Acid(mg/L)0.37 0.23 0.26 0.12Table 10^Extended Elicitation from Early Growth (Culture Series 261)  (cont.)102451/44, 41.rz .- -42t424%,.• 4.6No...V41,4.40261-1 (10/1.0%)261-2 (10/1.0%) -261-3^.1.33701.3360RI1.33501.33401.3330 •^••^.,.•^..,•^a^I^•^I^•^• •0 5^10^15 20 25Culture Age (days)Figure 34 Growth Curves of TRP 4a Culture Series 261  Refractive index at 25.0°C.Legend symbols are followed by the age and level (v/v) at which Botrytis was added tocultures. 20TriterpeneLevel(mg/L)10--o— A—a-- BC--fit– 012^14^16^18^20^22^24Culture Age (days)Figure 35^Triterpene Levels from Culture Series 261  Oleanane triterpenes A-D,oleanolic acid (0), polpunonic acid (P). Culture 261-3 is a control with no additionswhile 261-1 and 261-2 are elicited with 1.0% Botrytis (v/v) on day 10. (cont'd next page)10322 243020TriterpeneLevel(mg/L)10I^I^I^114^16^18^20Culture Age (days)010i1222 243020TriterpeneLevel(mg/L)100 I^I^I^i10^12^14^16^18^20Culture Age (days)—0-- A—D-- B—.-- CD— —* — 0P—0— A—CI-- B--IP-- CD— —* — 0PFigure 35^Triterpene Levels from Culture Series 261 Oleanane triterpenes A-D,oleanolic acid (0), polpunonic acid (P). Culture 261-3 is a control with no additionswhile 261-1 and 261-2 are elicited with 1.0% Botrytis (v/v) on day 10.104The elicitation conditions of culture series 262 repeated those used for series 261. Elicitorwas added at 1.0% in early growth (day 8, RI 1.3360). The results are presented in table 11 andin figures 36, 37. Triterpene levels in the control culture (262-2) remained below 4 mg/L, fallingfrom initially high levels which may have been stimulated by the osmotic stress of inoculation tofresh medium at day 0. The triterpene levels in the elicited cultures were first determined at thetime of Botrytis addition. From initially low levels, the triterpenes increased greatly in the first 72hours and continued to rise over the entire growth of the culture. The final triterpene levels werehigher than the maximum levels of series 262. One apparent difference between 262 and 261 wasthe more rapid growth of 262 cultures (18 days to the end of growth compared to 22 days for 261cultures). The more vigorous growth of series 262 may have allowed continued triterpeneproduction instead of the diminishing synthesis seen in the late growth of series 261.105Culture 262-1 262-2Initial Culture 12.5 12.5Volume (L)Inoculum Level(v/v) 9.0% 9.0%[ Age (d) / RI ] [16 / 1.3332] [16 / 1.3332]Age atElicitation (d) 8 -[ RI ] [1.3360]Elicitor Level(v/v)1.0 % not addedCulture^262-1Elicitation Period/ Age^(d)0 / 8 4 / 12 7 / 15 10 / 18RI of CultureMedium1.3360 1.3351 1.3343 1.3334Volume Sampled(ml)195 204 208 200EtOAc Extractweight (mg/L)349 559 586 975Triterpene A(ma)0.559 8.38 9.36 12.0Triterpene B(ma)2.54 34.7 42.2 58.3Triterpene C(mg,./1-)0.00 3.68 8.39 9.49Triterpene D(mg/I-)0.48 20.2 28.9 42.0Oleanolic Acid(mg/L)1.13 9.59 11.1 17.5Polpunonic Acid(mg/1-)1.09 6.64 18.2 21.7Table 11^Extended Elicitation from Early Growth (Culture Series 262)Triterpene analysis performed on crude esterified extracts of culture samples.Cultures originated with TRP 4a B inoculum. (cont. next page)106Culture^262-2Elicitation Period/ Age^(d)0 / 8 0 / 12 0 / 15 0 / 18RI of CultureMedium1.3360 1.3349 1.3338 1.3333Volume Sampled(ml)200 208 199 203EtOAc Extractweight (mg/L)354 316 291 280Triterpene A(mg/1-)0.830 0.26 0.23 0.17Triterpene B(mg/I-)3.97 2.23 1.68 1.81Triterpene C(mg/L)0.00 0.00 0.00 0.00Triterpene D(mg/L)1.08 0.538 0.35 0.31Oleanolic Acid(mg/L)1.78 0.909 0.774 0.887Polpunonic Acid(mg/L)1.50 0.47 0.43 0.36Table 11^Extended Elicitation from Early Growth (Culture Series 262)  (cont.)1071.33701.3360RI1.33501.3340♦^ 262-1 (8/1.0%)— 262-2.4ts.ss.s1.3330 • •0^5^10^15^20^25Culture Age (days)Figure 36 Growth Curves of TRP 4a Culture Series 262 Refractive index at 25.0°C.Legend symbols are followed by the age and level (v/v) at which Botrytis was added tocultures.6 ^5 -4TriterpeneLevel(mg/L)^32.......^........ .....^•.,... ..^%.*• ,,^....m. . .0• ^-K40TriterpeneLevel(mg/L)^30--o-- A-0- BC-^08^10^12^14^16^18Culture Age (days)8^10^12^14^16^18Culture Age (days)Figure 37^Triterpene Levels from Culture Series 262^Oleanane triterpenes A-D,oleanolic acid (0), polpunonic acid (P). Culture 262-2 is a control with no additions while262-1 is elicited with 1.0% Botrytis (v/v) on day 8. Note a tenfold decrease in scale forthe lower plot.109Culture 266-1 was designed to examine the effects of elicitation on very young cultures.For this purpose, 1.0% Botrytis was added at the time of inoculation (day 0, RI 1.3371). Tomatch the elicited cultures of series 261 and 262, culture 266-2 was treated with 1.0% Botrytis inearly growth (day 8, RI 1.3361). A control culture, 266-3, was maintained until close to the endof growth (day 18, RI 1.3335), but was then elicited with 1.0% Botrytis for 72 hours. Thisallowed a re-examination of late growth elicitation. The growth conditions and results arepresented in table 12 and figures 38, 39.All three elicitations resulted in large increases in triterpene production. Culture 266-2produced higher triterpene levels than the similar cultures 261-1, 261-2 and 262-1. While it hadbeen noted that increased triterpene production occurred in rapidly growing cultures, the growthrate of the 266 cultures was lower than that of the 262 cultures. This suggests that variations inthe growth rate and in the elicitation response of successive cultures are not directly related. Thecontrol triterpene levels (266-3) remained low until a remarkably rapid synthesis of triterpeneswas initiated immediately after elicitation. The 72 hour triterpene levels from this late elicitationwere much higher than the 72 hour levels from the parallel early growth elicitation (266-2) andyet previous cultures, series 252-259, had demonstrated more rapid triterpene synthesis followingearly growth elicitation. The unusually large elicitation response of culture 266-3 may haveresulted from elicitation at RI 1.3335 when the culture was not yet at the end of its growth phase(the RI decreased further to 1.3332 over 72 hours).The day 0 elicited culture, 266-1, did not show increased triterpene levels until the day 11analysis. At this time culture 266-2 displayed only slightly lower triterpene levels after just 72hours of elicitation. At the end of growth (day 21) the day 0 elicited culture contained loweramounts of triterpenes than the parallel day 8 elicited culture. While both cultures had contained1.0 % Botrytis from day 8 to the end of growth, a slower triterpene production from the day 0culture may have been the result of desensitization to the elicitor during the first week of growthwhen triterpene synthesis had remained at control levels.110Culture 266-1 266-2 266-3Initial Culture 13 13 13Volume (L)Inoculum Level(v/v) 8.3 % 8.3 % 8.3 %[ Age (d) / RI ] [17 / 1.3333] [17 / 1.3333] [17 / 1.3333]Age atElicitation (d) 0 8 18[ RI ] [1.3371] [1.3361] [1.3335]Elicitor Level(v/v)1.0 % 1.0 % 1.0 %Culture^266-1Elicitation Period/ Age^(d)5 / 5 8 / 8 11/11 14 /14 18/18 21/21RI of CultureMedium1.3367 1.3362 1.3358 1.3353 1.3344 1.3336Volume Sampled(ml)202 204 202 206 197 200EtOAc Extractweight (mg/L)165 240 400 586 708 749Triterpene A(mgJL)0.703 1.01 4.81 8.51 12.2 14.9Triterpene B(mg/L)4.32 6.06 23.0 39.9 59.1 93.1Triterpene C(mg/L)0.00 0.966 2.94 4.02 8.40 8.36Triterpene D(mg/L)1.25 2.31 8.98 14.1 24.3 27.5Oleanolic Acid(mg/L)1.29 3.13 10.2 21.2 28.6 36.5Polpunonic Acid(mg/L)1.62 3.04 5.97 12.1 17.1 24.1Table 12^Extended Elicitation from Early Growth and from Day 0 (Culture Series 266)Triterpene analysis performed on crude esterified extracts of culture samples.Cultures originated with TRP 4a B inoculum. (cont. next two pages)111Culture^266-2Elicitation Period/ Age^(d)0 / 5 0 / 8 3 / 11 6/14 10/18 13 /21RI of CultureMedium1.3363 1.3361 1.3357 1.3351 1.3343 1.3335Volume Sampled(ml)204 205 200 210 202 199EtOAc Extractweight (mg/L)238 299 452 688 983 1070Triterpene A(mg/L)0.45 0.634 3.57 16.1 16.6 29.4•Triterpene B(mg/L)3.03 3.86 15.7 56.4 82.3 117Triterpene C(mg/L)0.00 0.47 0.930 7.77 15.5 20.8Triterpene D(mg/L)1.00 1.40 7.66 32.2 51.5 68.2Oleanolic Acid(mg/L)2.07 2.01 4.86 16.2 23.2 31.2Polpunonic Acid(mg/L)1.46 1.95 1.88 12.4 24.9 38.2Table 12^Extended Elicitation from Early Growth and from Day 0 (Culture Series 266)(cont.)112.^Culture^266-3Elicitation Period/ Age^(d)0 / 5 0 / 8 0 / 11 0 / 14 0 / 18 3 / 21RI of CultureMedium1.3363 1.3361 1.3354 1.3348 1.3335 1.3332Volume Sampled(ml)202 196 200 196 199 200EtOAc Extractweight (mg/L)236 247 399 333 329 447Triterpene A(mg/I-)0.540 0.34 - 0.724 0.618 9.66Triterpene B(mg/L)2.75 3.18 8.52 4.99 4.27 43.4Triterpene C(mg/1-)0.00 0.11 0.41 0.21 0.40 3.11Triterpene D(mg/I-)0.639 0.806 1.81 0.893 0.854 26.9Oleanolic Acid(mg/L)1.41 2.66 2.41 2.64 2.02 14.3Polpunonic Acid(mg/L)1.28 1.37 1.14 0.842 0.533 9.73Table 12^Extended Elicitation from Early Growth and from Day 0 (Culture Series 266)(cont.)113•25"*....^•••• ... - .....^..*"...":***.?■.:'*. ` .1:1 .%...N:....:.:4.:..:^ti‘....*:ZZ:.• N*7.ZN.• ‘..•1.33701.3360RI1.33501.3340266-1 ( 0/1.0%)♦^ 266-2 ( 8/1.0%)— -0 - 266-3 (18/1.0%)1.3330 • • • • 1 "•• 1 ••• . 1 •• " 1 "0^5^10^15^20Culture Age (days)Figure 38 Growth Curves of TRP 4a Culture Series 266 Refractive index at 25.0°C.Legend symbols are followed by the age and level (v/v) at which Botrytis was added tocultures.1008060TriterpeneLevel(mg/L) 4020(266-1)-0- A-13- g--lb.-- CD— — at — 0P4^6^8^10 12 14 16 18 20 22Culture Age (days)Figure 39 Triterpene Levels from Culture Series 266^Oleanane triterpenes A-D,oleanolic acid (0), polpunonic acid (P). Cultures are elicited with 1.0% Botrytis (v/v)on day 0 (266-1), 8 (266-2), or 18 (266-3).^(cont'd next page)12010080TriterpeneLevel(mg/L) 6040(266-2)—0-- A—a— BC— — n— 04^6^8^10 12 14 16 18 20 22Culture Age (days)4^6^8^10 12 14 16 18 20 22Culture Age (days)504030TriterpeneLevel(mg/L) 2010(266-3)A—0— BC--)t— 0Figure 39 Triterpene Levels from Culture Series 266^Oleanane triterpenes A-D,oleanolic acid (0), polpunonic acid (P). Cultures are elicited with 1.0% Botrytis (v/v)on day 0 (266-1), 8 (266-2), or 18 (266-3).115(vii) Conclusions on Elicited Triterpene ProductionThe time course studies demonstrated that the maximum triterpene yield was obtainedfrom cultures elicited at the beginning of the rapid growth phase. Elicitation with 1.0% Botrytisproduced a steady increase in triterpene levels for at least six days, and usually until the end ofculture growth. The growth time for the early elicited cultures ranged from 18 to 22 days. Theseculture parameters provide the highest yielding and most rapid biosynthetic production of thetriterpene acids available.The rate of triterpene production during the first 72 hours of elicitation varied betweencultures. In some, a rapid and steady triterpene synthesis was initiated immediately afterelicitation while in others an initially moderate synthesis rate accelerated after 72 hours. Thisvariable time course of elicited triterpene production may have been a factor in the inconsistentelicitation results of culture series 252-259. An unpredictable delay before the induction of rapidtriterpene synthesis made the time course analysis far more reliable than a single 72 hour harvest.(viii) Further Studies on Triterpene ElicitationFurther studies of TRP 4a elicitation should first determine if freeze-dried preparations ofBotrytis are effective as elicitors. Adding measured weights of dry fungus would allow a moreprecise control of elicitor levels. Experiments to date have used a Botrytis culture grown to theend of its growth phase, homogenized, sterilized and added in liquid form. As each new batch ofBotrytis could have grown to a different final density, "1.0% Botrytis" was not an accuratestatement of the elicitor content. To further refine the elicitations, the fungus could befractionated to obtain the active principle.The triterpene acids A, B, C and D have the same carbon skeleton and due to theirsimilarity it is likely that they are formed from one precursor at a late stage in their biosynthesis.116Both epimers of the C22 hydroxyl group are present, the equatorial (a) hydroxyl of triterpenes Band D as well as the axial ((3) hydroxyl of triterpenes A and C. This indicates that control of theC22 oxidation lacks the high degree of stereoselectivity present in most enzymatic conversions.Hydration of a planar carbocation would be a mechanism consistent with the observed ratio ofproducts (B, D>>A, C) as the equatorial approach of water would be favoured over a hinderedaxial approach (inferred from the structures, figure 18).In contrast, the C3 triterpene alcohols produced by Tripterygium have only the equatorial(13) orientation. This is consistent with the biosynthesis of pentacyclic triterpenes fromsqualene-2,3-oxide93, 86, which is in turn formed through the stereoselective oxidation ofsqualene. The cyclization of squalene-2,3-oxide to the C3 equatorial alcohol is considered to be afinal common step in the biosynthetic production of both the triterpene acids and the steroids.In the laboratory, ketone acids A and B are converted completely to the dihydroxy acidsC and D by treatment with sodium borohydride25 . Conversely, the biosynthesis from squaleneoxide would produce C and D initially followed by an oxidation to produce A and B. The timecourse of elicited triterpene production showed simultaneous formation of both C3 oxidationlevels. These studies could not determine whether the more rapid synthesis of C3 ketones (A andB) over the C3 alcohols (C and D) was an indication of the actual order of biosyntheticproduction or a result of an equilibrium that favored the ketone form.If the TRP 4a cells are capable of interconverting the C3 oxidation levels, it may bepossible to influence the direction of the conversion. The enzymatic process would require aredox cofactor such as a nicotinamide or flavin nucleotide and the cellular pool of these cofactorscan be driven (a) to the oxidized state through glycolysis inhibition in the presence of oxygen, or(b) to the reduced state by oxygen deprivation in the presence of sugars. The oxidized statewould accumulate ketones A and B. This manipulation could be used to simplify the triterpenepurification or to shift production towards a desired form of the triterpenes.117(ix) Error Analysis for the Determination of Elicited Triterpene LevelsSource of ErrorContribution toReportedTriterpene LevelsMagnitudevariance betweensuccessive culturesincrease ordecreasepotentiallylargepeak overlap in GCchromatogram(series 261-266)increase 30 %chromatographylosses(series 252-259)decrease 10 - 20 %GC analysis increase ordecrease5 %Table 13^Consistency in the Elicitation Results for the Triterpene AcidsThe factors affecting the reproducibility of triterpene elicitations in successive cultureswere ranked in the above order. Cultures elicited under identical conditions did not alwaysproduce similar triterpene levels. The main component of this variation was eliminated inexperiments 261-266 by using a single line of TRP 4a inoculum. The remaining variation wasattributed to spontaneous changes in the inoculum as well as in the Botrytis cultures.While the triterpene ester peaks in the GC chromatogram of purified extracts were wellresolved, the crude extracts analyzed for series 261-266 provided a less stable baseline with somepeak overlap. A comparison of analysis results before and after purification (series 252-259)demonstrated that the crude extract triterpene levels were inflated by an average of 30 % (with arange of 0 - 80 %). For this reason, the crude extract analyses were only used in the time coursestudies where the relative levels from a single bioreactor could be compared.118Losses of 10 - 20 % during silica gel chromatography (culture series 252-259 only) weredetermined from the mass balance in Column separations of the partially purified triterpeneesters. The losses included irreversible binding to the silica gel as well as lactonization of thetriterpenes B and D. These losses subtracted from the elicitation response as the elicited cultureextracts were purified and not the control culture extracts.The GC analysis techniques provided a high level of accuracy as the esterificationproceeded cleanly and the extracts were assayed until an agreement of ± 5% was reached.Usually this was accomplished in two to three injections.(V) THE NATURAL PRODUCTS ENHANCED BY ELICITATIONThe majority of compounds observed in extracts of elicited cultures were triterpenes.These included (in the order of silica gel elution) tingenone (b), triterpene B lactone (wilforlideB,5), a mixture of sitosterols, triterpene D lactone (wilforlide A, 55), polpunonic acid (62),oleanolic acid(42), celastrol (0), salaspermic acid (0) and the triterpene acids A, B, C and D(51, 2, 2 and 51). All of these compounds except salaspermic acid had been observed inMSNA0.5K0.5 cultures of TRP 4a by Malcolm Roberts 25 , and were identified by TLC and GCcomparisons with pure standards as well as by NMR, IR and mass spectroscopy. Salaspermicacid had been previously observed only in whole plant extracts55 . The purification andidentification of the above compounds, save the triterpenes A-D, was performed by Mrs. HuifenGu in this laboratory (unpublished). The quinone methide triterpenes tingenone and celastrolwere produced by elicited cultures but were completely absent from control culture extracts. Thesame was true of salaspermic acid, which was elicited to approximately the same levels aspolpunonic acid. This was reasonable as the two compounds are closely related friedelanetriterpenes. The GC analysis could not resolve salaspermic acid from the large group of earlyeluting compounds and thus it was not assayed in the elicitation experiments.119Oleanane triterpenes have often been isolated as water and methanol soluble glycosidederivatives (attachment at the C3 hydroxy1) 87-91 . Using the established conditions for hydrolysis,samples of methanol extracts of culture series 261 and 266 were treated with aqueous H2SO4under reflux. This process caused considerable lactonization of triterpenes B and D but thetriterpene assay was not increased, indicating that the triterpene diols, C and D, were not storedas glycosides by the TRP 4a cultures. Triterpene glycosides may be present in the parent plant,Tripterygium, since its aqueous extracts are active as medicinal tonics.While the elicitation study has achieved good results with the production of triterpenes,conditions are still being sought for the elicitation of the diterpene compounds triptolide andtripdiolide. A screening for fungal elicitors and growth media that will support this process isplanned for future studies.(VI) PURIFICATION OF TRITERPENES A. B. C AND DTo accumulate the triterpene acids, ethyl acetate extracts of elicited cultures from series261, 262 and 266 were combined with the non-esterified portions of series 252-259 extracts. Thepooled extract was first subject to anion exchange chromatography in methanol / pyridine whichseparated an acid fraction. The acids were further purified using silica gel columnchromatography, [methylene chloride / methanol / acetic acid (100:2:1)], and final separation ofthe acids required columns eluted with 3% methanol in chloroform and with hexanes / ethylacetate / methanol / acetic acid (65:35:1:1).Triterpene acids B and D were dissolved in methanol and recrystallized from methanol /water (4:1). Triterpene acids A and C were moistened with a few drops of methanol whichallowed dissolution in methylene chloride. Recrystallization then proceeded following the partialevaporation of solvent.120Recrystallization provided 600 mg each of the triterpene acids B and D and 150 mg eachof the triterpene acids A and C, pure by GC and TLC. The bulk of the triterpene acids remainedin mixed column fractions and crystallization liquors (crystallization occurred in low yields andwas successful only with fairly pure samples). The pure triterpene acids were set aside forpharmacological testing in other groups equipped for such determinations.121SYNTHESIS OF PRECURSORS FOR BIOTRANSFORMATION TO THE TRIPTOLIDESElicitation had stimulated triterpene production but did not increase production of thetriptolides. The most promising avenue to the triptolides remained the incorporation of syntheticprecursors into the biosynthetic pathway used by Tripterygium cultures. To this end weundertook the synthesis of the butenolide 1. With the ring A functionality of triptolide in place,there was potential that this compound was identical to one of the natural biosyntheticintermediates and that triptolide producing cultures (or enzyme preparations from these cultures)could be used to complete the synthesis through oxidations at rings B and C. For this study it wasessential to insert an isotope into the skeleton of the precursor so that successful incorporationinto the triptolide product could be verified. A reasonable site for isotope replacement was thecarbonyl carbon of the butenolide which was the only additional carbon required in the synthesisfrom /-dehydroabietic acid (la The addition of a 14C-nitrile at carbon 3 introduces a label in thedesired position and at the appropriate oxidation level for hydrolysis to the butenolide product.In my research project, C3 nitrile synthesis was attempted using derivatives of the epoxy-alcohol a by a substitution with potassium cyanide (scheme I). Epoxidation was used to temperthe reactivity at C3 after all attempts at derivatization or displacement of the allylic hydroxyl hadyielded the diene or products of allylic rearrangement 25,97 . The alcohol 2. ^converted intothe stable mesylate 11 in high yield. If this compound incorporated cyanide at C3, the plannedroute to butenolide 7f (scheme I) continued with an epoxide opening to the allylic alcoholl° 13. Ontreatment with hydroxide, a ready formation of imino-lactone was expected due to the proximityof the hydroxyl to the nitrile. Hydrolysis to the butenolide would be carried out in mild aqueousacid.122NCEt2AINOP 02-.*Bz , 0°CNC1 ) KOH , Et0H/H 20 , A2) HCI , EtOH/H20CH3S02C1 , Et3N^).-CH2Cl2 , -10°C90%HeScheme IThis sequence was checked at the cyanide substitution where no reaction occurred atroom temperature and unwanted products were formed after heating (60°-80°C). The polarsolvent DMSO had been selected for this reaction as as it supported a high concentration ofpotassium cyanide and could not supply protons to promote addition to the epoxide. The additionof HMPA to the reaction mixture did not alter the outcome nor did the addition of tetra-n-butylammonium iodide or replacement of DMSO with dimethyl formamide. Treatment of themesylate in acetonitrile or benzene with a KCN-dicyclohexy1-18-crown-6 complex gave noreaction after 16 hours under reflux.123An examination of the KCN/DMSO reaction was carried out using 300 mg of themesylate and 3 equivalents of KCN under mild reaction conditions (60°C for 24 hours). Theelimination product la was recovered in 23% yield (it had formed a greater proportion of theproduct at higher temperatures) while 31% of the starting material remained unreacted. Theremainder of the starting material had been converted into three nitrile products. One wasisolated in a pure form (12 , 6%) while the other two could not be resolved ($(2, la; 35%). Thesecompounds were isomers of each other and of the nitriles a and b. produced in a mixture by aprevious synthesis*.79, 80, 81 a , bThe nitriles^B__Q and^had identical mass spectra and produced the same parent mass(M+) and (M+ - CI13) peaks formed by the nitriles a and b . All of the nitriles produced identicalinfrared spectra with the definitive stretches for nitrile (med, 2240 cm -1 ) and ketone (str, 1720-1710 cm -1 ). The isomers were readily distinguished using NMR spectroscopy with five uniquesets of signals clearly present. The integration of axial methyl signals provided an estimate of theisomeric ratio in mixed nitrile fractions:Compound^6 of axial methyl (ppm)^isomer ratio79 1.3980 , 81 1.31 / 1.03 1 : 2a , b 1.45 / 1.23 2 :^1Demers 101 had demonstrated that a transition from the A/B-trans to the A/B-cis ringjunction moves the axial methyl shift downfield (probably due to phenyl ring re-orientation).This effect could not be used to assign the structures of the nitrile isomers as it was observed that* prepared by Malcolm Roberts25 through conjugate addition to the a-methylenecyclohexanone124the A , h mixture contained one isomer with a downfield methyl shift in the presence of anAB-trans ring junction. Structural assignment of the nitriles using NMR was therefore notstraightforward; the shift of the axial methyl signals resulted from the combined anisotropicshielding influences of the carbonyl, nitrile and aromatic ring orientations.The formation of aldehyde 713 through elimination was expected as Malcolm Roberts 25had observed this product in a similar reaction. The mechanism of its production is almostcertainly an initial elimination followed by a pinacol-type rearrangement of the 0-2,3-epoxide:77^ 78The formation mechanism for the.nitrile products 79, 80 and 81 is less clear; I haveproposed one possible route that allows a migration of acetonitrile from C4 to C3 (see below).While C3 of the mesylate is likely to be the most electrophilic carbon, the exocyclic methylene iscertainly more accessible to cyanide. Addition to the epoxide generates the powerfully basicalkoxide with the correct geometry to allow proton abstraction a to the nitrile. SN2 substitutionto form a cyclopropane would then be facile due to the antiperiplanar relationship of thecarbanion to the mesylate leaving group*. After this, ring opening through base-catalyzedcollapse of the tertiary alcohol forms the resonance stabilized a-nitrilate anion. Proton transfer toeither of two enolate isomers followed by the aqueous work-up allows a maximum of four nitrileisomers; three of these were observed in the product . The AB-trans-C3 a compound is likelythe missing isomer as it is the only isomer that cannot adopt a chair conformation with the C3substituent equatorial.* A cyclization analogous to epoxide formation from an a-bromohydrin.125An alternate strategy was formation of the triflate derivative of epoxy-alcohol 76 toencourage cyanide attack at C3. Although stable epoxy-triflates have been reported 102 , allattempts at triflation of 76 (triflic anhydride, pyridine, dichloromethane, -78°---) 0°C) led to theformation of many products. This was not altered by the addition of KCN/crown ether to thereaction mixture at low temperature. Cyanide substitution of epoxy-alcohol derivatives was notcontinued beyond this point.While cyanide could not be incorporated (at C3) through substitution, it was found to bereadily incorporated through addition to the carbonyl of epoxy-ketone 82 (scheme II).Cyanohydrin formation 103 was accomplished in a two-phase mixture of dichloromethane andwater containing KCN and sodium bicarbonate*. The reaction produced a mixture of thecyanohydrin 83 with 5-10 % of the unreacted ketone. The cyanohydrin was stable in neutral andmildly acidic solutions but reverted to the ketone in the presence of bases and during silica gelchromatography.* functioning here as a weak proton donor126A mesylate (8I) was readily prepared in 70% yield from ketone. The strategy at this pointwas induction of the pinacol-type rearrangement of epoxide to aldehyde followed by eliminationof the mesylate group and selective reduction to the allylic alcohol appearing in scheme I.KCN , NaHCO3^lo-H2O/CH2Cl2r.t. , 90%CH3S02C1, Et3NCH2Cl2 , -10°C78 % B F3' Et20benzene , AC H 3 S 03 CH 3S03Li N(iPr)2THE , -78°—) 0° CNaBH 4NC CeCI3EtOH/H20Scheme HThe rearrangement of k4  was attempted using excess boron trifluoride etherate inbenzene 104 . No reaction was observed at room temperature or after 24 hours of at 40°C. Heatingto reflux caused decomposition of the starting material to many products. The lack of epoxideopening was attributed to the presence of electron withdrawing groups adjacent to the epoxidewhich would suppress carbocation formation. The route was not continued beyond this point.127The formation of an epoxy-cyanohydrin was the first successful incorporation of cyanideat C3. Using this addition strategy, another route to the butenolide was explored. The synthesis ofallylic cyanohydrin 16 was attempted as this compound would allow several alternate approachesto the butenolide (scheme III).The first step of scheme III, formation of cyanohydrin 86 from the methylene-cyclohexanone , was accomplished in high yield. While KCN• crown ether in DMF favouredthe conjugate addition product25 , a repetition of the two-phase cyanide addition (KCN, NaHCO3,H2O, CH2C12) produced a small quantity of cyanohydrin (with a majority of the starting materialunreacted). When sodium bicarbonate was replaced with the more acidic ammonium chlorideand ether used instead of dichloromethane 105 a 90% conversion to cyanohydrin was achievedwith no conjugate addition product. A high concentration of cyanide was essential for high yieldand KCN was used at 3 equivalents with respect to ketone.The presence of water may bring about 1,2-addition through several influences. As thehydrated cyanide ion is a much less powerful nucleophile than the "naked" cyanide produced byKCN• crown ether in aprotic solvents 106, it is likely that conjugate addition can occur much lessreadily in the aqueous system. Electron withdrawal by oxygen creates a higher density ofpositive charge at C3 than at the exocyclic methylene thus if conjugate addition is not rapid, ionicattraction can draw the cyanide anion towards a 1,2-addition. This becomes very relevant in thepresence of water as the accumulation of positive charge at the carbonyl carbon is expected to beincreased due to a delocalization of oxy-anion charge resulting from hydrogen bonding.128CH3S02C1 , Et3NCH2C12 , -10° C88%1) KOH , Et0H/H20 , A2) HCI , Et0H/H20Pd(CH3CN)2C12THF , A.---..-....--'^1) KOH , Et0H/H20 , A.*.^2) HCI , Et0H/H20NCScheme III129Another reason for the exclusive 1,2-addition of aqueous cyanide may be an incompleteconjugation in the enone system of B5, increasing the energy required for 1,4-addition. This wasinferred from the infrared spectrum where the carbonyl stretching frequency was not low enoughto have been produced by a fully conjugated enone. The following compounds illustrate theeffect of 7c-overlap on the carbonyl infrared absorbance:ao82^cyclohexanone^85^(+)-pulegone1715 cm -1^1710 cm -1^1695 cm -1^1675 cm -1The enone 85 falls mid-way between the standard absorbance of ketone and enone.Decreased conjugation most likely indicates that the enone system is not planar (allowing less7c-overlap). This is likely a result of torsional forces from the rigid AB-trans ring junction whichtend to drive the A-ring towards a chair conformation with the carbonyl directed downwárd. Aswell as suppressing congugate addition, this shields the upper face of the carbonyl with themethylene group so as to allow cyanide attack only from the lower face. (A boat conformationmay also be available to the A-ring in which the upper face of the carbonyl is brought up close toan axial methyl, again allowing cyanide approach only from below (from the convex face)).The proton NMR spectrum revealed that the cyanohydrin 86 was formed as a singleisomer. An epimer would have been readily detected as the nitrile group exerts strong anisotropicshielding that would alter the A-ring shifts. Unfortunately, there was no readily available methodto determine which isomer had been produced. Small nucleophiles are known to favour an axialapproach to cyclohexanones which, as 85 , lack axial substitution in the 3 position 107 . At thispoint I will assume an axial attack by cyanide (with ring A in the chair conformation) to yield thea—nitrile. This assignment receives support from the reactivity of several cyanohydrinderivatives that will be discussed shortly.130The a-Nitrile CyanohydrinIn the first strategy to butenolide (scheme III), the cyanohydrin mesylate $2 was preparedin 88% yield from the cyanohydrin. The allylic mesylate was considered an ideal candidate forSN2' displacement 108 with an oxygen nucleophile to produce an allylic alcohol that wouldhydrolyze to the butenolide. Treatment with potassium hydroxide in tetrahydrofuran at room .temperature for 60 minutes caused complete conversion to a more polar (TLC) compound. Whilethe mesylate group was clearly missing (NMR and IR spectra) this was not the expected product.It was assigned this structure:The NMR spectrum revealed 3 vinyl protons, twofrom the exocyclic methylene (8 5.85 and 5.21 ppm,o O.^doublets, J = 1 Hz; coupling removed throughH2N^89^irradiation of either signal) and the third attributedto the C2 proton (8 5.37 ppm, broad singlet).The amide (N-H) protons were revealed as a broad singlet (8 4.38 ppm, 2H) which was removedby deuterium exchange. In the infrared spectrum the N-H stretching frequencies characteristic ofamides were observed (3590, 3495 and 3400 cm -1 ) along with a strong amide carbonyl stretch(1644 cm -1 ).The SN2' displacement with mesylate 87 was then attempted using less basic oxygennucleophiles. Reaction at room temperature with: (i) cesium acetate in DMF 109 , (ii) cesiumacetate in acetone, (i) and(ii) containing potassium iodide or (iii) sodium acetate in acetic acid ledto a recovery of starting material. When these reaction mixtures were heated the major product131NCformed was the nitrile 90 which resulted fromelimination. This compound was not converted tothe amide $2 in KOH/THF, indicating that amide90^formation involved nitrile hydrolysis prior to theelimination of the mesylate group.While the route was halted here, a conjugate addition to C2 (hydride or hydroxyl or a Birchreduction) followed by epoxidation of the exocyclic olefin and then epoxide opening with basewould produce an allylic alcohol similar to the originally desired intermediate of scheme III.Subsequently, nitrile hydrolysis would yield the butenolide.As the SN2' reaction with an external nucleophile was unsuccessful, a second pathwayinvolving intramolecular 3,3-rearrangement was attempted. This route hinged on activation ofthe exocyclic olefin with a bis-acetonitrile palladium (II) chloride complex. Palladium catalystshave been used to initiate rearrangement of many allylic acetates 110 including a-cyanoallylicacetates 111 . The reactions had shown poor results with 0-substituted allyl systems, but it wasexpected that conjugation with the nitrile would drive a conversion to the rearranged product.Cyanohydrin B_¢ was converted to a stable acetate 88 in high yield, but when the acetate wastreated with the palladium complex no rearrangement was observed (reflux in dry THE underargon). A similar type of rearrangement was attempted through treating the free cyanohydrinwith thionyl chloride in ether. No reaction took place at room temperature and heating caused adecomposition to many products (including the ketone precursor 85 ). This was somewhatsurprising in light of the high yielding rearrangement of the corresponding hydroxy-acid methylester under the same conditions which was reported by Tokoroyama et al. 98 (fig. 14, route A, p.55). The stereochemistry of this hydroxy-ester was not determined and the compound may havebeen inverted at C3 with respect to cyanohydrin $6 .The lack of facile allylic rearrangement provided evidence towards the assignment of thecyanohydrin as the a-nitrile isomer. Studies of many rearrangements have indicated that allylic132(1)HO"(see figures)11 and 14CI(2)(3 )( see figure 11 )110,108 .displacements are favoured by a syn-diaxial relationship of nucleophile and leaving groupIn the cyanohydrin^, the A-ring is conformationally rigid with the C3-(3 substituent equatorialand the C3-a substituent axial. While the equatorial bond lies on the same plane as the exocyclicmethylene, the axial bond is at an angle of just over 90°. Some rearrangement would thereforehave been expected if the cyanohydrin contained an a (axial) hydroxyl.Allylic rearrangement between C3 and the exocyclic methylene is well precedented forthis ring system in cases involving an axial C3 substituent. The following reactions illustrate thisprocess:In each of these examples an axial C3 bond is formed or broken. Although reactions (2)and (3) are reversals of the attempted cyanohydrin rearrangement and are 2,3 rather than 3,3processes, a cyclic transition state is still involved and a lack of 0-addition to C3 indicates that anequatorial bond is not reactive in the rearrangements. In these two examples, free rotation of themethylene substituent provided equal access to the upper and lower faces of the A 3,4-olefin133CH3plane yet the migrating groups moved exclusively into the more hindered axial position. As stericfactors were not responsible for the geometry of approach, the most probable explanation of theobserved results is that the rearrangements were governed by electronic factors.If the cyanohydrin had been formed with the 13-nitrile geometry, an acetate derivativewould be expected to rearrange through the cyclic intermediate pictured below.Of particular import is the alignment between thevacant a* orbital of the C3 - oxygen bond and theoccupied 71 orbital of the exocyclic olefin. This shouldallow electron donation from the olefin to C3 to formthe 2,3-7t bond of the rearranged product. This orbitalalignment is minimal in the a-nitrile epimer,providing an explanation of why this compoundwould not readily undergo allylic rearrangement.The same electronic factors would govern the SN2' reaction as the geometry of the leavinggroup rather than that of the approaching nucleophile is attributed with control over thereactivity. The observed lack of allylic rearrangement is the basis on which the nitrile wasassigned to the a position in cyanohydrin 86 . The experimental results support these mechanisticconsiderations which indicate that the allylic rearrangement of cyanohydrin derivatives can notbe used to access the butenolide product.At this point, the most promising route remaining was through the dienes^or 2Q thatwere formed from elimination of the allylic cyanohydrin mesylate 87 . This strategy was outlinedearlier and remains to be explored. The synthesis of tripdiolide (31) had been carried out byTokoroyama et al. using a similar diene intermediate (figure 14, route B, p. 55).134One attempt to gain more substantial proof of the stereochemistry of the allylic cyanohydrin$¢ involved synthesis of the methoxymethyl derivative 91 . The reaction proceeded in 92% yieldusing methylal (formaldehyde dimethyl acetal) andthe acid catalyst phosphorus pentoxide (P205) 112 .This compound was subject to NOE experiments withH 3c0CH20NC^91^irradiation of the methylene signals of the protectinggroup. No enhancements were observed and thusno information was revealed with regards to the position of the oxygen substituent. Carefulcrystallization of one of the derivatives would allow an X-ray analysis for conclusive structuraldetermination. The allylic cyanohydrin mesylate would be suitable as it recrystallized fairlyreadily.Origin of the Starting Materials The starting materials for these syntheses were prepared as illustrated in scheme IV. Theyields are reported for purified products. Using the methods of vanTamelen and Demers et06,101, dehydroabietic acid (45.) was purified from mixed resin acids through recrystallizationof the ethanolamine salt; the exocyclic olefin 92 was obtained from the regenerated acid throughCurtius rearrangement and Cope elimination. Further elaborations were carried out as reportedby Malcolm Roberts25 . Allylic oxidation (Sharpless) of 92 produced the a-alcohol 93*.Treatment of this alcohol with peroxy-acid produced a single epoxide 76 which had beendemonstrated to be the a isomer through NOE enhancements between the axial methyl and theexocyclic methylene protons. A mild oxidation (Corey) then produced a fair yield of the epoxyketone $.2 . The allylic alcohol 22 was oxidized to the methylenecyclohexanone $5 using theSwern oxidation. The enone was stable for several weeks at -20°C but decomposed to manyproducts after a few days at room temperature.* In the NMR spectrum the C3 proton produces a broad singlet, w112 = 5 Hz, (S 4.37 ppm) indicating its equatorialposition which is p on C3.135^1.-HO''''(i) oxalyl chloride , DMSOCH2Cl2 -60°(ii) Et3N , -60°-- 25°^60%Se02 , HOAc , t-BuOOHCH2Cl2 65%mCPBA , CH 2Cl2 95%PCC , Na0AcCH2Cl2 85%82HO' s 7645(1) SOCl2 , Bz/DMF, 50° [acyl chloride](2) NaN3 , H20/acetone, 0° [acyl azide](3) toluene , 100° [isocyanate](4) LiAIH4 , THF, reflux [2° amine](5) HCO2H/H2CO(aq) , reflux [3° amine](6) (i) mCPBA, CHCI 3 , -20° (ii) Et3N, - 20° to reflux70% overallScheme IV136EXPERIMENTAL(I) IDENTIFICATION OF TRP 4A METABOLITES1 H NMR spectra were recorded with a Bruker WH 400 spectrometer (400 MHz).Chemical shifts are reported in ppm relative to tetramethyl silane. Infrared spectra were recordedwith a Perkin Elmer 710B infrared spectrometer from KBr pellets or from 5% solutions (sampleminus reference). Ultraviolet spectra were recorded with a Unicam SP 800B spectrophotometer.Melting points were taken with a Reichert apparatus (heated stage) and are uncorrected. Electronimpact low and high resolution mass spectra were recorded with a Kratos MS 50 spectrometer .(the Kratos MS 80 and 902 were also used for low resolution spectra). Elemental analyses werecarried out in this department by Mr. P. Borda.Silica gel column chromatographies were run with open or low pressure glass columnsusing Merck silica gel 60, 230-400 mesh. BDH OmnisolveTM grade solvents and BDH reagentgrade hexanes were used for elution. Routine TLC analysis was carried out with Merck silica gelplates (0.2 mm, F-254, glass backed) sprayed with molybdic acid and heated at 110°C forvisualization. A special colour reagent was used to visualize triterpenes: the developed plateswere coated first with a spray of 30% conc. H2SO4 in glacial acetic acid and second with a sprayof 5% anisaldehyde in isopropanol and were heated at 110°C for 5 minutes. This produceddifferent colours for the various triterpenes (Appendix IV).(i) Metabolite Production from Cell CulturesCell culture methods were developed by Gary Hewitt of the Biological Services sectionof our department. Mr. Hewitt supervised the growth of all cultures with the assistance of Mr.David Chen, Ms. Elizabeth Bugante and myself. TRP 4a suspension cultures 71 were grown in500 ml stocks on a rotary shaker or in 12 L glass/stainless steel air-lift bioreactors. Cultures wereraised in the dark at 26°C using a liquid PRL-4 medium with 2.0 mg/L 2,4-diclorophenoxyaceticacid and 10 % qv coconut milk (from freshly picked young coconuts with husks). Cultures were137initiated with an 8% inoculum (v/v). Growth was monitored through the refractive index ofsamples removed at 72 hour intervals (Galileo refractometer, 25.0°C). The pH and microscopicpurity were also determined from these samples.For the fungal elicitor, a culture of Botrytis sp. (PRL# 2042) was grown on solid nutrientagar (0B5) in Roux bottles. The mycelial mass was suspended in Aerosol OT solution (4501.11/L,Fisher Scientific) and fragmented aseptically with an Ultra Turrax tissue homogenizer (28,000rpm for 30 seconds, F25 head). The homogenate was used to inoculate fresh PRL-4 liquidmedium and the culture was grown to maturity at 26°C on a rotary shaker(130-140 rpm, 7-10days). At harvest, the culture was treated with the tissue homogenizer for 5 minutes and thenautoclaved at 121°C for 20 minutes. The elicitor preparation was stored frozen at -20°C untilneeded and was added to TRP 4a cultures at 1.0 ml/L (0.1%), 10.0 ml/L (1.0%) or 50.0 ml/L(5.0%).TRP 4a cultures were harvested rapidly by a mild suction filtration using miracloth(Calbiochem) followed by a rinse with distilled water (10% v/v). Fresh cells and medium werefrozen immediately and stored at -20°C. The medium was freeze-dried in the bulk trays of anFTS Systems apparatus.Prior to metabolite extraction, cells were thawed and vacuum filtered with Whatman #1paper. The cell solid was extracted through suspension in ethyl acetate (at least an equal volume)and treatment with the tissue homogenizer for 2 minutes, followed by vacuum filtration. Thiswas repeated three times. The cell liquid was extracted four times with ethyl acetate. The freeze-dried medium was also extracted four times after it was dissolved in a small volume of distilledwater. The cells aid medium were made slightly acidic (pH 4) by the addition of citric acidbefore extraction. This was done to insure complete protonation of the organic acids and was notdone with samples assayed for tripdiolide, a compound degraded by acid. In the liquid-liquidextractions, emulsions were broken by Celite addition and vacuum filtration or by the addition ofsolid NaCl. All ethyl acetate extracts were washed with water then brine, followed by a drying138over anhydrous sodium sulphate. Solvent removal was accomplished by rotary evaporation andthe extracts were stored for 24 hours under high vacuum.(ii) Tripdiolide AnalysisThe tripdiolide assay published by this group99 was used for determinations. Sevensamples, accurately diluted in ethyl acetate, were spotted onto Eastman/Kodak Chromagramsheets (plastic backed silica gel, 20 cm x 20 cm, stock #13181) at 2 cm intervals. Each plate heldfour extract samples which alternated with tripdiolide standards (2.0, 4.0 and 6.0 jig,recrystallized from ethanol/water 4:1). After development with 4% methanol in chloroform, theplates were air-dried then sprayed evenly with 5% v/v ceric sulphate in 10% aqueous H2SO4.Heating for exactly 5 minutes at 110°C produced faint brown spots at the location of tripdiolide ;most other compounds appeared as faint purple spots. A horizontal band, 1 cm x 20 cm, wascentered on the tripdiolide spots and masked off with black vinyl tape. Fluorescence within theband was then recorded (30 minutes after the plates had been heated) using a Turner fluorometerfitted with a Camag plate scanner. The analysis was repeated with more dilute extract sampleswhen peak integrations exceeded the range of the three point calibration generated by thetripdiolide standards on each plate.(iii) Triterpene AnalysisMethyl esters were prepared from carboxylic acids by dissolving a culture extract or pureacid in methanol, cooling to 0°C with stirring and adding diazomethane in ether. Diazomethanewas prepared from DiazaldTM according to the Aldrich method supplied with the reagent.Diazomethane was added in ten-fold excess or until bubbling had ceased. The samples werecovered and stirred for one hour and then a second addition was made to insure that excessdiazomethane was present. The solvent and reagent were allowed to evaporate overnight in thefume hood and the samples were dried by rotary evaporation followed by 24 hours in highvacuum.139The treated samples were purified by silica gel column chromatography using a step-gradient of ethyl acetate in toluene (1:19 .4 1:3). This provided the fractions for the gaschromatography analysis. Further purification of the esters could be carried out using columnseluted with toluene/ethyl acetate 4:1. The pure esters were obtained by recrystallization frommethanol/water 4:1.For the GC calibration, pure ester samples were accurately diluted with ethyl acetatecontaining 1.000 mg/ml methyl cholate. (Cholic acid was treated with diazomethane andrecrystallized from ethanol/water. ) Calibration factors were calculated from the linear responserange of serial dilutions using the methyl cholate as an internal standard. The extract samples,accurately diluted with ethyl acetate, were injected in 5.00 Ill volumes Dilutions were oftennecessary to bring sample concentrations within the calibrated range.Analysis was carried out with the HP 5840A gas chromatograph fitted with a HewlettPackard HP 1 column (12m x 0.2mm i.d. x 0.51.tm film thickness; cross-linked dimethylpolysiloxane gum). The elution was isothermal at 280°C with a 290°C cleaning ramp after eachanalysis. Flame ionization detection was used (300°C). The injector was set at 300°C and thecarrier gas was helium.The retention times for all compounds analyzed are listed in Appendix V. Triterpene Band D were determined from the sum of the lactone and the ester peak integrations.140(iv) Isolation of Triterpene Acids A, B, C and DIn the initial stage of triterpene acid purification, acid fractions were obtained from crude cultureextracts through anion exchange. This was carried out using BioRad AG-1-X4 resin (OH - form)in a 3 cm x 30 cm glass column. Samples were added in methanol (5% pyridine), washed withmethanol (5% toluene) and eluted with 0.5 M formic acid in methanol (5% toluene). The eluantfractions were concentrated by rotary evaporation, diluted with ethyl acetate, washed with water,dried with brine and Na2SO4, concentrated again and dried in vacuo. Pure triterpene acids wereobtained from the acid fractions using silica gel column chromatography followed byrecrystallization. The methods that were used are described in the discussion section (VI).22D-Hydroxy-3-oxoolean-12-en-29-oic acid  (51):Triterpene A. Recrystallized from CH2C12 / CH3OH. m.p. 267-270°C. UV(CH3OH) X max217 (E 775). IR (CHC13; cm -1 ): 3615, 2937, 2640, 1698, 1463, 1368, 1217, 724. 1 H NMR(CDC13) 8: 0.91 (3H, s), 1.04 (3H, s), 1.07 (3H, s), 1.09 (3H, s), 1.11 (3H, s), 1,41 (3H, s), 0.85-2.35 (m), 2.39 (1H, ddd, J=4,6,16 Hz), 2.55 (1H, ddd, J=4,11,16 Hz), 3.48 (1H, s, OH), 3.58 (1H,dd, J=3.6,7 Hz), 5.33 (1H, t, J=3.5 Hz). LRMS m/z (rel intensity): 470 (3.8, M -9, 452 (7.0), 437(2.7), 426 (4.4), 424 (5.5), 408 (15.7), 391 (3.2), 340 (11.1), 325 (4.1), 264 (43.7), 246 (67.8),217 (79.9), 205 (57.3), 201 (38.3), 189 (46.0), 171 (44.5), 159 (42.7), 147 (55.9), 135 (82.3), 119(95.1), 107 (90.5), 81 (89.8), 69 (71.4), 55 (100), 43 (53.4). Elemental Analysis, calc. forC30114604: C 76.55, H 9.85; found: C 76.02, H 9.97 .22a-Hydroxy-3-oxoolean-12-en-29-oic acid (52):Triterpene B. Recrystallized from CH3OH / H2O. m.p. 287-289°C. UV(CH3OH) Xmax212 (6 1882). IR (CHC13; cm -1 ): 3617, 2976, 2630, 1698, 1461, 1386, 1219, 1027, 910, 715. 1 HNMR (CDCI3) 8: 1.02 (3H, s), 1.04 (3H, s), 1.07 (3H, s), 1.08 (3H, s), 1.10 (3H, s), 1.18 (3H, s),1.29 (3H, s), 1.27-2.29 (m), 2.38 (1H, ddd, J=4,6,16 Hz), 2.55 (1H, ddd, J=4,11,16 Hz), 3.60141(1H, dd, J=4,12 Hz), 5.29 (1H, t, J=3.5 Hz). LRMS m/z (rel intensity): 470 (0.6, Mt), 452 (11.8),437 (2.0), 408 (1.5), 391 (1.3), 353 (0.6), 340 (0.5), 326 (1.7), 299 (1.2), 285 (2.2), 264 (3.7), 246(100), 228 (12.2), 218 (25.1), 205 (35.8), 201 (20.0), 185 (20.9), 173 (14.2), 159 (16.9), 145(23.6), 131 (31.0), 119 (38.0), 107 (29.3), 95 (34.3), 81 (22.1), 69 (14.4), 55 (24.6), 43 (21.7).Elemental Analysis, calc. for C30114604: C 76.55, H 9.85; found: C 76.40, H 9.90 .30,220-Dihydroxyolean-12-en-29-oic acid (52):Triterpene C. Recrystallized from CH2C12 / CH3OH. m.p. 282-284°C. UV(CH3OH) Xmax216 (8 840). IR (KBr pellet; cm -1 ): 3470, 3421, 2976, 2640, 1698, 1471, 1393, 1298, 1240, 1048,1032, 1005. 1 H NMR (C5D5N) 8: 0.76 (3H, s), 0.85 (6H, s), 1.02 (3H, s), 1.03 (3H, s), 1.08 (3H,s), 1.63 (3H, s), 0.65-2.63 (m), 3.22 (1H, dd, J=6,9.5 Hz), 3.80 (1H, dbroad, J=4.5 Hz), 5.21 (1H,t, J=3 Hz). LRMS m/z (rel intensity): 472 (6.1, Nit), 454 (5.0), 439 (2.6), 426 (2.0), 411 (2.1),393 (1.5), 264 (99.7), 246 (53.0), 231 (15.4), 217 (100), 207 (59.9), 190 (45.2), 175 (39.3), 161(22.7), 147 (37.9), 135 (74.1), 119 (49.9), 107 (48.2), 95 (52.4), 81 (49.9), 69 (48.6), 55 (48.7),43 (30.7). Elemental Analysis, calc. for C301 -14804•H20: C 73.43, H 10.27; found: C 73.53,H 10.40 .313,22a-Dihydroxyolean-12-en-29-oic acid  (54):Triterpene D. Recrystallized from CH3OH / H2O. m.p. 292-295°C. UV(CH3OH) ,max212 (E 1617). IR (KBr pellet; cm -1 ): 3475, 3387, 2946, 2550, 1698, 1467, 1384, 1235, 1038. 1HNMR (C5D5N) 8: 0.98 (3H, s), 1.03 (3H, s), 1.04 (3H, s), 1.23 (3H, s), 1.28 (3H, s), 1.37 (3H, s),1.58 (3H, s), 0.82-2.73 (m), 3.42 (1H, dd, J=6,10 Hz), 4.01 (1H, dd, J=5.5,13 Hz), 5.38 (1H, t,J=3 Hz). LRMS m/z (rel intensity): 472 (0.2, Mt), 454 (2.6), 436 (1.7), 421 (1.3), 410 (0.6), 393(1.2), 342 (1.1), 325 (0.6), 314 (0.9), 299 (2.0), 264 (2.7), 246 (100), 218 (21.6), 207 (18.1), 201(15.5), 190 (35.6), 175 (20.3), 159 (14.8), 145 (20.1), 131 (22.5), 119 (32.2), 107 (23.2), 95(25.9), 81 (17.7), 69 (12.2), 55 (14.7). Elemental Analysis, calc. for C30H4804: C 76.23, H10.23; found: C 76.10, H 10.33 .142(II) SYNTHETIC REACTIONS TOWARD 18(4- 3)-ISODEHYDROABIETENOLIDEChromatography and spectroscopy as described in section I. Dry solvents (BDHOmnisolveTM or freshly distilled) were stored over 4A sieves while pyridine and triethylaminewere stored over KOH pellets. Reagents were from Aldrich save potassium cyanide (BDHanalaRTm), methane sulphonyl chloride (BDH, 98%) and crude (1)-dehydroabietic acid (ICN).Solvent removal was carried out by rotary evaporation followed by a period of at least 16 hoursin a high vacuum desiccator. Phosphate buffer (0.50 M, pH 7.0) was prepared by the addition ofKOH to a solution of KH2PO4 in glass-distilled water.3a-methane sulphonaty1-4(19)a-epoxy-18-norabieta-8,11,13-triene ( 77 )Triethyl amine (415 41, 2.98 mmol) and methane sulphonyl chloride (130^1.68 mmol) wereadded to a stirred solution of epoxy-alcohol a (425 mg, 1.48 mmol) in dry dichloromethane(8 ml) with stirring under argon on an ice/salt bath. After 20 minutes the excess reagent wasquenched with phosphate buffer (pH 7) The reaction mixture was diluted with ether (50 ml) andextracted with phosphate buffer, water and brine then dried over sodium sulphate. After solventremoval the product precipitated from a 1:1 mixture of hexanes and benzene to provide themesylate 71 (485 mg, 1.33 mmol, 90%) as a white powder. Melting point 121°-123°C (decomp.).IR (CHC13; cm-1 ): 2950 (str), 2860 (sh), 1502 (w), 1355 (str, 0-S-0), 1180 (str, 0-S-0), 1117(med), 940 (str), 908 (med). 1 H NMR (CDC13) 6: 7.21 (1H, d, J= 8 Hz), 7.04 (1H, d, J= 8 Hz),6.95 (1H, s), 4.42 (1H, t, J= 2.5 Hz), 3.10 (3H, s), 2.95 (1H, d, J= 4 Hz), 2.88 (3H, mult), 2.79(1H, d/d, J= 4,1 Hz), 2.52 (1H, d/d, J= 13,2 Hz), 2.29 (1H, d/mult, J= 14 Hz), 2.21 (1H, d/mult,J= 14 Hz), 2.11 (1H, d/mult, J= 14 Hz), 1.97 (1H, t/d, J= 13,5 Hz), 1.68 (1H d/mult, J= 13 Hz),1.43-1.30 (1H, mult), 1.24 (6H, d, J= 7 Hz), 1.17 (3, s). LRMS m/z (rel intensity): 364 (6, M+),349 (0.2), 268 (48), 253 (76), 237 (33), 222 (41), 211 (39), 193 (51), 181 (100), 164 (61), 141(53), 129 (44), 115 (41), 91 (31), 77 (24), 55 (20), 43 (66). Elemental Analysis, calc. forC20H28O4S (364.50) : C 65.90, H 7.74; found: C 65.84, H 7.60 .143Cyanide AdditionMesylate 2/ (300 mg, 0.823 mmol) was dissolved in dry dimethyl sulphoxide (3 ml). Finelypowdered potassium cyanide (163 mg, 2.50 mmol) was added and the mixture was heated on anoil bath (60°- 70°C) with stirring under nitrogen for 48 hours. The yellow solution was dilutedwith ether (200 ml) and extracted with water and brine then dried over sodium sulphate. Solventremoval gave 290 mg of a brown oil. Column chromatography using ethyl acetate (10) /chloroform (10) / hexanes (80) provided aldehyde 7. (52 mg, 0.19 mmol, 23%, a colourless oilwhich decomposed in air to a dark mixture beyond several days), a mixture of nitriles andunreacted 22 (92 mg, 0.25 mmol, 31 %). The nitrile mixture was partially resolved by columnchromatography using ethyl acetate (7.5%) in a 1:1 mixture of toluene and hexanes to provide 22(15 mg, 0.055 mmol, 6 %) and a mixture of aQ and a (85 mg, 0.288 mmol, 35%) whichprecipitated together from chloroform/hexanes.19-norabieta-2,8,11,13-tetraen-18-al (^)IR (CC14; cm -1 ): 2960 (str), 2910 (str), 2850 (str),1722 (med),1500 (w),1460 (med),1380(med),1353 (med),1282 (w),1120 (str),1080 (med). 1H NMR (CDC13) 6: 9.57 (1H, d, J= 4 Hz),7.21 (1H, d, J= 8 Hz), 7.05 (1H, d, J= 8 Hz), 6.93 (1H, s), 6.02 (1H, mult), 5.62 (1H, d/mult, J=10 Hz), 2.85 (4H, mult), 2.61 (1H, d/d, J= 18, 6 Hz), 2.25 (1H, d/mult, J= 18 Hz), 2.05 (1H, t/d,J= 12, 3 Hz), 1.78 (1H, mult), 1.70 (1H, mult), 1.24 (6H, d, J= 7 Hz), 1.15 (3H, s). LRMS m/z(rel intensity): 268(55, M+), 253(79), 239(16), 235(24), 225(30), 223(28), 211(34), 193(47),186(72), 181(100), 177(51), 171(45), 164(60), 159(25), 152(22), 141(44), 129(44), 115(41),105(32), 91(44), 83(85), 77(29), 65(14). 55(31), 43(75). HRMS, caic. for CI9H240: 268.1827 ;found: 268.1828 .Nitrile 22Yellow oil. IR (CC14; cm-1 ): 2955(str), 2860(sh), 2240(med), 1740(sh), 1710(str), 1617(w),1503(med), 1456(str), 1428(med), 1380(med), 1352(w), 1335(w), 1310(med), 1243(med),1185(w), 1105(med), 1076(med), 918(med), 897(med), 832(med). 1 H NMR (CDC13) 8: 7.12144(1H, d, J= 8 Hz), 6.97 (1H, d, J= 8 Hz), 6.88 (1H, s), 3.10 (1H, quint/mull, J= 9 Hz), 2.81 (1H,sept, J= 7 Hz), 2.75-2.64 (4H, mult), 2.48 (1H, t, J= 3 Hz), 2.34 (1H, quar/mult, J= 7 Hz), 2.21-2.09 (2H, mult), 1.98 (2H, t/mult, J= 13 Hz), 1.39 (3H, s, H3C-20), 1.33 (1H, d/d, J= 13, 2 Hz),1.21 (6H, d, J= 7 Hz). Calc. for C20H250N : 295.4236 .Nitriles lif2 , .81White solid, melting point 139°- 142°C. IR (CHC13; cm -1): 2995(sh), 2950(str), 2842(sh),2240(med), 1710(str), 1605(w), 1500(med), 1465(med), 1419(med), 1384(med), 1105(w),1068(w), 897(w), 830(med). 1H NMR (CDC13) 8: 7.21 (d, J= 8 Hz), 7.19 (d, J= 8 Hz), 7.07 (d,J= 8 Hz), 7.05 (d, J= 8 Hz), 6.96 (s), 6.94 (s), 2.97-2.68 (mult), 2.65 (d/d, J= 12, 2 Hz), 2.58-2.42(mult), 2.29-2.15 (mult), 2.10-1.96 (mult), 1.95-1.71 (mult), 1.31 (1H, s, H3C-20), 1.25 (d, J= 7Hz), 1.24 (d, J= 7 Hz), 1.03 (2H, s, H3C-20). LRMS m/z (rel intensity): 295(36, MI), 280(100),262(14), 253(13), 238(38), 221(17), 213(29), 202(9), 193(23), 179(39), 169(22), 155(21),141(45), 128(47), 115(40), 91(24), 77(24), 55(24), 51(16), 43(56). Calc. for C201 -1250N :295.4236 .3-hydroxy-4(19)a-epoxy-18(4-43)-abeoabieta-8,11,13-triene-18-nitrile ( 83 )Aqueous potassium cyanide (3 ml, 373 mg KCN, 5.73 mmol) containing sodium bicarbonate(480 mg, 5.71 mmol) was added to a rapidly stirred solution of epoxy-ketone K (400 mg, 1.41mmol) in dichloromethane (12 ml) at room temperature. A 95% conversion (TLC) was achievedafter 3 hours and the mixture was taken up in ether (100 ml) and extracted with water and brinethen dried over sodium sulphate. Solvent removal gave 410 mg of a white solid which was 90%cyanohydrin lia and 10% starting material (NMR). The mixture was not purified as exposure tosilica gel caused a reversion to ketone $2 . IR (CHC13; cm -1 ): 3410(med, broad), 2940(str),2850(sh), 1717(med), 1610(w), 1500(med), 1459(med), 1381(med), 1363(med), 1110(str, broad),987(med), 925(med), 885(med), 827(med). 1H NMR (CDC13) 8: 718 (1H, d, J= 8 Hz), 7.05 (1H,d, J= 8 Hz), 6.96 (1H, s), 3.17 (1H, d, J= 4 Hz), 3.10 (1H, s, D20 exch.), 2.92-2.83 (3H, mult),2.81 (1H, d, J= 4 Hz), 2.42 (3H, d/mult, J= 11 Hz), 2.07 (1H, t/d, J= 14, 4 Hz), 1.95 (1H, t/d, J=14514, 4 Hz), 1.78 (1H, d/mult, J= 12 Hz), 1.48-1.35 (1H, mult), 1.23 (6H, d, J= 7 Hz), 1.15 (3H, s).LRMS as starting material. Cale. for C20H2502N : 311.423 .3-methane sulphonaty1 -4(19)a-epoxy-18(4-33) -abeoabieta -8,11,13-triene-18-nitrile ( 84 )Triethyl amine (340 pl, 2.44 mmol) and methane sulphonyl chloride (130 p.1, 1.67 mmol) wereadded to a stirred solution of cyanohydrin $3. (250 mg, 0.803 mmol, containing additional ketone$2) in dry dichloromethane (10 ml) with stirring under argon on an ice/salt bath. After 2 hoursthe excess reagent was quenched with phosphate buffer (pH 7) The reaction mixture was dilutedwith ether (100 ml) and extracted with phosphate buffer, water and brine then dried over sodiumsulphate. Solvent removal gave 312 mg of a yellow oil and chromatography in ethyl acetate(2) /toluene(98) provided the mesylateli4 (244 mg, 0.626 mmol, 78%) as a white solid. lit (CC14;cm-1 ): 2944(str), 2905(sh), 2850(sh), 1607(w), 1500(med), 1450(med), 1418(med), 1380(str),1332(med), 1190(str), 1080(med), 1070(med), 1020(med), 962(str), 920(med), 888(med),855(med). 1 H NMR (CDC13) 5: 7.17 (1H, d, J= 8 Hz), 7.06 (1H, d, J= 8 Hz), 6.97 (1H, s), 3.26(1H, d, J= 4 Hz), 3.23 (3H, s), 2.98 (1H, d/mult, J= 13 Hz), 2.94-2.87 (3H, mult), 2.86 (1H, d, J=4 Hz), 2.50-2.35 (3H, mult), 1.96 (1H, t/d, J= 13, 4 Hz), 1.80 (1H, d/mult, J= 13 Hz), 1.48-1.36(1H, mult), 1.24 (6H, d, J= 7 Hz), 1.19 (3H, s). LRMS miz (rel intensity): 389(24, m+), 374(35),344(4), 295(43), 293(22), 280(70), 278(100), 262(7), 248(28), 236(46), 220(21), 206(55),193(28), 179(26), 165(27), 141(27), 128(32), 117(32), 97(52), 91(31), 76(23), 43(74). Calc. forC211-12704NS : 389.51 .3-hydroxy -18(4--)3) -abeoabieta -4(19),8,11,13 -tetraene-18-nitrile ( 86 )Aqueous solutions of potassium cyanide (2.5 ml, 1020 mg KCN, 15.7 mmol) and ammoniumchloride (2.5 ml, 892 mg NH4C1, 16.7 mmol) were added to a rapidly stirred solution of enone .a.d(1.40 g, 5.22 mmol) in diethyl ether (5.0 ml) with cooling on an ice bath. After 90 minutes thereaction mixture was taken up in ether (200 ml) and extracted with water and brine then driedover sodium sulphate. Solvent removal gave 1.54 g of white solid which was 90% cyanohydrin13.6 and 10% starting material (NMR). The mixture was not purified as exposure to silica gel146caused a reversion to ketone $1 . IR (CC14; cm-1 ): 3580(sh), 3405(str, broad), 2945(str),2850(sh), 2205(w), 1720(w), 1684(med), 1653(w), 1612(w), 1500(str), 1460(med), 1378(med),1184(w), 1124(med), 1075(med), 1007(w), 920(str), 828(med). 1 H NMR (CDC13) 8: 7.22 (1H, d,J= 8 Hz), 7.07 (1H, d, J= 8 Hz), 7.01 (1H, s), 5.48 (1H, d, J= 1 Hz), 5.04 (1H, d, J= 1 Hz), 3.05-2.93 (3H, mult), 2.88 (1H, sept, J= 7 Hz), 2.59 (1H, did, J= 11, 2 Hz), 2.43-2.35 (2H, mult), 2.14-2.00 (1H, mult), 1.98 (1H, d, J= 10 Hz), 1.94-1.82 (2H, mult), 1.27 (6H, d, J= 7 Hz), 1.02 (3H, s),D20 exchange is not observed. LRMS as starting material. Calc. for C201 -1250N : 295.42 .3-methane sulphonaty1-18(4-43)-abeoabieta-4(19),8,11,13-tetraene-18-nitrile ( 87 )Triethyl amine (1.12 ml, 8.46 mmol) and methane sulphonyl chloride (0.475 ml, 6.09 mmol)were added to a stirred solution of cyanohydrin B..¢ (1.00 g, 3.38 mmol, containing an additionalquantity of ketone aa) in dry dichloromethane (20 ml) with stirring under argon on an ice/saltbath. After one hour the excess reagent was quenched with phosphate buffer (pH 7) The reactionmixture was diluted with ether (200 ml) and extracted with phosphate buffer, water and brinethen dried over sodium sulphate. After solvent removal the product (1.41 g as an oil) wasdissolved in chloroform (5m1). The addition of carbon tetrachloride (10m1) caused precipitationof the pure mesylate (0.561 g) as a white powder The remaining product was chromatographedwith ethyl acetate (10%) in hexanes to provide a total of 1.11 g of mesylate 131 (2.97 mmol,88%). Melting point 124°- 126°C (decomp.). IR (CHC13; cm -1 ): 3000(sh), 2950(str), 2850(sh),1645(w), 1604(w), 1497(med), 1457(med), 1417(med), 1375(str), 1332(sh), 1187(str),1100(med), 1068(med), 1000(med), 960(str), 922(med), 828(med). 1H NMR (CDC13) 8: 7.18(1H, d, J= 8 Hz), 7.05 (1H, d, J= 8 Hz), 6.98 (1H, s), 5.56 (1H, s), 5.11 (1H, s), 3.29 (3H, s),3.02-2.93 (2H, mult), 2.93-2.81 (2H, mult), 2.64 (1H, did, J= 11, 3 Hz), 2.41 (1H, d/t, J= 14, 3Hz), 2.31 (1H, t/d, J= 14, 4 Hz), 2.00 (1H, t/d, J= 14, 4 Hz), 1.93-1.83 (2H, mult), 1.25 (6H, d, J=7 Hz), 1.03 (3H, s). LRMS m/z (rel intensity): 373(13, M+), 358(3), 279(20), 277(37), 262(78),249(5), 234(32), 220(100), 199(20), 193(33), 186(12), 171(14), 143(15), 129(15), 117(14),91(12), 79(7), 43(21). Elemental Analysis, calc. for C21H2703NS (373.51): C 67.53, H 7.29, N3.75; found: C 67.36, H 7.29, N 3.65 .14718(4-3)-abeoabieta-2,4,(19),8,11,13-pentaen-18-amide ( 89 )Potassium hydroxide (5% w/v in methanol, 0.5 ml, 0.45 mmol) was added to a solution ofmesylate (40 mg, 0.11 mmol) in THE (1.00 ml). The mixture was stirred at 22°C for one hourand was then acidified with 10% HCl aq and extracted into ether (3 x 25 ml). The solvent wasextracted with water and brine then dried over sodium sulphate. Solvent removal gave 35 mg of amore polar compound (TLC) as a brown oil. Preparative TLC using ethyl acetate (30) / toluene(70) provided $2 (22 mg, 0.074 mmol, 68%) as a colourless oil.IR (CHC13; cm -1 ): 3590(w), 3495(med, N-H stretch), 3400(med, N-H, stretch), 3350(sh),2980(sh), 2900(str), 2845(sh), 1644(str, C=0 stretch; amide), 1598(med), 1500(w), 1442(w),1380(w), 1320(str), 1224(str), 1155(str), 1102(w), 1002(med), 967(w), 900(med), 832(med). 1 HNMR (CDC13) 8: 7.22 (1H, d, J= 8 Hz), 7.06 (1H, d, J= 8 Hz), 6.98 (1H, s), 5.85 (1H, d, J= 1Hz), 5.37 (1H, s; broad; C2-H), 5.21 (1H, d, J= 1 Hz), 4.38 (2H, s; broad; D20 exchange; NH2),3.04-2.93 (2H, mult), 2.86 (1H, sept, J= 7 Hz), 2.54 (1H, d/t, J= 14,5 Hz), 2.48-2.39 (3H, mult),2.04 (1H, d/mult, J= 13 Hz), 1.90-1.78 (3H, mult), 1.24 (6H, d, J= 7 Hz), 1.11 (3H, s). LRMSm/z (rel intensity): 220(0.3), 211(0.2), 205(1.5), 160(2), 149(0.6), 110(6), 95(11), 82(36),67(52), 57(100), 43(57), 41(70). Calc. for Ca:a-1250N : 295.42 .18(4—>3)-abeoabieta-2,4,(19),8,11,13-pentaene-18-nitrile ( 90 )Cesium acetate (412 mg, 2.14 mmol; from cesium carbonate and acetic acid )was added to asolution of the mesylate E (200 mg, 0.535 mmol) in dry DMF (9 ml). The mixture was stirredunder nitrogen for 72 hours (room temperature) without reaction (TLC). When the mixture washeated to 60°C for 8 hours a new product was formed (less polar on TLC). The mixture wasdiluted with ether (150 ml) and extracted with water and brine then dried over sodium sulphate.Solvent removal gave a brown oil (190 mg). Preparative TLC using ethyl acetate (20) / hexanes(80) provided the unsaturated nitrile 2f2 (85 mg, 0.31 mmol, 57%) and unreacted starting material(75 mg, 37%). [Treatment of 2Q with methanolic KOH in THE provided a mixture of two newcompounds which were slightly less polar than the amide .a.2 (TLC). ]1481 H NMR (CDC13) 8: 7.19 (1H, d, J= 8 Hz), 7.07 (1H, d, J= 8 Hz), 6.97 (1H, s), 6.71 (1H, d, J= 6Hz), 5.58 (1H, s), 5.29 (1H, s), 2.98-2.82 (4H, mult), 2.56-2.47 (1H, mult), 2.43 (1H, d/d, J= 12,1Hz), 2.15 (1H, d/t, J= 12,2 Hz), 1.78-1.66 (1H, mult), 1.25 (6H, d, J= 7 Hz), 1.02 (3H, s). LRMSm/z (rel intensity): 277(34, m+), 262(40), 246(23), 234(24), 220(100), 204(12), 192(13),186(18), 178(10), 171(25), 165(10), 153(8), 143(22), 129(22), 117(21), 115(20), 91(15), 77(11),43(39). Calc. for C201-123N : 277.408 .3-acetoxy -18(4—>3) -abeoabieta -4(19),8,11,13 -tetraene - 18 -nitrile ( 88 )Triethyl amine (0.283 ml, 2.03 mmol), acetic anhydride (0.287 ml, 3.05 mmol) and 4-dimethyl-aminopyridine (12 mg, 0.10 mmol) were added to cyanohydrin 86 (300 mg, 1.02 mmol;containing an additional quantity of ketone B1) in dry dichloromethane (5 ml) with stirring underargon on an ice bath. After 40 minutes the reaction was allowed to warm to room temperatureand after a further 20 minutes the reaction mixture was quenched with aqueous phosphate buffer(pH 7). The mixture was extracted with ether (150 ml) and washed with phosphate buffer, waterand brine then dried over sodium sulphate. Solvent removal gave 348 mg of a white solid whichwas chromatographed in ethyl acetate(15) / hexanes(85) to provide the acetate fia (288 mg, 0.853mmol, 84%). IR (CC14; cm -1 ): 2950(str), 2860(sh), 1763(str), 1650(w), 1502(med), 1460(med),1377(str), 1230(str), 1187(med), 1110(med), 1045(med), 1020(w), 930(med), 835(w). 1H NMR(CDC13) 8: 7.19 (1H, d, J= 8 Hz), 7.05 (1H, d, J= 8 Hz), 6.98 (1H, s), 5.37 (1H, d, J= 1 Hz), 5.03(1H, d, J= 1 Hz), 3.05-2.92 (2H, mult), 2.90-2.82 (2H, mult), 2.70 (1H, did, J= 12,1 Hz), 2.35(1H, d/t, J= 13,3 Hz), 2.23 (3H, s, COCH3), 2.04 (1H, t/d, J= 13,2 Hz), 1.98-1.82 (3H, mult),1.24 (6H, d, J= 7 Hz), 1.02 (3H, s). Calc. for C22H2702N : 337.46 .3-methoxymethoxy - 18(4 —)3) -abeoabieta -4(19),8,11,13 - tetraene -18-nitrile ( 91 )Phosphorus pentoxide (P2O5, 4.80 g, 33.8 mmol) was slowly added to a rapidly stirred solutionof methylal (formaldehyde dimethyl acetal; 24 ml, 270 mmol) and cyanohydrin 1.6 (1.00 g, 3.38mmol, containing additional ketone in dry chloroform (20 ml) at room temperature under ablanket of argon. The mixture separated into an upper layer of pale yellow, transparent solution149and a lower layer of viscous red oil. The two phases were stirred together for five hours at whichtime the mixture was poured into an ice-cold solution of sodium carbonate (50 ml, 10% aqueous)with rapid stirring. This resulted in complete removal of the red material (P205/methylal). Thechloroform was removed and the aqueous material extracted with ether (3x50 ml). The pooledsolvent was extracted with water and brine then dried over sodium sulphate. Solvent removalgave 1.15 g of a yellow oil which was chromatographed with ethyl acetate(5) / hexanes(95) toprovide the methoxymethyl ether 21 (1.057 g, 3.11 mmol, 92%) as a colourless oil.IR (CC14; cm -1 ): 2940(str), 1725(w), 1646(med), 1611(w), 1500(med), 1460(str), 1440(sh),1378(med), 1302(w), 1218(med), 1182(str), 1120(str), 1057(sh), 1035(str), 982(med), 924(med),897(med), 827(med). 1 H NMR (CDC13) 5: 7.20 (1H, d, J= 8 Hz), 7.05 (1H, d, J= 8 Hz), 6.98(1H, s), 5.55 (1H, s), 5.14 (1H, d, J= 7 Hz, OCH2O), 5.01 (1H, s), 5.00 (1H, d, J= 7 Hz,OCH2O), 3.51 (3H, s), 3.02-2.95 (2H, mult), 2.86 (1H, sept, J= 7 Hz), 2.62 (1H, d/d, J= 10,3 Hz),2.53 (1H, d/d, J= 9,3 Hz), 2.36 (1H, d/d, J= 9,3 Hz), 1.96 (2H, d, J= 10 Hz), 1.93-1.82 (2H,mult), 1.25 (6H, d, J= 7 Hz), 1.02 (3H, s). Caic. for C22 H2902N : 339.45 .150REFERENCES^1^S. Z. Qian, Contraception 36 (3) 335-345 (1987)^2^X.-L. Tao, Y. Sun, Y. Dong, Y.-L. Xiao, D.-W. Hu, Y.-P. Shi, Q.-L. Thu, H. Dai,N.-Z. 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Fuji, S. Nakano, E. Fujita, Synthesis (April) 276-277 (1975)156172018 (4 --) 3) abeo-abietane( triptolides )181619APPENDIXAPPENDIX I30^2924Terpene Skeleton NumberingAPPENDIX IIAge(d)261-1 261-2 261-3 262-1 262-2 266-1 266-2 266-30 1.3371 1.3371 1.3371 1.3371 1.3371 1.3371 l^1.3371 1.33711234 1.3366 1.3367 1.33665 1.3364 1.3363 1.3367 1.3363 1.3363678 1.3360 1.3360 1.3362 1.3361 1.3361910 1.3358 1.3358 1.335911 1.3358 1.3357 1.335412 1.3351 1.334913 1.3352 1.3352 1.335114 1.3353 1.3351 1.334815 1.3343 1.3338161718 1.3334 1.3333 1.3344 1.3343 ,^1.333519 1.3340 1.3339 1.33382021 1.3336 1.3335 1.333222 1.3335 1.3335 1.3335Growth Curves for TRP 4-a in 12 L Aerated Bioreactors. RI25°C of filtered broth fromexperiments 266, 262, 261, 259, 258, 256, 255, 254, 252. Where not recorded, initial RI isestimated (1.3371). Double outlines mark the addition of Botrytis.^[plotted in figure 17]158APPENDIX II (cont.)Age(d)256-1 256-2 256-3 258-1 258-2 259-1 259-2 259-30 (1.3371) 1.3371 1.3371 1.3371 1.3371123456 1.3363 1.3365 1.33647 1.3362 1.3364 1.33638 1.33709 1.336610 1.3360 1.3360 1.3360 1.3360 1.3360^_. 1.336011 1.3360 1.33601213 1.3354 1.3353 1.3354 1.3355 1.3356 1.335814 1.3352 1.335215 1.3351 1.33501617 1.334818 1.3348 1.33441920 1.333921 1.334022 1.33342324 1.3336159APPENDIX II (cont.)Age(d)252-1 252-2 254-1 254-2 254-3 255-1 255-2 255-30 (1.3371)12345678 1.336291011 1.3359 1.3360 1.335912 1.3340 1.3349 1.33541314 1.3355 1.3353 1.335315 1.3349 1.3349 1.334916 1.3332 1.333617 1.3350 1.3349 1.335018 1.334019 1.3334 1.3333 1.3337 1.3334^I_ 1.3335202122 1.3335 1.3334 1.3335160APPENDIX IIICulture Age(days)Cell Fresh Weight(0--)Cell Dry Weight(g/1--,)Water Content(%)266-114 74.1 5.50 92.618 107 8.25 92.3^.21 113 11.6 89.7266-214 158 7.59 95.218 141 9.42 93.321 177 13.2 92.5266-314 185 10.2 94.518 228 16.7 92.721 226 15.8 93.0Culture Dry Weight in TRP 4a Series 266. Dry weights from the freeze-dried cells of 25.0 mlculture samples. The mean water content (± standard deviation) is 92.9 ±1.5 %.161ttp B lactone (blue)sitosterols (pink)ttp D lactone (purple)polpunonic acid (yellow)ttp A ester (pink)ttp B ester (blue)ttp C ester (pink)ttp D ester (purple)oleanolic acid (pink)salaspermic acid (yellow)ttp A acid (pink)ttp B acid (blue)ttp C acid (pink)ttp D acid (purple)APPENDIX IVReference TLC Chromatogram(a)^Diterpene standardsSamples:^(b)^Elicited Culture Extract Before Spray Reagent(c)^Elicited Culture Extract After Spray ReagentA silica gel TLC plate developed in toluene (35): chloroform (16):ethyl acetate (15): formic acid (1) and sprayed with:(i) 30% H2SO4 in glacial acetic acid(ii) 5% anisaldehyde in isopropanolfollowed by heating 5 minutes at 110°C. The triptolide spots are brownand the orange/brown quinone methides disappear after the spray is applied.162APPENDIX VCompound Parent Acid Retention Time (min.)methyl cholate cholic acid 17.6methyl oleanolate 42 22.0methyl polpunonate 62 25.2ttp B lactone 55. 28.2ttp D lactone 56 29.7ttp A ester a 30.9ttp B ester 52 32.1ttp C ester 51 32.8ttp D ester 54 34.1Retention Times in the Triterpene Analysis using Gas Chromatography1 methyl cholate2 up B lactone *3 ttp D lactone *4 ttp A ester' 5 ttp B ester6 ttp C ester7 ttp D ester* Triterpene lactones areformed from the methylesters on injection.Retention times vary withthe age of the column butpeak intervals are quiteconstant.APPENDIX V(continued)GC Chromatogram of a Mixture of Triterpene Esters (A.B.C.D)Co-injected with a Cell Culture Extract164

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