Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Biotransformation studies on tobacco cembranoids using plant cell cultures Li, Kai 1991

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1992_spring_li_kai.pdf [ 6.55MB ]
Metadata
JSON: 831-1.0061784.json
JSON-LD: 831-1.0061784-ld.json
RDF/XML (Pretty): 831-1.0061784-rdf.xml
RDF/JSON: 831-1.0061784-rdf.json
Turtle: 831-1.0061784-turtle.txt
N-Triples: 831-1.0061784-rdf-ntriples.txt
Original Record: 831-1.0061784-source.json
Full Text
831-1.0061784-fulltext.txt
Citation
831-1.0061784.ris

Full Text

Biotransformation Studies on Tobacco Cembranoidsusing Plant Cell CulturesByKai LiB.Sc. Jiangsu Teachers' College, China, 1982M.Sc., Suzhou University, China, 1985A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF CHEMISTRYWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIANovember 1991© K. Li, 1991In 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.(Signature)Department of Chemistry The University of British ColumbiaVancouver, CanadaDate: 30 November. 1991 AbstractThis thesis deals with the biotransformation studies on tobacco cembranoids using theplant cell culture lines coded as TRP4a and T-43-T which are derived from Tripterygiumwilfordii , an important Chinese herbal plant, and Nicotiana sylvestris, respectively.The studies are divided into three parts: 1) Biotransformation of the diols 1 and 2 usingthe TRP4a cell line. 2) Biotransformation of the diol 1 using the T-43-T cell line. 3)Biotransformation of cembranoid analogues using the T-43-T cell line.Incubation of the diol 1 with the growing cells of TRP4a and with cells resuspended inphosphate (pH 6.3) or TrisHC1 (pH 7.5) buffers for varying time periods affords five products4, 8, 92, 93 and 94. The epoxide 4 is the major product occurring in about 50% yield. Theallylic alcohols 92 and 93 are assigned as a pair of diastereoisomers with different chiralities atC-10 but their absolute configurations at this centre have not been established. Similarly, whenthe diol 2 is incubated with the growing cells of TRP4a, the epoxide 95 is obtained as a majorproduct in about 50% yield. Therefore, it is confirmed that the 11,12 double bond in the diols 1or 2 is the most active site for oxidative reactions. The reaction parameters such as cell age,buffer, pH, incubation time, substrate concentration and substrate administration methods havebeen investigated.No significant biotransformations are achieved when the cell free extract (CFE) and thecell homogenate prepared from the TRP4a cells are involved and the substrate diol 1 isrecovered in each case. However, the pellet fractions obtained during the centrifugation in thepreparation of CFE, when resuspended in phosphate buffer (pH 6.6) and with addition ofhydrogen peroxide, FMN and manganous chloride as cofactors, are capable of transforming thediol 1 into the epoxide 4 in about 40% yield.Biotransformation studies using the T-43-T cell line derived from Nicotiana sylvestris, atobacco species in which the diols 1 and 2 as well as many other cembranoids are encountered,indicate that the growing cells are capable of transforming the diol 1 into the epoxide 4 and theallylic alcohols, but only in low yields when compared to the data with TRP4a. The epoxide 4,which is a major product in studies with the TRP4a cell line, is a minor component (about 20%)in the product mixture resulting from experiments with T-43-T cell line. On the other hand, theC-10 and C-12 alcohols, 93 and 8 respectively, are obtained in relatively higher yields (20-29%).However, in contrast to the data obtained with TRP4a, when such cofactors as hydrogenperoxide, FMN and manganese chloride are added, both the T-43-T cell homogenate and thepellets resuspended in phosphate buffer (pH 6.6) afford good yields of the epoxide 4 (72% and62%, respectively) in biotransformation experiments with the diol 1.Biotransformation of cembranoid analogues using the T-43-T cell line was alsoinvestigated. The growing cells can transform the epoxide 4 into the triol 109, a product formedby hydroxylation at the unactivated methine position C-15, in 55% yield.In experiments with cell homogenates obtained from the T-43-T cell line, epoxidation isobserved in the enone 43 and the seco-diketone 44 and epoxides 7 and 107 are obtainedrespectively. On the other hand, the aldehyde 32 is reduced selectively to the alcohol 110,which in turn, undergoes a cyclization to the ether 111 in a SN2'-like manner in the presence ofsuch cofactors as hydrogen peroxide, manganous chloride and FMN.The tetrol 104, when exposed to cell homogenate, is oxidized at C-6 and the latterintermediate undergoes a spontaneous intra-molecular Michael addition to give the ether 112 inapproximately 20% yield. In addition, the ethers 113 and 114 are obtained in very smallamounts.In conclusion, the T-43-T cell line is capable of performing both oxidation and reductionreactions, namely selective epoxidation of the 11,12 double bond, hydroxylation at the allylicpositions (C-10 and C-12), hydroxylation at a non-activated methine position (C-15) andselective reduction of aldehyde to the corresponding alcohol.iii43().\ --\ ... z(),,/^/ \789495 107 109110^ 111^ 112^113114ivTable of ContentsTitle page^Abstract IITable of Contents VList of Schemes^ VIIIList of Figures IXList of Tables IXList of abbreviation^ XIAcknowledgements XIII1 INTRODUCTION 11.1 Tobacco cembranoids^  11.1.1^Structures and Nomenclatures^  31.1.2^Biological considerations 41.1.3^Biogenesis ^  51.1.4^Synthesis of cembranoids^ 71.2 Biogenetic considerations for cembranoid transformations^ 101.2.1^Oxidation of the 11,12 double bond^  111.2.2^Oxidation of the 7,8 double bond  151.2.3^Oxidation of the hydroxyl group at C-6  161.2.4^Acid-induced reactions^ 201.2.5 Degraded cembranoids 221.3 Plant tissue cultures^ 301.3.1^Applications of plant tissue cultures in biotransformation studies^ 301.3.2^Oxidative reactions in biotransformation studies using plant cell cultures^ 371.3.3^Reductive reactions in biotransformation studies using plant cell cultures^ 421.3.4^Plant cell lines developed and available in our laboratory^ 431.4 Objectives of the project^ 442 RESULTS AND DISCUSSION 462.1 Biotransformation of cembranoids using the TRP4a cell line^ 462.1.1^Biotransformation of the diols 1 and 2 using the whole cells ^ 542.1.2^Chemical studies on the cembranoids: preparation of cembranoid analogues ^ 702.1.3^Biotransformation of the diol 1 using cell free extract (CFE) ^ 742.1.4^Biotransformation of the diol 1 using the cell homogenate and the pellet ^ 782.2 Biotransformation of cembranoids using the T-43-T cell line^ 842.2.1^Biotransformation of the diol 1 using the whole cells  842.2.2 Biotransformation of the diol 1 using the CFE prepared fromthe T-43-T cell line^  86^2.2.3^Biotransformation of the diol 1 using the cell homogenate andthe resuspended pellet prepared from the T-43-T cell line^ 872.3 Biotransformation of cembranoid analogues using the T-43-T cell line 922.3.1^Biotransformtion of the epoxide 4 using the T-43-T whole cells^ 922.3.2^Biotransformation of the enone 43 using the cell homogenate 952.3.3^Biotransformation of the seco-diketone 44 and seco-epoxide 107using the cell homogenate^ 972.3.4^Biotransformation of the seco-aldehyde 32 using the cell homogenate^ 992.3.5^Biotransformation of the tetrol 104 using the cell homogenate^ 1032.4 Overall conclusions^  1062.5 Further Research directions  1063 EXPERIMENTAL 1073.1 Chemical conversions of diol 1 to cembranoid analogues^ 1083.1.1^Conversion of diol 1 into (1S, 2E, 4S, 6R, 7E, 11E)-6-acetoxy-2,7,11-cembratriene-4-ol 97 and (1S, 2E, 4S, 6R, 7E, 11S, 12S)-6-acetoxy-2,7-cembradiene-4,11,12-triol 98 ^  1083.1.2^Conversion of triol 98 to (4E, 6R, 8S, 9E, 11S)-6-acetoxy-4, 8-dimethy1-8-hydroxy-11-isopropy1-14-oxo-4, 9-pentadecadienal 99 ^ 1093.1.3^Conversion of 99 to methyl (4E, 6R, 8S, 9E, 11S)-6-acetoxy-4, 8-dimethy1-8-hydroxy-11-isopropy1-14-oxo-4, 9-pentadecadienoate 100 ^ 1103.1.4^Conversion of 100 to methyl (4E, 6R, 8S, 9E, 11S)-6,8-dihydroxy-4, 8-dimethy1-11-isopropy1-14-oxo-4, 9-pentadecadienoate 101 ^ 1113.1.5^Conversion of 101 to methyl 4-methyl-6-oxo-heptene-4-oate 103 and(3E, 5S)-5-isopropyl-3-nonene-2,8-dione, norsolanadione, 78 ^ 1123.1.6^Conversion of diol 1 to (1S, 2E, 4S, 6R, 7E, 11S, 12S)-2,7-cembradiene-4,6,11,12-tetrol 104 ^  1133.1.7^Conversion of tetrol 104 to (4E, 6R, 8S, 9E, 11S)-4,8-dimethy1-6,8-dihydroxy-11-isopropy1-14-oxo-4,9-pentadecadienal 32 ^ 1143.1.8^Conversion of 32 to (4E, 8S, 9E, 11S)-4,8-dimethy1-8-hydroxy-11-isopropy1-6,14-dioxo-4,9-pentadecadienal 106 and nor-solanadione 78^ 1153.1.9^Conversion of diol 1 to (15, 2E, 4S, 7E, 11E)-2,7,11-cembratriene-4-o1-6-one 43 ^  1163.1.10 Conversion of 43 to (3E, 7E, 11S, 12E)-11-isopropy1-4,8-dimethy1-3,7,12-pentadecatriene-2,14-dione 44 ^  116vi3.1.11 Conversion of 32 to (4E, 6R, 8S, 9E, 11S)-4,8-dimethy1-11-isopropy1-14-oxo-4,9-pentadecadiene-1,6,8-triol 110^  1173.1.12 Conversion of epoxide 4 to (1S, 2E, 4S, 7E, 11S, 12S)-11,12-epoxy-4-hydroxy-2,7-cembradien-6-one 7  1183.1.13 Conversion of 7 to (3E, 7S, 8S, 11S, 12E)-4, 8-dimethy1-7,8-epoxy-11-isopropy1-3, 7, 12-pentadecadiene-2, 14-dione 107^ 1193.2^Propagation of the plant cell cultures^  120^3.2.1^Propagation of the TRP4a cell culture  1203.2.2^Propagation of the T-43-T cell culture  1203.3 Biotransformation using the TRP4a cell line^  1213.3.1^Biotransformation using the whole cells  1213.3.2^Biotransformation using the CFE 1283.3.3^Biotransformation using the cell homogenate, resuspended pellet andsupernatant (CFE) with cofactors^  1303.4 Biotransformation using the T-43-T cell line^  1323.4.1^Typical procedure for biotransformation with batchwise addition of the diol 1to the growing cell suspension culture  1323.4.2^Typical procedure for biotransformation with addition of the diol 1 to thegrowing cells via peristaltic pump^ 1333.4.3^Typical procedure for biotransformation of the diol 1 usingthe cell homogenate ^  1333.4.4^Typical procedure for biotransformation of the diol 1 using the CFE^ 1343.4.5^Typical procedure for biotransformation of the diol 1 using theresuspended pellet^  1343.5 Biotransformation of cembranoid analogues using the T-43-T cell line^ 1343.5.1^Biotransformation of the epoxide 4 using the whole cells  1343.5.2^Biotransformation of (1S, 2E, 4S, 7E, 11E)-4-hydroxy-2,7,11-cembratriene-6-one 43 using the cell homogenate ^  1353.5.3^Biotransformation of (3E, 7E, 11S, 12E)-11-isopropy1-4,8-dimethy1-3,7,12-pentadecatriene-2,14-dione 44 using the cell homogenate ^ 1363.5.4^Biotransformation of (4E, 6R, 8S, 9E, 11S)-48-dimethy1-68-dihydroxy-11-isopropy1-14-oxo-4,9-pentadecadienal 32 using the cell homogenate^ 1373.5.5^Biotransformation of (15, 2E, 4S, 6R, 7E, 11S, 12S)-2,7-cembradiene-4,6,11,12-tetrol 104 using the cell homogenate^  138APPENDIX 140REFERENCES ^ 147viiList of SchemesScheme 1, Proposed biosynthetic pathways for the diols 1 and 2 ^ 6Scheme 2, Synthesis of a-diol 1 ^ 8Scheme 3, Proposed biogenetic pathways leading to the triols 8 and 9 ^ 12Scheme 4, Products obtained from photooxygenation of the diol 1 followed byreduction with triethylphosphite^  13Scheme 5, Epoxidation of the 11,12 double bond in the diol 1 ^ 13Scheme 6, Formation and acid-induced reaction of the epoxide 4  14Scheme 7, Epoxidation of the 7,8 double bond in the diol 1  15Scheme 8, Proposed biogenetic pathways related to the oxidation at C-6 ^ 17Scheme 9, Products obtained from photooxygenation of the ketol 43 followed byreduction with triethyl phosphite^  18Scheme 10, Products obtained from acid-induced rearrangement of the epoxide 7^ 19Scheme 11, Proposed pathways via acid-induced rearrangements of the diol 1 ^ 20Scheme 12, Products obtained in acid-induced rearrangements of the diol 1  21Scheme 13, Formation of the (8S, 11R)-8,11-epoxy-diol 57 ^ 22Scheme 14, Formation of the C19 compound 61 ^  24Scheme 15, Formation of the C18 compound 63  24Scheme 16, Formation of the C15 compounds  26Scheme 17, Formation of the C14 compounds ^  27Scheme 18, Formation of solanone 73 and the other C13 compounds^ 28Scheme 19, Formation of nor-solanadione 78 and the other C12 compounds ^ 29Scheme 20, The development of a plant cell suspension culture  32Scheme 21, The preparation of cell free extract (CFE) ^  35Scheme 22, Production of 3',4'-anhydrovinblastine 91 by CFE from C. roseus ^ 37Scheme 23, Some examples of allylic hydroxylation using plant cell cultures  41Scheme 24, Some examples of reduction using plant cell cultures ^ 43Scheme 25, Oxidative coupling leading to lignan 118 ^  44Scheme 26, Proposed objective for cembranoid biotransformation ^ 45Scheme 27, Biotransformation of the diol 1 using TRP4a cell line 47Scheme 28, The mass spectrometric fragmentation pattern of the diol 1^ 51Scheme 29, The mass spectrometric fragmentation pattern of the triol 92 52Scheme 30, Biotransformation of the diol 2 using TRP4a cell line ^ 54Scheme 31, Procedures for biotransformation using re-suspension culture of TRP4a ^ 55Scheme 32, Chemical conversion of the diol 1 to norsolanadione 78 ^ 71VIIIScheme 33, Alternative way to norsolanadione 78 ^ 72Scheme 34, Conversion of the diol 1 to seco-diketone 44 73Scheme 35, Conversion of the epoxide 4 to 7 and 107 73Scheme 36, Conversion of the seco-aldehyde 32 to alcohol 110 ^ 74Scheme 37, Preparation of CFE and biotransformation using CFE 76Scheme 38, Preparation of cell homogenate and resuspended pellet andbiotransformation^ 79Scheme 39, Biotransformation of the epoxide 4 to 109 ^ 93Scheme 40, Chemical conversion of the diol 1 to enone 43 and biotransformationof 43 to 7 ^ 95Scheme 41, Chemical conversion of 43 into 44 and biotransformation of 44 to 107 ^ 97Scheme 42, Chemical conversion of the epoxide 4 to 107^ 99Scheme 43, Biotransformation of the seco-aldehyde 32 into 110 and 111 ^ 100Scheme 44, Biotransformation of the tetrol 104 ^  103List of FiguresFigure 1, Some cembranoids isolated from tobacco ^ 2Figure 2, Fundamental carbon skeletons of cembranoid 10, labdanoid 11 andcarotenoid 12 ^ 2Figure 3, Degradation patterns of cembranoids ^ 23Figure 4, Structure of iron protoporphyrin IX and catalytic cycle of cytochrome P-450 ^ 38Figure 5, 1H NMR spectrum of the diol 1 in CDC13 (400 MHz)^ 140Figure 6, 1H NMR spectrum of nor-sorlanadione 78 in CDC13 (400 MHz)^ 141Figure 7, 1H NMR spectrum of the triol 92 in CDC13 (400 MHz) 142Figure 8, 1H NMR spectrum of the trio! 93 in CDC13 (400 MHz)^ 143Figure 9, 1H NMR spectrum of the triol 94 in CDC13 (400 MHz) 144Figure 10, 1H NMR spectrum of the seco-alcohol 110 in CDC13 (400 MHz)^ 145Figure 11, 1H NMR spectrum of the epoxide 109 in CDC13 (200 MHz) 146List of TablesTable 1, Concentrations of the diols 1 and 2 in the leaf surface gum of differentN. tabacum varieties ^ 4Table 2, Concentrations of diols 1 and 2 in N. tobacum organs ^ 5Table 3, 13 C NMR chemical shifts (ppm) of cembranoids in CDC13 48ixTable 4, Effect of pH on biotransformation of the diol 1 ^ 57Table 5, Effect of pH on biotransformation of the diol 2 57Table 6, Effect of increasing substrate concentration on biotransformation of the diol 1^ 59Table 7, Effect of cell age on the biotransformation of the diol 1 with direct substrate ^ 62Table 8, Effects of substrate concentration on biotransforamtion of the diol 1 ^ 63Table 9, Biotransformation by batchwise addition of the diol 1 to cell suspensionculture ^ 64Table 10, Biotransformation by semi-continual addition of diol 1 via a peristaltic pump ^ 65Table 11, GC conditions and retention times^ 67Table 12, GC results of biotransformation of diol 1 67Table 13, Biotransformation of diol 1 with the CFE prepared from TRP4a cell line^ 78Table 14, Biotransformation of diol 1 with the cell homogenate, pellet and supernatantprepared from TRP4a cell line^  81Table 15, The effects of manganese chloride and FMN on biotransformation of diol 1 ^with resuspended pellet 83Table 16, Biotransformation with batch-wise addition of substrate to T-43-T whole cells ^ 85Table 17, Biotransformation of the diol 1 with semi-continual addition of substrate toT-43-T whole cells via peristaltic pump ^ 86Table 18, Biotransformation of the diol 1 with the CFE prepared from the T-43-T cellline ^ 87Table 19, Biotransformation of the diol 1 with the cell homogenate and resuspendedpellet prepared from T-43-T cell line^ 89Table 20, Biotransformation of diol 1 with cell homogenate at a longer incubation time ^ 90Table 21, Biotransformation with the T-43-T cell homogenate without co-factors. ^ 91Table 22, Further biotransformation of the epoxide 4 with the T-43-T whole cells ^ 93Table 23, 13C NMR Chemical shifts (CDC13) of the epoxide 4 and the triol 109 ^ 94Table 24, Biotransformation of the enone 43 with the T-43-T cell homogenate^ 96Table 25, 13C NMR chemical shifts (ppm) in CDC13^ 96Table 26, Biotransformation of the seco-diketone 44 with the T-43-T cell homogenate ^ 98Table 27, Biotransformation of the seco-aldehyde 32 with the T-43-T cell homogenate ^ 101Table 28, 13C NMR chemical shifts (ppm) in CDC13^ 102Table 29, Biotransformation of the tetrol 104 with the T-43-T cell homogenate ^ 104List of abbreviations[alD^Specific optical rotation recorded at ambient temperature (230C)using sodium D-lineAc^acetylAPT^attached proton testbr broadc^concentration (g/100m1)CFE^cell free extractsCI-MS^mass spectrum with chemical ionizationcm-1^wave number8 chemical shift relative to TMSd^doubletdd doublet of doubletsdt^doublet of tripletsEI-MS^mass spectrum with electron impacteq. equivalentGC^gas-liquid chromatographyh hoursHRMS^high resolution mass spectroscopyHz hertzIR^infraredJ coupling constantL^litreLRMS^low resolution mass spectroscopyM molarM+^molecular ionmCPBA^meta-chloroperoxybenzoic acidximg^milligramsMHz^megahertzml millilitermmol^millimolemp melting pointMS^mass spectroscopyMS medium^Murashige and Skoog's nutrient mediumm/z^mass to charge ratio25nr,^refractive index recorded at 25°C using sodium D-linenm^nanometerNMR^nuclear magnetic resonancePCC^pyridinium chlorochromateppm^parts per millionPRDCo^PRL-4 medium[89] supplemented with 2,4-dichlorophenoxyaceticacid (D) (2 mg/L) and coconut milk (100 ml/L)rpm^rotation per minutes singlett^tripletTBHP^t-butyl hydroperoxideTLC^thin layer chromatographyTMS^tetramethylsilaneTrizma®^tris(hydroxymethyl)aminomethaneTRP4a^plant cell line derived from Tripterygium wilfordiiT-43-T^plant cell line derived from Nicotiana sylvestrisxiiACKNOWLEDGEMENTSI wish to express my appreciation to Professor James P. Kutney for his valuableadvice, both during the progress of this research and in the preparation of this thesis.I would also like to thank Dr. Alexis W. L. Chu, Ms. Radka Milanova, Dr.Hiroyuki Nakata, Dr. Yoshi Okada and Dr. Tadashi Tsuda for their collaboration and help.Thanks must also go to Mr. Gary M. Hewitt, Ms. Fay Hutton and Mr. David Y. P.Chen for their expertises, technical help and willingness to provide the various plant celllines, to Mr. Francisco Kuri-Brena and Mr. Caries Cirera for their suggestions, discussionand proofreading.At the same time, I wish to thank all the members (both past and present) of Dr.Kutney's group for their friendship.Financial aid from the People's Republc of China, the Universaity of BritishColumbia and the Swedish Tobacco Company, Stockholm, Sweden is gratefullyacknowledged.Finally, I am deeply indebted to my wife for her support and encouragement.1 INTRODUCTION1.1 Tobacco cembranoidsApart from cotton and food-providing plants, tobacco is one of the most importantcultivated plants in the world. Even though there are more than sixty different species, most ofthe commercial tobacco products are prepared from one species only, Nicotiana tabacum which isa hybrid between N. sylvestris and N. tomentostformis .[ 1] N. tabacum has not been foundgrowing in a wild environment but only as a cultivated species. Due to their economic importanceboth in producing and consuming countries, together with the health factors associated with theiruses, tobacco plants have been subjected to detailed chemical and biological studies.The extensive studies lead to the results that, besides nicotine which is a well knownpharmacologically active compound, about 2500 chemical constituents have been isolated andidentified so far from tobacco and its smoke.[ 2,3] Among them are isoprenoids, one of the majorclasses of tobacco chemical constituents. The content of isoprenoids has been examinedthoroughly and the results show that mono-, sesqui-, di- and triterpenoids, carotenoids or evenhigher isoprenoids have been found. [3, 4]As far as tobacco aromas are concerned, carotenoids, labdanoids and cembranoids are thethree major sources in the isoprenoid family.[3,4] It is a well known fact that the typical aroma ofthe tobacco leaf is created during the post-harvest treatment, which involves air-, fire-, or sun-curing and aging. These processes lead to very substantial chemical changes and thus account forthe generation of flavorants. Among the three major sources, the 14-membered macrocycliccembranoids, which belong to the diterpenoid class, have received considerable attention becausethese compounds are prone to undergo biodegradation and give rise to a variety of volatile, lowmolecular weight compounds, many of which are important flavour substances therebyproviding significant contributions to the aromas of commercial tobaccos.1 511HO1 24 5 61 0117HO8Figure 1, Some cembranoids isolated from tobaccoHOsoOH9II I2Figure 2, Fundamental carbon skeletons of cembranoid 10, labdanoid 11 and carotenoid 12III1.1.1 Structures and NomenclaturesThe discovery of cembranoids in tobacco dates back to the early 1960's.[ 61 Sincethen, more than fifty cembranoids have been reported as constituents of tobacco. (See Figure1 for representative examples). All of these natural products, with the exception of 3, have(1S, 2E) configuration, indicating that a close biogenetic relationship exists within the group.Among the cembranoids isolated so far from tobacco, two epimeric diols, 1 and 2, are themajor components and, in fact, the first examples of macrocyclic diterpenes containing the14-membered ring system.[7] These two diols were first isolated in 1962. Their relativestereochemistry and absolute configurations were solved by X-ray analysis [ 8] , ozonolyticdegradations [9] and chemical correlations [ 10].HOHOOH^ OH3181 2 1 0Cembrane nomenclature, which is based on the structure of cembrane 10, is usedthroughout this thesis. Thus, the name for 1 is (1S, 2E, 4S, 6R, 7E, 11E)-2,7,11-cembratriene-4,6-diol, or "a-diol" according to the orientation of the hydroxyl group at C-4.In the literature, this compound is also named as (1S, 2E, 4S, 6R, 7E, 11E)-2,7,11-thunbergatriene-4,6-diol and a-4,8,13-duvantriene-1,3-diol based on the thunbergane andduvane nomenclature for its parent saturated compound 10. The former name derives fromPinus thunbergii, a species name for pine, the latter is a Serbian word for tobacco. InChemical Abstracts, the current index name for the diol 1 is 1S-(1R*, 3S*, 4E, 8E, 12R*,13E)-1,5,9-trimethy1-12-(1-methylethyl)-4,8,13-cyclotetradecatriene-1,3-diol. All of thesenames can be found in the literature. The diol 2 differs from the diol 1 only in thestereochemistry at C-4 and is referred to, in abbreviated form, as "0-diol".41.1.2 Biological considerationsDepending on the genetic background, tobacco plants produce cembranoids,labdanoids, or both types of cyclic diterpenoids. For example, Oriental tobaccos usuallyproduce both cembranoids and labdanoids while Virginia and Burley tobaccos synthesizecembranoids exclusively.[11] These genetic differences relate back to the fact that Nicotianatabacum is a hybrid between N. sylvestris (female) and N. tomentosiformis (male). Thesespecies synthesize cembranoids and labdanoids, respectively.[Lill It is found that thecembranoids are synthesized in the glandular heads of the trichome on the surface of theleaf and in the flower [12, 131 and are present in the gummy exudates. They can be isolatedin high yield from cuticular wax extracts, where they co-occur with aliphatic hydrocarbons,fatty alcohols, wax esters and sucrose esters.[ 14] The exudate of tobacco leaves can accountfor 0.5-10% of the fresh weight of the leaves, depending on variety [ 11] and thecembranoid content in the leaf exudate may be as high as 75%.[ 15] It is reported that thediols 1 and 2 constitute 0.01% and 0.005%, respectively, of the dry burley leaf.[6] As amater of fact, the abundance of these cembranoids depends on the genetic background aswell as the edaphic conditions, even the leaf positions. [15, 16] post-harvest treatment alsocan affect its compositions.[17]Table 1, The concentrations of diols 1 and 2 in the leaf surface gum of different N.tabacum varieties [151N. tabacum variety (1 + 2)%Ti 1112 0.5Virgin Bright 5NC 2326 7Vinica 8Burley B5 8Amarelinho 25Kurztag 75Table 2, Concentrations of the diols 1 and 2 in N. tabacum organs [ 161Plant organ Fresh Weight of % Ratioweight (g) 1 & 2 (mg) (1 : 2)Young up haves 50.1 73 0.15 43:57Old lower leaves 47.1 20 0.04 39:61Immature petals 3.7 18 0.49 76:24Mature petals 10.5 61 0.58 75:25Mature calyces 3.0 70 2.33 76:24Fertilized calyces 2.5 63 2.52 60:40While the physiological functions of the tobacco cembranoids still have not beenestablished, it has been shown that both 1 and 2 possess the abilities to inhibit plant growth[18] and tumor promotion [ 191 , and to repel insects.[20] It is also reported that they canstimulate oviposition activity of tobacco budworm [21] and inhibit germination of fungalspores [22]. These properties could play an important role in the development of pest-resistant high-flavor tobaccos.1.1.3^BiogenesisLittle is known about the biogenesis of the tobacco cembranoids compared to that oftobacco alkaloids. This could be due to the fact that the tobacco cembranoids aresynthesized in the leaf trichome, a site difficult to access for exogenous labelled precursors.Biogenesis of diols 1 and 2 is under investigation and according to recent studies usingisotopically labelled precursors [ 16], these diols seem to arise from geranylgeraniol 13, anaccepted precursor in terpenoid synthesis. The mechanism by which geranylgeraniol 13 iscoverted into the cyclopropane ent-casbene 14 remains unclear although thestereochemistry of the process has been studied and a possible mechanism proposed.[231 Itis considered that in the formation of the cyclopropane ring, the pro-S hydrogen at C-1 of51\^9 7\ 516 14 12^10 8^6^4 OHIIII16III 16Acetate Mevalonic acid13 1442Scheme 1, Proposed biosynthetic pathways for diols 1 and 2geranylgeranyl pyrophosphate is eliminated and the methyl groups on the cyclopropanering are formed by suprafacial attack on the re -re face of the 14,15 double bond of thegeranylgeranyl pyrophosphate. Ent-casbene 14 thus formed is then converted to cembrene15 (also a constituent of tobacco), via 5-hydroxylation, solvolysis and reduction. Anstereospecific enzymatic hydroxylation of cembrene 15 at the allylic C-6 position gives2,4,7-cembratetraene-6-ol 16 and hydration of the 4,5 double bond leads to the finalproducts, 1 and 2.Geranylgeraniol 13 itself can be formed through the usual mevalonate pathway [24].It has been found that all the radiolabelled sodium [1- 14g-acetate, [2- 14C)-mevalonate, all-(E)-[2- 14C]-geranylgeraniol and [3- 14 C]-cembrene can be incorporated intocembranoids .[ 16]1.1.4 Synthesis of cembranoidsRecently, significant advances have been made in the synthesis of cembranoids andseveral new approaches have been reported.[25] One of the synthetic routes to the diol 1developed by Marshall et al. [26, 27, 28] is via the 17-membered ether 23, which is readilyprepared from the acetate of trans, trans -farnesol 17. By selective allylic oxidation of the E-isopropylidene methyl group using selenium dioxide and tert-butyl hydroperoxide,silylation of the newly formed allylic alcohol with tert-butyldimethylsilyl chloride (TBSC1)and 4-N, N-dimethylaminopyridine (DMAP) followed by cleavage of acetate, the alcohol18 is obtained.[29] Swern oxidation [30] using oxalyl chloride and dimethyl sulfoxide leadsto aldehyde 19. Addition of propargylmagnesium bromide followed by alcohol protectionwith 2,3-dihydropyran (DHP) affords the alkyne 20. The latter, upon desilylation withtert-butylammonium fluoride in THF, affords an alcohol which is converted to the allylicchloride 21 by treatment with methanesulfonyl chloride (MsC1) and lithium chloride (LiC1)according to the Collington-Mayers' procedure.[ 31 ] Treatment of the lithiated acetylide with7OTBSOTBS1)BrMgCH2C--=- CHEt20, 96% 2) DHP, CH2Cl2 , 96%0N1) n-BuLi,TMEDAOH^90%2) PPTS, Me0H4 ..,..<^85%3) TBSC1,DMF, ImROTHPO24 R=THP25 R=TBS 231) Bu4NF, THF98%2) LiCI, MsCI84%Cl n-BuLi,(CH 20)nTHF, 83%19EtMgBrTHF-HMPA84%0^VTHPO2 1221) Se02/t-BuO0HCH2 C1 2 , 25% OH^OTBS82) TBSCI/DMAP3) K2CO 3/Me0H85% THPO17 18(C0C1)2/DMS0Et3 N/CH2C1 292%Scheme 2, Synthesis of a -diol 1TBSOOH (COC1)2DMSO, CH2C1 226Me2CuLiEt2O90%TBSO1) (iBu)2A1HTHF, 96%2) (Ph3P)3R h ClC6H 6, EtOHH2, 69% 272591) VO(acac)2, t-BtOOH, 88%2) CH3S 0 2C1,C 511 5N 92%3) Bu4NF,THF, 92%Na, NH3THF,75 %29^ 1Scheme 2, Synthesis of a -diol 1 (continued)paraformaldehyde gives the chloroalcohol 22 which cyclizes to the ether 23 upon lowtemperature (0°C) addition of 1 equivalent of ethylmagnesium bromide in tetrahydrofuranand hexamethylphosphorylamide (THF-HMPA) followed by reflux.The THP ether 23 readily undergoes a [2,31-Wittig rearrangement with n-butyllithium in a mixture of THF, pentane and N,N,1•1 1 ,1T-tetramethylethane-1,2-diamine(TMEDA) at -78°C and gives the ring contraction product 24 as a major diastereomer.[26]Hydrolysis of 24 with pyridinium p-toluenesulfonate (PPTS) in methanol followed bysilylation with tert-butyldimethylsilyl chloride (TBSC1) converts 24 into 25, the latterbeing a better substrate than 24 for further reactions.Swern oxidation of 25 gives the ketone 26. Treatment of 26 with lithium dimethylcopper yields the enone 27 which is then reduced with di-isobutyl aluminium hydride andfinally hydrogenated selectively with Wilkinson's catalyst to afford alcohol 28 in 69%yield. Epoxidation of the allylic alcohol 28 with vanadyl acetylacetonate and tert-butylhydroperoxide affords a single epoxide which is then converted to mesylate 29 throughtreatment with methanesulfonyl chloride in pyridine followed by desilylation. Reductiveelimination with sodium ammonia affords the final product, a-diol 1. The (3-diol 2 can alsobe synthesized in a similar manner. [27]1.2 Biogenetic considerations for cembranoid transformationFrom the results obtained during structure elucidation of compounds isolated fromtobacco and "biomimetic" interconversions, it was recognized that the diols 1 and 2 are thekey intermediates in the biogenesis of the majority of the other tobacco cembranoids. Themetabolic transformations are postulated to be initiated by1) oxidation of the 11,12 double bond2) oxidation of the 7,8 double bond10113) oxidation of the hydroxyl group at C-64) acid-induced rearrangements1.2.1 Oxidation of the 11,12 double bondFor the sake of clarity, only the 4S epimer, diol 1, which is the more abundantepimer of the two epimeric diols in tobacco, is discussed below.Oxidation of the trisubstituted 11,12 double bond in the diol 1 is a major routeleading to the formation of a number of tobacco constituents .By the first of the two principal pathways (Scheme 3), the diol 1 is converted intothe 11- and 12-hydroperoxides 5 and 6. This could occur either by photo-oxygenation or,more probably, through the assistance of an oxygenase, because both processes can lead tothe formation of hydroperoxides. The most direct evidence is that these twohydroperoxides have been isolated from the flowers of Greek tobacco.[ 32] They areobvious intermediates in the biogenesis of the 4, 6, 11-triol 8 and the 4, 6, 12-triol 9.The 11- and 12-hydroperoxides 5 and 6 are also likely to cleave the 11,12 bond, assuggested by the isolation of the seco-aldehyde 32.[ 33 ] This compound could undergooxidation to give the seco-acid 33, which has been known as a component of tobacco.[ 341In agreement with this, the diol 1 reacts smoothly with singlet oxygen at the 11, 12-double bond, giving the two 4,6,11-triols, 9, 34, and the two 4,6,12-triols, 8, 35, in theratio of 63:1:31:5 after reduction of the initially generated hydroperoxides using triethylphosphite (Scheme 4).[35] This sensitized photooxygenation proceeds by a preferentialattack of singlet oxygen on the trisubstituted 11,12 double bond both regio- andstereospecifically, leaving the trisubstituted 7,8 double bond, which is less active due to thedeactivating effect of the hydroxyl group at C-6, unattacked.Epoxidation of the 11,12 double bond in the diol 1 is the second principal pathwayand leads to the formation of the (11S, 12S)-11,12-epoxide 4, which is an abundant1I I1218 9Scheme 3, Proposed biogenetic pathways leading to triols 8 and 9a+1 +13b3943 4^ 351% 5%Scheme 4, Products obtained from photooxygenation of the diol 1followed by reduction with triethylphosphiteScheme 5, Epoxidation of the 11,12 double bond in the diol 1IIImCPBA1 4IIIa39IIIIII<HO 3 7Scheme 6, Formation and acid-induced reaction of epoxide 43638tobacco cembranoid (Scheme 5).[ 10] This reaction can be accomplished by oxidation of thediol 1 with m-chloroperoxybenzoic acid (m-CPBA) and shows a regioselectivity reminiscentof that exhibited by its reaction with singlet oxygen [ 36]. The epoxide 4 undergoes an acid-induced rearrangement to form 8, 11- and 8, 12-epoxy-diols 36 and 37 which have beenisolated from Greek tobacco.[37, 38] Subsequent dehydration leads to the formation ofcompounds 38 and 39, which have also been found in various tobaccos.[ 9, 39]The validity of the biogenetic pathway shown in Scheme 5 has been reinforced bybiomimetic experiments.[36] Thus, treatment of the epoxide 4 with dilute hydrochloric acid indioxane-water affords four major products 36, 37, 38 and 39 (Scheme 6).141 41 4276% 24%...< ButOOHVO(acac) 215The generation of 36 is explicable by the anti-addition of water to the 11,12-epoxide group followed by an attack of the newly formed 11-hydroxyl group on the 7,8double bond and the simultaneous elimination of the hydroxyl group at C-6 by an SN2'mechanism. An analogous attack of the 12-hydroxyl group on the 7,8 double bond wouldbe involved in the reaction leading to the 8,12-epoxy-diol 37. Elimination of water from36 leads to the alcohols 38 and 39.1.2.2 Oxidation of the 7,8 double bondOxidation of the 7,8 double bond in the diol 1 is less common than that of the11,12 bond and only two compounds in which the vulnerable 11,12 double bond isretained have so far been isolated from tobacco in minute quantities. These two compoundshave been identified as the (7S, 8S)-7,8-epoxide 41 and the (7R, 8R)-7,8-epoxides 42 byX-ray analysis and synthesis.[ 401 Thus, treatment of the diol 1 with t-butyl hydroperoxideand a catalytic amount of vanadyl acetylacetonate results in a regiospecific epoxidation ofthe 7,8 double bond and affords 41 and 42 in a 76:24 ratio (Scheme 7).Scheme 7, Epoxidation of the 7,8 double bond in the diol 11.2.3 Oxidation of the hydroxyl group at C -6Oxidation of the hydroxyl group at C-6 converts the diol 1 into the ketol 43, and thelatter could serve as a precursor of the seco-diketone 44 via a retro-aldol type of reaction.Both 43 and 44 have been isolated from dark-fired tobacco (Scheme 8).[ 41 ] Oxidation of the11,12 double bond in the ketol 43 provides a route for the biogenesis of 6-oxo-compounds7, 30 and 45, which could be obtained alternatively by oxidation of the hydroxyl groups atC-6 in epoxide 4, (4, 6, 12)-triol 8 and (4, 6, 11)-triol 9, respectively. The epoxide 7 can beconverted to the 8,11-epoxy-diol 46 via an acid induced rearrangement and the diol 46, inturn, may serve as the precursor of the dehydration products 47 and 48. These threecompounds are the only 8, 11-epoxy-compounds containing 6-oxo group so far isolatedfrom tobacco [42],Experimental results support the viability of the biogenetic pathways outlined inScheme 8. Ketol 43 reacts smoothly with singlet oxygen to give four major products, 30,45, 49 and 50, after reduction of the hydroperoxides initially generated. The product 49,which still has not been isolated from tobacco, could be formed by spontaneous cyclizationof the (4S, 11R)-diol, a product not isolated from the reaction mixture (Scheme 9). [42]The routes proposed for the biogenesis of the 8,11-oxy-diols 46, 47 and 48 were alsoexplored experimentally by treatment of the epoxide 7 with dilute sulfuric acid in dioxane-water(3:1) (Scheme 10).[42] Among the six products obtained, the major one was found to beidentical to 46 in addition to two minor ones 47 and 48. The three remaining products wereformulated as 51, 52 and 53. The generation of these products may be accounted for asshown in Scheme 10. An initial anti-addition of water to the 11,12-epoxy group in 7 yields thetriol 31 which undergoes protonation of the oxo-group at C-6, migration of the 7,8 doublebond, and attack of the 11-hydroxyl group on C-8 to give two diols, 46 and 51. These twodiols are plausible precursors of 47, 48 and 52. Since the product 53 is an (8R, 12S)-8,12-epoxy-diol, it is likely to be formed by a mechanism that involves a C-12 carbocation as anintermediate. [42]161 1744^48III43III4647Scheme 8, Proposed biogenetic pathways related to the oxidation at C-6 I II +3n1 8+I4 34%(84,11R), 49IIIScheme 9, Products obtained from photooxygenation of the ketol 43 followedby reduction with triethyl phosphiteI I46III— H+QC, pHIIIHO531 1 III1947^48^52Scheme 10, Products obtained from acid-induced rearrangement of 75R21201.2.4^Acid-induced reactionsAn acid-induced reaction, leading to fragmentation or allylic rearrangement of the 4,6-diol, explains the generation of the seco-aldehyde 54 and the 4, 8-diol 55. Thesecompounds have long been known as tobacco constituents (Scheme 11).[ 43, 441The 4, 8-diol 55 is susceptible to dehydration and oxidation of the 11,12 doublebond. This is illustrated by the isolation of the (4S)-mono-ol 56 [45], the (4S, 8S, 11S)-triol 58 [35] and the (8S, 11R)-8,11-epoxy-diol 57 in tobacco.[461Scheme 11, Proposed pathways via acid-induced rearrangements of the diol 1The results of acid-induced reaction of the diol 1 using dilute sulfuric acid indioxane-water show that, in addition to the starting diol 1, five major products are formed(Scheme 12){471. It can be concluded that, under weakly acidic conditions, the diol 1 issubjected to competing allylic rearrangement, epimerization and dehydrative fragmentationreactions. Compound 55 can also be obtained in 20% yield when passing the diol 1through a chromatographic column packed with acid-washed alumina.rn+WI10%5515%5921+24% 3%1 44 +12%25%54Scheme 12, Products obtained in acid-induced rearrangements of the diol 1OH^ OH^ OHo o 0HCICHCI 3mCPB AHO"The (8S, 11R)-8,11-epoxy-diol 57 in Scheme 11 is the only cembranoid of thisstereochemistry that has been encountered so far.[ 461 The synthesis of 57 is shown inScheme 13. Thus treatment of the (4S, 8S)-diol 55 with m-chloroperoxybenzoic acidaffords the corresponding (11S,12S)-11,12-epoxide 59. When exposed to a trace ofaqueous hydrochloric acid in chloroform, the epoxide 59 undergoes a facile SN2 type ofepoxide ring opening at the secondary carbon C-11 by attack of the hydroxyl group at C-8to give the (8S, 11R)-8,11-epoxy-diol 57 .[46}55^ 59^ 57Scheme 13, Formation of (8S, 11R)-8,11 -epoxy-diol 571.2.5 Degraded cembranoidsDetailed studies have revealed that, in addition to cembranoids, tobacco contains asubstantial amount of volatile compounds which may be classified as degradedcembranoids. About sixty of such compounds have been isolated from tobacco.[ 3} Thecharacteristic features of these compounds are their irregular isoprenoid skeleton containingan isopropyl group and consisting of 8 to 19 carbon atoms. With a few exceptions they arecarboacyclic. Like their precursors, most of them have only been obtained from tobacco.This implies that these compounds could be specific to this plant.It has been suggested that the degraded cembranoids are generated by ruptures ofthe bonds in the parent cembranoid skeleton as shown in Figure 3 even though the mode ofbiogenesis has not yet been verified by labelling studies.[3, 4, 51 The key metabolites thus221 0120C19C12C13C14C15C18C13C14 C121816C18, C1513151712formed have 12, 13, 14, 15, 18 and 19 carbon atoms. They may undergo subsequentchemical alternations involving loss of carbon atoms to give further degraded products.Since the concentration of degraded cembranoids in the aroma fractions obtainedfrom cured leaves is high (it has been estimated that they constitute about 10% of the totalvolatile material of Burley tobacco [ 48]), it can be concluded that these bond ruptures arefavored reactions. It should be emphasized, however, that although some degradedcembranoids are generated during the curing processes of the tobacco leaves, many of themare present in green leaves and fresh flowers, although at a lower concentration.[ 17] Thisindicates that these degradation reactions also occur in the growing tobacco plants and theyare not mere artefacts during the post-harvest treatments.Figure 3, Degradation patterns of cembranoidsThe cleavage of the 12,20 bond leads to the C19 key metabolite 62, a compoundisolated from tobacco flowers.1471 It might occur by the biogenetic pathway shown inScheme 14, although not validated experimentally. The latter process involves the triol 60231— HOand the corresponding epoxide 61 as intermediates. The latter undergoes an acid-inducedrearrangement, leading to the loss of carbon at C-20 and the formation of the ketone 62.The C18 key metabolite, prenylsolanone 63, is the sole C18 representativeencountered to date.[491 Its biosynthesis would involve rupture of the 5,6 and 7,8 bonds inthe parent cembranoid. This process has previously been suggested to take place by a retro-aldol type fragmentation from seco-aldehyde 54 as shown in Scheme 15.[4]24HO620^(-0H+ CH2-ei61Scheme 14, Formation of the C19 compound 61 01, (45)2, (4R) 5463Scheme 15, Formation of the C18 compound 63The biogenesis of C15 compounds is explicable by breakage of the 7,8 and 11,12bonds in parent cembranoids. As shown in Scheme 16, the C15 key metabolites 66, analdehyde not yet encountered in tobacco, may arise via the 7,11- and/or 7,12-dihydroperoxides 64 and 65, which can suffer the prerequisite ruptures of the 11,12 bond.The 2,10-diol 67, derivable by reduction of 66 followed by cyclization, had been isolatedfrom Japanese SUIFU tobacco.[50]The predicted C14 key metabolite 68 may also be generated through the 7,11-and/or 7,12-dihydroperoxides, but via a route illustrated in Scheme 17, The resulting C14aldehyde 68 is the precursor of the diol 69 and the hydroxy-acid 70. The hydroxy-acid70, in turn, would give the acids 71 and 72 by dehydration. Both the aldehyde 68 and itsdaughter compounds 69-72 have been isolated from tobacco.[51 ]As shown in Scheme 18, oxidative cleavage of the 6,7 double bond in seco-aldehyde 54 [49] or an acid-induced cleavage of the 6,7 double bond in 33 [34] leads to theC13 key metabolite, solanone 73, which is an abundant and important flavor constituent intobacco. Subsequent metabolism via hydration and epoxidation leads to the hydroxy-ketone 74 and the epoxide 75. The epoxide 75 undergoes a stereo-controlledrearrangement to give the dioxabicyclo-[3.2.1]-octane 76 and the dioxabicyclo-[3.3.1}-nonane 77 [4, 52].Ruptures of the 4,5 and 11,12 bonds in parent cembranoids are the key steps in theformation of C12 degraded cembranoids. Among the C12 metabolites, norsolanadione 78 isa predominant constituent of the tobacco volatiles and an important precursor of many otherC12 constituents [53, 54]. For instance, the formation of 78 can take place by oxidativebreakage of the 11,12 double bond in a pre-formed seco-diketone 44 (Scheme 8 and 19).The norsolanadione 78 undergoes epoxidation to afford the epoxide 80 which canrearrange to the dioxabicyclo-[3.2.1]-octanes 84 and 85 [4, 53]. The epoxy-diol 83, acompound isolated from Greek tobacco [ 51 ], may be generated by reduction of the epoxy-2 5+HOO HOOHOOI6 4_^ IHO=^*OH126MI66HO OH H+O- ^HO —/.0H67Scheme 16, Formation of the C15 compoundsA 1 1I+I)cx0 OHH>1111‹0, /N.f684277 0 69\COOHA72Scheme 17, Formation of the C14 compoundsHO54O/73IC0r33>< ^IScheme 18, Formation of solanone 73 and the other C13 compounds28OH^OHdione 80. Norsolanadione 78 is also the precursor of several C8 to C11 tobaccocompounds, such as the recently encountered degraded products 82 and 86 [ 551.It is noteworthy that 82 and 86 are the first degraded cembranoids encountered intobacco in which the isopropyl-bearing carbon atom also carries oxygen.290/N7944O/78ZN80OHOOCV\/N. 81 86Scheme 19, Formation of nor-solanadione 78 and other C12 compounds1.3 Plant tissue culture1.3.1 Applications of plant tissue cultures in biotransformation studiesPlants are not only the most important sources for foods, oils and fibres but also animmense repository of chemicals including flavours, essences, pigments, fine chemicals,pharmaceuticals and novel biologically active substances. Plants and their extracts havebeen used by mankind for centuries and thousands of organic compounds whose chemicalsyntheses are really a challenge to organic chemists have now been directly isolated fromplants. Vinblastine 87 and taxol 88, for example, are two important anticancer drugswhich could be isolated from Catharanthus roseus and Taxus brevifolia, respectively. Butmost of these compounds occur in the plants only in minute amounts and localize strictly inspecific organs of whole plants, such as roots, flowers and leaves. Their production byfield plantation is highly dependent on environmental factors such as climate, season aswell as destruction by pests and disease, all of which can influence plant growth and thebuild-up of specific metabolites in the plants. The isolation processes for such naturalproducts from other co-occurring substances are usually very difficult, lengthy and costlyas they involve the purification of small amount of substances from a very large mass ofplant material. In addition, many plants grow in some inaccessible regions and may takeyears to reach maturity or accumulate metabolites. This can also severely limit productivity.Consequently the final product is often very costly if such a process is to be economicallyviable. In summary all of these factors make it undesirable to obtain such expensive finechemicals from field plants and often inappropriate for commercial production.Chemical syntheses can provide an approach for obtaining supplies of thesecompounds. The modern spectroscopic techniques have aided structural determination andfacilitated the elucidation of biochemical pathways. However, even in the case wheresynthetic routes are well established, the total syntheses of the desired product cannot30NHH3COOCCH 0 OCOCH2COOCH 31CH 3OHTaxol, 880..•OH....NH0= Calways be achieved economically. Actually, they are rarely useful in any practicalproduction of such complex natural products, often required in large scale for their use asdrugs, etc.Vinblastine, 87Fortunately, many studies indicate that plant natural products can be obtainedalternatively from in vitro culture systems, including plantlets, specific plant organs or cellsgrowing in solid or liquid nutrient medium [ 56]. Also there is evidence that such plant cell3 1cultures retain an ability to transform specific exogenous substrates administered to the cellcultures [57]. Therefore, plant cell cultures, if successively developed for large scalefermentation, can be considered not only as substitutes for some field plants, but also serveas useful "tools" to transform inexpensive and plentiful substances into rare and expensivesubstances.Cell cultures have now been established for many plants. Scheme 20 gives anoutline of the processes involved in establishing a typical plant cell culture.Whole plant IitExplant^ICallus cultureITSuspensionculture Scheme 20, The development of a plant cell suspension cultureThe initial stage for preparing such a culture involves the removal of a section ofplant tissue under aseptic conditions. This material, known as explant, is then placed onnutrient agar which consists of carbohydrates such as sucrose, inorganic salts and smallamounts of plant growth auxins. The explant may be a piece of root, leaf or other parts ofthe plant. Successful growth of the plant tissue on the solid nutrient medium results in a32callus culture which is a mass of disorganized cells. The callus culture can be maintainedfor a long time through sub-culturing, a process in which the sample of callus is removedand placed on a fresh agar medium where they continue to grow. If a sample of callus isplaced into a liquid nutrient medium, a suspension culture can be obtained. The liquidnutrient medium is prepared using similar constituents which are initially used to producethe nutrient agar. The cell suspension cultures can be sub-cultured in the same way as thecallus cultures.Once a cell culture has been established, growth may be rapid, and environmentalfactors such as light, temperature and aeration can be controlled to promote cell growth andmetabolite accumulation. Compared with field crops, cell cultures offer the following majoradvantages: [58]a) Compounds could be produced year-round under controlled laboratoryconditions, assuring a steady supply without seasonal fluctuation.b) Metabolic processes can be regulated and thus, yields of the compounds ofinterest can be maximized.c)^Cells could be genetically modified and thus accumulate specificintermediates or other metabolites through biosynthesis or biotransformationThe potential of plant cell cultures has been demonstrated by the production of thecardiac drug ubiquinone-10 using suspension culture of Nicotiana tabacum in amounts of15 mg/L or about 1.9 mg/g dry cell. This yield is ten times greater than that in the intactplant [59]. As a matter of fact, Shikonin, an antiseptic and dyestuff used in Asia sinceancient times, is currently being produced commercially in Japan by using cell culture ofLithospermum erythrorhizon . In this instance, yields exceeding 2 g/L (12.4% of dryweight) after 14 days of cultivation have been achieved.[60]The use of plant cells as a type of biochemical reagent or enzyme system is a veryexciting prospect. In these so called biotransformation studies, plant cell suspension33cultures are very often used. In preparation of such a culture, as mentioned above, theundifferentiated callus tissue is dissociated into a fine cell suspension by putting the callusinto liquid nutrient medium and allowing it to grow in rotary shakers. The cells will growto a stationary phase after an appropriate time period and are then ready for use.A typical biotransformation experiment involves addition of substrate to the plantcell culture under sterile conditions, incubation for a certain period of time, harvest andisolation of products. If the products are present in the culture broth, the cells can befiltered and products can then be extracted from the broth. However, if the products aretrapped in the cells, the cell material must be homogenized to break up the cells so that theproducts can be extracted. The advantages of using whole cell suspensions include thehomogeneity of the culture environment, the ease of manipulation of culture conditions andthe rapid generation of large volumes of relatively uniform tissue. Also, the cofactorsnecessary for the various enzyme functions are generated in situ and need not to be addedexternally.Despite the tremendous versatility of the system, some problems still can arisewhen this whole cell biotransformation technique is used. The lack of specific enzymes isfrequently responsible for the inability of the cell culture to biotransform the foreignsubstrates. Also the substrate must have a certain degree of solubility in the cell culturegrowth medium, and must be able to diffuse or transport through the cell membrane. Inaddition, the isolation of products may be difficult if they remain inside the cells and do notdiffuse into the medium. Since there are many enzymes present in the cell culture, theproduction of the desired product by specific enzymes may be complicated if competingenzymes can transform the substrate to undesirable end products. Many other reactions andsubsequent degradation can occur to produce undesirable by-products. Despite theseobstacles, this methodology holds much promise as shown by the semi-continuousconversion of 13-methyldigitoxin, a highly toxic byproduct obtained in the extraction ofcardenolides from Digitalis lanata , to the highly valuable cardiac drug D-methyldigoxin by34Digitalis lanata cell culture. In the latter study, an 80% yield was obtained in a 300 litreairlift-bioreactor.{62, 63].Cell free systems provide another approach used in biotransformation studies. Thisinvolves the isolation and use of the enzymes, thus maintenance of the biomass is notrequired and any problems connected with cell membrane impermeability are alsoeliminated. Typically, the cell culture is filtered and the cells are disrupted byhomogenization in the presence of a buffer at a low temperature. This homogenate is thencentrifuged to remove the cell debris. The supernatant thus obtained is called cell freeextract (CFE) and can be considered as a solution of enzymes originally present in theculture but now in a cell-free form (Scheme 21). The purification is often limited to aremoval of buffer-insoluble material, such as cell walls, but it may also includechromatography to isolate the required enzymes.Cell suspension cultureFiltrationCells IHomogenizationin bufferHomogenate ICentrifugation Supernatant(Cell Free Extract)CFEPellet(Cell debris)35Scheme 21, The preparation of Cell Free Extract (CFE)The advantage is that the reaction condition can be determined precisely because it iscarried out in a homogeneous solution rather than inside a cell. The isolation of products isrelatively easier than that in whole cell biotransformation because much less cell material ispresent in this case. It is evident that the substrate and any necessary cofactors for thereaction must be supplied directly to the CFE mixture as these can no longer be synthesizedin situ. This requirement is a major consideration for a potential commercial process. Forexample, biotransformation employing costly nicotinamide adenine dinucleotide phosphate(NADPH), a cofactor for the oxidative enzyme cytochrome P-450, would be practical onlyif particularly expensive final products were involved.Other drawbacks of this approach include the possibility of loss of enzymaticactivity during CFE preparation as many enzymes are very labile and can be denatured byosmotic shock, pH change or temperature. The complexity of the overall procedure maynot make it as suitable for industrial-scale operation as whole cell biotransformation.However, the latter problem could be partly avoided by using the CFE enzymes in apurified and immobilized form which would permit their recovery and re-use after eachbiotransformation.To date over 30 classes of compounds have been produced or transformed by usingplant cell cultures including steroids, alkaloids, terpenoids and quinones [ 56]. These datasuggest a bright future for this technology. An illustration of such a biotransformation inour laboratory was the preparation of 3',4'-anhydrovinblastine 91, a precursor of theanticancer drug vinblastine 87, using Catharanthus roseus cell culture (Scheme 22).Incubation of catharanthine 89 and vindoline 90 with C. roseus CFE in the presence ofhydrogen peroxide as a cofactor gave 3',4'-anhydrovinblastine 91 in 25% yield [64]. Thisyield increased to 38% when immobilized enzymes from the same culture were employed[65] .36+CH 30C. roseus CFEH202NH^OCOCH 31 cH3 COOCH 3Vindoline 90Catharanthine 89VNHH3COOC \NC30^N1 HCH3OCOCH 3COOCH 3373',4'-anhydrovinblastine 91Scheme 22, Production of 3 1 ,4 1-anhydrovinblastine 91 by CFE from C. roseus1.3.2^Oxidative reactions in biotransformation studies usingplant cell culturesAs mentioned above, plant cell cultures are capable of producing and/ortransforming a variety of chemical compounds. This is due to the fact that different kinds ofenzymes are present in plant cells and act as catalysts for those reactions leading to specificproducts. Among those bio-reactions, the oxidative processes are very often encountered.As oxidative reactions are concerned, it is worth to mention cytochrome P-450. Thecytochromes P-450, abbreviated from "Pigment with an absorption at 450 nm", arehemoproteins with a characteristic absorbance at 450 nm in the UV spectra of their COadducts and are well known enzymes common to many plants and animals. They play animportant role in such processes as steroid metabolism, drug detoxification and thecarcinogenic activation of polycyclic aromatic hydrocarbons. The levels of thesehemoproteins can be influenced by many chemicals and the enzymes, in turn, are capableof metabolizing many compounds. Because of the diversity of substrates and the variety oftransformations that these enzymes execute, this family of cytochromes has attracted theattention of researchers in many fields, including organic and inorganic chemistry,biochemistry and pharmacology.Extensive research has led to the isolation and characterization of many apparentlydistinct forms of cytochrome P-450. Now the complete primary structures of many P-450shave been elucidated through protein and DNA sequences. The active site of P-450 haslong been known to contain the ferroprotoporphyrin IX prosthetic group. (Figure 4).38Figure 4, Structure of iron protoporphyrin IX and catalytic cycle of cytochrome P-450A general accepted catalytic cycle for P-450 is shown in Figure 4 [ 661. The featuresinclude:I. Binding of the substrate to give a ferric complexII. One-electron reduction of the iron to the iron(11) stateIII. Binding of oxygen to generate the oxy form SFe3+02 -IV. A second one-electron reduction to yield the iron peroxo species Fe 3+022-V. Formal heterolysis of the 0-0 bond with generation of the reactive oxidant[FeO]3+ and a molecule of waterVI. A two electron oxidation of substrate to produce SO and regenerate the ferricstate of the enzymeDuring metabolism or biotransformation, cytochrome P-450 behaves as a mono-oxygenase. The mono-oxygenation occurs according to Equation 1 and involves theincorporation of one oxygen atom into the substrate. Obviously, the reaction requires theinput of two electrons. In other words, the enzyme system needs a reducing co-substrate(AH2) as indicated in Equation 2.S + 02 + 2e- + 2H+ ^> SO + H2O^(1)S + AH2 + 02 ^>S0 + H2O + A (2)Nicotinamide adenine dinucleotide phosphate (NADPH) is a usual reducingcofactor. Due to the fact that the pyridine nucleotides are two electron donors, while P-450can only accept one electron at a time, other transfer agents must be involved in theprocess. Such agents usually are flavin mononucleotide (FMN) or flavoadeninedinucleotide (FAD). These flavoproteins can undergo a single electron transfer to the P-450system which is then able to oxidize the substrate.Cytochrome P-450s are responsible for many oxidative reactions and the reportedresults can be clas , ified into six categories.[67]391. Carbon hydroxylation: formation of an alcohol at a methyl, methylene, ormethine position.2. Heteroatom release: oxidative cleavage of the heteroatomic portion of a molecule3. Heteroatom oxygenation: conversion of a heteroatom-containing substrate to itscorresponding heteroatom oxide4. Epoxidation: formation of oxirane derivatives of olefins and aromatic compounds5. Oxidative group transfer: a 1, 2 carbon shift of a group with concomitantincorporation of oxygen as a carbonyl at the C-1 position6. Olefinic suicide destruction: inactivation of the heme of cytochrome P-450 by anenzyme product or an enzyme intermediateFor instance, cytochrome P-450pB_B [68] can catalyze all six types of oxidativereactions with certain substrates, i.e., carbon hydroxylation (e.g.,cyclohexane [ 69]),heteroatom oxygenation (e.g., azoprocarbazine [70]), heteroatom release (e.g.,benzphetamine [68]), epoxidation (e.g.,arachidonate [71 ]), oxidative group transfer (e.g.,trichloroethylene [72]), and olefinic suicide destruction (e.g., vinyl chloride [ 73]).Among the six types of reactions mentioned above, epoxidation and hydroxylationare two most important and very often encountered reactions. These reactions have receivedconsiderable attention in biotransformation studies. It was reported that the suspensioncultures of Nicotiana tabacum have the ability to hydroxylate the trans-methyl group in theisopropylidene moiety of linalool 115 and its acetate 116 to give the corresponding 8-hydroxyderivatives 117 and 118.[74] Such an ability was also investigated with themonoterpenoids having terminal, endocyclic and exocyclic C-C double bonds, such as a-terpineol 119 and its acetate 120, f3-terpineol 126 and its acetate 127 and y-terpinylacetate 133 as showed in Scheme 23. The terpineols were hydroxylated at the carbonatoms allylic to the C-C double bond to yield the corresponding allylic alcohols.40117, R=OH118, R=OAc115, R=OH116, R=OAc++HO,,+133 134 135 1 3 6119, R=OH120, R=OAc121, R=OH122, R=OAc123, R=OH124, R=OAc 125, R=OAc126, R=OH127, R=OAc128, R=OH129, R=OAc130, R=OH131, R=OAc 132, R=OAc+OAc^OAc+OH RI41Scheme 23, Some examples of allylic hydroxylation using plant cell culturesOn the other hand, terpinyl acetates were hydroxylated, not only at the allylic positions, butalso at the C-C double bond to give glycols as the major productsP 41The process of glycol formation was investigated in the biotransformation of a-terpinyl acetate 120 and y-terpinyl acetate 133 in the cultured cells of N. tabacum. It wasfound that glycols were formed from epoxidation of the C-C double bond, followed byhydrolysis of the resulting epoxides.[75]1.3.3 Reductive reactions in biotransformation studies usingplant cell culturesThe reductions of C-C double bonds and of carbonyls to alcohols are very popularin biogenesis and biotransformations. These reactions are catalyzed by dehydrogenasesutilizing pyridine or flavin coenzymes and considered as the reverse processes of theoxidations using dehydrogenases. Therefore, these reactions obey the specificityrequirements of the dehydrogenases.S + AH2 > SH2 + A (3)There are many reports on the reductions of ketones and aldehydes to thecorresponding alcohols with plant cell cultures. One of these examples is thebiotransformation of monoterpenoids by plant cell suspension cultures of Lavandulaangustifolia. [76] It was found that this plant cell culture can reduce monoterpenoidaldehydes and structurally related compounds to the corresponding primary alcohols. Someof the structures are given in Scheme 24.Citronellal 137, geranial 139 and perillaldehyde 141 were reduced to thecorresponding alcohols 138,140 and 142. It was also found that acyclic monoterpenoidalcohols 138 and 140, once formed, disappeared from the cultures over about a 15 hrperiod and were metabolized into unidentified compounds. In contrast, the cyclicmonoterpenoid alcohol, perillyl alcohol 142, remained unmetabolized in the cultures over a72h period.42) .,CHO137CHO43OH138OH139^ 140CHO 141^ 142Scheme 24, Some examples of reduction using plant cell cultures1.3.4 Plant cell lines developed and available in our laboratoryTremendous efforts have been directed in our laboratory towards the production ofpharmaceutically important chemicals from plant tissue cultures. As a result, several stablecell lines have been established. For example, the AC3 cell line derived from Catharanthusroseus is capable of producing the indole alkaloid vinblastine 87, a clinically used drug forcancer chemotherapy, and biotransforming the indole alkaloids catharanthine 89 andvindoline 90 into 3',4'-anhydrovinblastine 91, a precursor of vinblastine 87 [77]. TheTRP4a cell line derived from Tripterygium wilfordii , a well known Chinese herbal plant,can produce the diterpene tripdiolide [78] which is a cytotoxic compound and revealssignificant male contraceptive activity. A cell line derived from Podophyllum peltatum isused to produce podophyllotoxin and demethylpodophyllotoxin, which could be utilized asintermediates in the production of the anti-cancer drug etoposide and its analogues [ 79].These cell lines have also been used in biotransformation studies on different substrates andseveral promising results have been achieved. For example, an oxidative coupling reactionof 117 catalyzed by enzymes in CFE prepared from C. roseus and in the presence ofhydrogen peroxide as cofactor affords an essentially quantitative yield of the ring-closedproduct 118 (Scheme 25), thereby providing a route for the syntheses of lignans [971 .HOMeOHOCFE^MeO 0111^C. roseusH20 2CH3O^OCH3^CH3OOH117Scheme 25, Oxidative coupling leading to lignan 1181.4 Objectives of the projectSince the chemical investigations on tobacco cembranoids reported so far werebased exclusively on isolation, structure elucidation and chemical transformation, nobiotransformation studies on tobacco cembranoids using plant cell cultures had beenreported. Therefore, it was of considerable interest to examine if similar interconversionscould be executed, more regiospecifically and hopefully in high yield, by enzymaticprocesses. With various plant cell lines and biotransformation technology in hands, ourintention to perform biotransformation studies on tobacco cembranoids was initiated. Oneof the aims of this project was to test the hypotheses of the proposed metabolic pathways asdiscussed previously. Another important objective was to see if it is possible to produce44OH11841.HO71077Rmolecules which had potential use in the area of aroma and fragrance chemicals, forexample, norsolanadione 78, via metabolism of the major cembranoids, diols 1 or 2, usingour plant cell cultures (Scheme 26).Scheme 26, Proposed objective for cembranoid biotransformation452 RESULTS AND DISCUSSION2.1 Biotransformation of cembranoids using the TRP4a cell lineThe major reasons for selecting the TRP4a cell line in a first attempt to perform thebiotransformation studies on the tobacco cembranoids were not only the stability andavailability of this plant cell line in our laboratory, but also the documented capability tocarry out oxidative degradations.[78, 80, 81] This plant cell line was well established in ourlaboratory and was well characterized in terms of its ability to generate peroxidase (oxidase)activity. With the hope that oxidative degradation could lead to ring cleavage of thecembranoids, biotransformation experiments using this cell line were performed.As discussed in the Introduction of this thesis, three olefinic linkages are present inthe starting diols 1 and 2. From the chemical point of view, the disubstituted 2,3 doublebond is expected to be less reactive for oxidation than the trisubstituted 7,8 and 11,12double bonds and, based on previously published studies involving chemical conversions[3] , the 11,12 double bond should react more readily than the 7,8 double bond. Ourbiotransformation studies confirmed such a proposal.The suspension culture of TRP4a showed a high degree of regio-selectivity towardtobacco cembranoids. It reacted only with the 11,12 double bond to give the epoxide as themajor product when either diol 1 or diol 2 was used as substrate.The biotransformation results showed that incubation of diol 1 with growing cellsof the TRP4a cell suspension culture and with cells resuspended in phosphate or TrisHC1buffers afforded recovered diol 1 and products 8, 4, 92 ,92 and 93 in order of increasingpolarity as determined by thin layer chromatography (Scheme 27). The (11S, 12S)-epoxide4 was the predominant product in 40-50% yield in our optimized conditions while the othercompounds were obtained as minor products in less than 10% yield. Among the fiveproducts, triols 92, 93 and 94 are new compounds not previously isolated from the living46147TRP4a8^ 492^93^94Scheme 27, Biotransformation of the diol 1 using TRP4a cell cultureplants of Nicotiana species, the triols 92 and 93 were assigned as a pair of diastereomers withstereochemistry at C-10 undefined at present.The structure elucidations of the products were accomplished largely on the informationobtained from 1H, 13C NMR and MS spectra. The mass spectra are informative even thoughthe ions in the high-mass region were often of very low abundance [ 82].With product 4, the mass spectrum indicated an increase of 16 mass units relative to thestarting diol 1 and a molecular ion peak at m/z 322 was observed. This implied that one oxygenatom was introduced into the starting material. In its 1H NMR spectrum, there were three methylgroups (singlets at 5 1.20, 1.37 and 1.78) and an isopropyl group (two doublets at 5 0.80 and4 80.85), characteristic signals of cembranoids. The 13C NMR spectrum of 4 revealed that therewere two double bonds, one double bond was disubstituted while another one was trisubstituted(three downward signals and one upward signal in APT spectrum), thereby indicating that one ofthe trisubstituted double bonds, (7,8 or 11,12), had been involved in the bioconversion andstrongly suggesting epoxide formation. Since the 1 H NMR spectrum indicated that the 7,8double bond was still present (a doublet of triplets at 8 4.48 for H-6), it was clear that the 11,12double bond was involved in epoxide formation. In accord with this assignment, both signalsnormally arising from the proton and carbon atoms of the 11,12 double bond were now shiftedupfield in the corresponding NMR spectra. A signal due to H-11 was observed at 8 2.88 as adoublet of doublets in the 1H NMR spectrum of 4 and two signals due to C-11 and C-12 werealso observed at 8 61.3 and 60.1 in the 13C NMR spectrum. By direct comparison of the spectraldata with the published results [10] , it was found that this biotransformation product was identicalto (1S, 2E, 4S, 6R, 7E, 11S, 12S)-11,12-epoxy-2,7-cembradiene-4,6-diol and was thusidentified as 4.Table 3, 13C NMR chemical shifts (ppm) of cembranoids in CDC13C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C10(1) 46.4 127.7 137.5 72.4 52.2 66.2 130.6 136.6 38.8 23.3(4) 47.1 127.7 138.4 72.3 52.9 66.1 132.7 135.2 35.6 24.9(8) 50.9 127.4 138.3 74.2 47.2 69.6 128.3 135.2 40.8 124.7(92) 47.5 127.7 137.8 72.8 50.0 66.8 129.7 138.6 45.6 67.2(93) 46.3 127.7 137.4 72.4 52.0 65.0 132.6 137.9 48.3 65.8(94) 46.2 127.5 137.4 72.4 52.7 66.1 131.3 136.2 38.7 22.9C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20(1) 124.4 133.3 36.8 27.9 33.0 19.3 20.7 30.1 16.1 15.0(4) 61.3 60.1 36.6 28.6 33.1 19.1 20.9 30.0 16.0 16.3(8) 138.9 73.9 40.1 26.4 30.2 17.8 21.9 31.8 18.1 30.1(92) 128.4 134.5 39.2 28.8 34.0 19.2 20.4 30.9 16.8 19.0(93) 129.0 133.6 36.3 27.2 33.0 19.4 20.5 30.1 16.6 14.7(94) 128.1 135.2 77.4 35.7 33.2 19.5 20.4 30.1 15.9 9.6Epoxide 4 has been isolated from various tobacco plant sources [36] and postulatedas the key biogenetic intermediate for the metabolism of cembranoids.[ 3] The isolation ofthe epoxide 4 as a major biotransformation product confirmed that the enzymaticepoxidation was a principal biogenetic pathway in the metabolism of tobacco cembranoids.The mass spectrum of another product, the tertiary alcohol 8, also showed amolecular ion at m/z 322. The 13C NMR spectrum indicated that there were three doublebonds and two tertiary hydroxyl groups (5 73.9 and 74.2 ppm) in the molecule. It alsoshowed that the two double bonds were disubstituted while another one was trisubstituted(five downward signals and one upward signal in APT spectrum), implying that a newlyformed double bond, possibly a disubstituted one, was the result of the biotransformationreaction. Because the signal at 5 4.80 (t, J=7 Hz) due to 11-6 was still observed in the 111NMR spectrum, the 7,8 double bond was unattacked. Therefore the newly introducedtertiary hydroxyl group was assigned to be located on C-12 and the 11,12 trisubstituteddouble bond was shifted to form a disubstituted 9,10 double bond. In accord with thisassignment, a signal at 5 2.70 (due to two diastereomeric protons at C-9) was observed asa doublet of doublets in the 1 H NMR spectrum. It was found that the spectroscopicproperties for this biotransformation product were in good agreement with those of (1S,2E, 4S, 6R, 7E, 10E, 12S)-2,7,10-cembratriene-4,6,12-triol, which had also been isolatedfrom tobacco [83]. This compound can be obtained by photooxygenation of the diol 1 withsinglet oxygen followed by reduction with triethyl phosphite.[ 10] In conclusion, thisproduct was assigned structure 8.The biotransformation products 92, 93 and 94 had not yet been isolated fromnatural sources so direct comparison with published data was not possible. The massspectra of these compounds were very similar and showed a molecular ion at m/z 322.Because both the isopropyl group and the three methyl groups, as well as the three doublebonds (two trisubstituted and one disubstituted) were still present, these three compoundswere novel hydroxylated products of diol 1.4950Product 92 formed a diacetate on treatment with acetic anhydride in pyridine. Since italready contained a tertiary hydroxyl group (5 72.8 for C-4 in 13C NMR), this product couldbe a triol with two secondary hydroxyl groups. This suggestion was reflected in its 13C NMRspectrum since an additional signal at 5 67.2 ppm could be seen. In comparing its MSfragmentation patern (Scheme 29) with that of diol 1 (Scheme 28), especially thefragmentation pathway of m/z 322 --> m/z 304 --> m/z 223 vs. m/z 306 --> m/z 288 --> m/z207, it was clear that the fragment ions at m/z 304, m/z 221 and m/z 223 carry the newlyintroduced hydroxyl function. Since the presence of identical fragment ions at m/z 81, m/z 83,m/z 123 and m/z 137 were noted in the mass spectra of 1 and 92, the placement of the newlyintroduced hydroxyl group at the C-10 position was most reasonable. Conformation for suchplacement came from the NMR data. In agreement with this assignment, the C-10 signal at 523.3 ppm present in diol 1 was shifted to 67.2 ppm in 92 and a new one-proton signal,attributed to H-10, was observed in the 1H NMR spectrum at 8 4.54 ppm (dt, J=2 and 8 Hz).It was found later that the 1 H NMR spectrum of the biotransformation product 92 wasidentical with that of the synthetic sample 92 obtained in an independent synthetic program atthe Swedish Tobacco Company, Stockholm.[84] Based on the available information, thestereochemistry at C-10 cannot be defined at the present time and the product was identified as(1S, 2E, 4S, 6R, 7E, 1041, 11E)-2,7,11-cembradiene-4,6,10-triol 92.Spectroscopic data for 93 indicated that the newly introduced hydroxyl group wasalso located at C-10 and the signals in the 13C NMR were very similar to those in product92 except for differences in the chemical shifts for C-10 (67.2 ppm in 92 and 65.0 in 93)and C-20 (19.0 ppm in 92 and 14.7 ppm in 93), suggesting that 93 might be adiastereomer of 92 with opposite stereochemistry at C-10. The differences in the 13C NMRchemical shifts for C-10 in 92 and 93 are obviously due to the stereochemical orientationof the newly introduced C-10 hydroxyl function. Therefore, product 93 was identified as(1S, 2E, 4S, 6R, 7E, 1042, 11E)-2,7,11-cembradiene-4,6,10-triol at this stage. Althoughthe above data established the structures of 92 and 93, the specific stereochemical01C6H9+m/z=81C14H230m/z=207m/z=288-CH3-CH3COm/z=273 m/z=245McLaffertyrearrangement)(,)c^>^<1 'C911 1 40m/z=138assignment at C-10 remains ambiguous. Various attempts to obtain a satisfactory crystal forX-ray analysis failed.51Im/z=3061 -H2O+.m/z=288+.C51170-H2O miz=83m/z=123m/z=270-C3H7H ,li --/ •m/z=245 m/z=43C9H15m/z=205^m/z=137Ci5H25 m/z=288 C101-1 17McLaffertyrearrangementm/z=227,'N__>^ <-1 ÷C101-116m/z=136-CH3^-CH3C0m/z=123^m/z=95 m/z=121 nil-L=93Scheme 28, The mass spectrometric fragmentation pattern of diol 1OHOHm/z=261 m/z=43 +C51170m/z=83OHC15H250m/z=221-H201Ci5H23m/z=203Clairm/z=304 m/z=137C91115m/z=123McLaffertyrearrangement)\___>^ <-1 +C101116m/z=13652C6H9+m/z=819 2m/z=3221 -H20m/z=304-H20m/z=286m/z=223CI4H23%-/2 -^( -1^CH3m/z=304-CH3COm/z=261McLafferty^01414210^rearrangementm/z=205ON >^<1 , .)C91-1140m/z=138-H20  m/z=289-C3H7m/z=243m/z=121^m/z=93Scheme 29, The mass spectrometric fragmentation pattern of triol 92Product 94 also formed a diacetate on treatment with acetic anhydride in pyridine.Therefore, this product could be also a triol with two secondary hydroxyl groups. Thehydroxylation of the cembrene skeleton was evident from the molecular ion at m/z 322 andthe fragment ions at m/z 304 and m/z 223. Even though the signal at 8 77.4 ppm arisingfrom the carbon bearing the newly introduced hydroxyl group was not easily observed dueto the solvent peaks (a triplet at 8 76.64, 77.07 and 77.49 due to CDC13), it was clearlyseen in an APT experiment. This downward signal indicated that the newly introducedhydroxyl group was attached to a secondary carbon atom. Because a new one-proton signalat 8 3.98 ppm as a doublet of doublets was also observed, the newly introduced hydroxylgroup was assigned to be attached at C-13. In accord with this assignment, the C-13 signalat 8 36.8 ppm in the 13 C NMR spectrum of diol 1 was shifted to 77.4 ppm in 94.Furthermore, the influence of the C-13 hydroxyl function on the chemical shift of the C-20signal in the 13 C NMR spectrum (9.6 ppm vs 15.0 ppm in diol 1) provided furtherevidence for the placement of the hydroxyl function at C-13. However, the above data donot establish the stereochemistry at C-13 and it remains unknown.In contrast to allylic hydroxylation at C-10 which afforded two isomeric viols 92and 93, only one C-13 hydroxylated product 94 was obtained. Fortunately, it was foundthat the spectral data of biotransformation product 94 were identical with those of a recentsynthetic sample 94 provided by the Swedish Tobacco Company [K. The newly createdcarbinol centre was then assigned to have the S-configuration.The mechanisms for the formation of the epoxide 4 and the allylic alcohols 92, 93and 94 are not clear. They could be cytochrome P-450-catalyzed reactions since these typesof enzymes are generally considered for these types of functional group introductions.When 13-diol 2 was allowed to react with the cell suspension, only the 11,12 doublebond was oxidized to give the epoxide 95 in approximately 50% yield (Scheme 30). Itsidentity was established by comparing the spectral data of 95 with the published data.[361The remaining fractions of the chromatography, constituting approximately 20% of theoriginal mixture, was an inseparable mixture of several compounds with identical retentiontimes on a TLC plate. Attempts to separate the mixture met with failure and eventually thecompounds decomposed.[ 85153TRP4a54 2^ 95Scheme 30, Biotransformation of (3-diol 2 using TRP4a cell culture2.1.1 Biotransformation of the diols 1 and 2 using the whole cellsSince the abilities of the TRP4a cell suspension culture to execute oxidativereactions vary considerably with the reaction parameters, a series of experiments werecarried out where conditions such as cell age, buffer, pH, incubation time and substrateconcentration were altered. In the biotransformation experiments performed, the substratewas dissolved in ethanol and added to the cell culture in one of the following four differentways:1) addition of substrate to the cells resuspended in the buffer.2) addition of substrate to the growing cell suspension culture in one batch.3) addition of substrate to the growing cell suspension culture in several batches.4) semi-continual addition of substrate to the growing cell suspension culture via aperistaltic pump.2.1.1.1 Addition of substrate to the cells resuspended in bufferSince plant cells are capable of producing various kinds of natural chemicalsubstances, the presence of these metabolites in the culture broth could interfere with theanalysis of the biotransformation results. Therefore the cell resuspension method was firstconsidered. In this series of experiments, TRP4a cell suspension cultures grown tostationary phase (monitored by pH and refractive index of the spent medium ) in PRDComedium were selected. The broth of the cell suspension culture was removed by filtrationand the cells were then resuspended in a buffered medium (phosphate or TrisHC1)containing sucrose (8%) as an osmotic balancing agent. The substrate dissolved in ethanolwas added in one batch to the above resuspended cell culture. The mixture was thenCell suspension culture1)Filtration through Miracloth2) Wash with distilled water3) Resuspension in buffer (pH 6.3)4) Substrate addition5) incubationWork-upFiltrationthrough Miracloth551)Homogenization inEtOAc, (24,000rpm)2) Filtration 1EtOAcextractsaqueous layer(discard)FiltrateEtOAcextractionCell materialCell material^EtOAc(discard) extracts1)Wash with water, brine2) Dry over anhydrous Na2SO43) Concentration in vacco4) Chromatographic separationProductsScheme 31, Procedures for biotransformation using resuspended culture of TRP4aincubated on a rotary shaker at 26°C and 135 rpm without illumination for an appropriatetime. The procedures are outlined in Scheme 31. Steps 1-5 (prior to workup) wereperformed under aseptic conditions in a sterile room.Progress of the reaction was monitored by TLC. After an appropriate incubationtime, the biotransformation mixture was harvested by filtration, followed by extraction ofbroth and cells with ethyl acetate as outlined in Scheme 31. Chromatography of the crudeextracts on silica gel afforded recovered substrate and biotransformation products.In order to isolate some polar products which may not be extracted by ethyl acetateand remain in the aqueous layer, the aqueous layer, after extraction, was subjected tofreeze-drying in vacuo and the solid material thus obtained was extracted with methanol.However, TLC analysis revealed no products of interest and in subsequent studies, theaqueous layer was not processed.2.1.1.1.1 Differences in reactivities between diol 1 and diol 2Structurally, diol 1 differs from diol 2 only in the chirality at C-4, however,marked differences in reactivities towards the TRP4a cell suspension culture wereobserved. The a-diol 1 was found to react with the cell suspension at a much slower ratewhen compared with its D-epimer, diol 2. The results are summarized in Tables 4 and 5.For example, under similar conditions, only 8.4 % of unreacted diol 2 was isolated after120 h of incubation (Entry 10), whereas more than 20% of diol 1 was recovered (Entry 1).2.1.1.1.2 Effect of pH on biotransformationDue the fact that both diols 1 and 2 were unstable under acidic conditions asdiscussed in the Introduction [ 47], TrisHC1 buffer at pH 7.5 was chosen for our initialstudies. Later on, it was found that the biotransforamtion could be carried out moreefficiently at a lower pH and the incubation time was reduced dramatically without anychange in product yields (Tables 4 and 5). Therefore, subsequent biotransformation studies5657were carried out in a phosphate buffered medium at pH 6.3. In all studies, TrisHC1 andphosphate buffers contained 8% of sucrose as an osmotic balancing agent.Table 4, Effect of pH on biotransformation of diol 1Expt No.Cell age (days)pH25nDSubstrate 1 /EtOH (mg/ ml )Volume of culture (L)1^2^319^19^195.20^5.50^5.151.3330^1.3330^1.3331186/15^22.5/5^45/51.5^0.5^0.54195.501.333125/50.55^616^195.68^5.611.3336^1.3332314/20^400/202.5^2.0Resuspension medium :^type TrisHC1^Phosph. TrisHC1 TrisHC1 TrisHC1 Phosph.pH 7.5^6.3^7.5 9.0 7.5^6.3Incubation time (h) 120^72^144 144 168^144Yield (%)^1 20.5^0^5.8 35.6 21.7^4.64 33.9^50.2^41.6 29.8 28.7^3592 0^0^0 0 9.1^12.393 12.5^24.0^14.9 20.4 8.9^13.894 12.3^13.0^12.2 8.0 4.1^5.2Total recovery (%) 79.2^87.6^74.4 93.8 72.4^70.9Table 5, Effect of pH on biotransformation of diol 2Expt. No. 7^8 9 10Cell age (days) 19 19 19 19pHn255.50^5.15 5.25 5.20D 1.3330^1.3331 1.3330 1.3330Substrate 2/EtOH(mg/ ml) 25/5^50/5 25/5 196/15Volume of culture (L) 0.5 0.5 0.5 1.5Resuspension medium :^type Phosphate^TrisHC1 TrisHC1 TrisHC1pH 6.3^7.5 9.0 7.5Incubation time (h) 72 144 144 120Yield (%)^2 0^0 30.4 8.495 53.6^52.6 36.8 48.7mixt 16.8^24.8 9.2 19.7Total recovery (%) 70.4^77.4 76.4 76.3It was found that biotransformations using phosphate buffer at pH 6.3 proceededmuch faster when compared to those using TrisHCl buffer at pH 7.5 and 9. In anexperiment with 200 mg of diol 1 per litre of cell suspension with a phosphate buffer at pH6.3 (Entry 6), only 4.6% of the starting material was recovered after 144 h. However, inanother experiment with a substrate level of 126 mg per litre of cell suspension withTrisHC1 buffer at pH 7.5 (Entry 5), 21.7% of starting material was obtained after 168 h.Product yields in these two cases remained unchanged. The reaction rate was furtherretarded when TrisHC1 buffer at pH 9 was used (Entry 4). A similar result was obtainedwhen diol 2 was used as substrate.2.1.1.1.3 Effect of substrate concentration on biotransformationIn the next series of experiments, the amounts of substrate administrated to the cellsuspension cultures were increased progressively in order to assess the optimum substrateconcentration for the cells to biotransform. When the amount of substrate was increased tothe level of above 600 mg per litre of cell suspension, a significant portion of the startingmaterial was found to be unreacted after 5-6 days. With the hope that prolonged reactiontime could lead to a complete substrate consumption, the incubation time was thereforeextended. Unfortunately, when the reaction was allowed to continue beyond 8-10 days,undesirable results were obtained. Not only the recovery of material and the yields for theusual products were found to be lower, but also a mixture of unidentified biotransformationproducts was obtained (Table 6). In the worst case, when 1000 mg of diol 1 was incubatedwith 1 L of cell suspension (1000 mg/L) at pH 6.3 for 240 h (Entry 14), 1, 4, 92, 93,and 94 were obtained in 18.2%, 23.2%, 4.5%, 3.9% and 4.0%, respectively, giving onlya total recovery of 53.9% (in general, recovery of material was between 75-85%). Acomplex mixture , not extracted in the usual procedure, was also obtained in addition to thenormal product mixture. It was possible that the initial products formed were further58biotransformed to a mixture of unidentified products. Therefore, the optimum conditionsfor the biotransformation of diols 1 and 2 seemed to be a reaction using 16-19 days oldcells with a substrate concentration below 400 mg per litre of cell suspension at pH 6.3 andan incubation time not exceeding 120 h.In summary, the technique of using resuspended cells in TrisHC1 and phosphatebuffers does provide the opportunity to achieve regioselective enzymatic attack of the diol 1to afford products 4, 92, 93 and 94. With 16-19 days old cells in which the enzymesystems were properly developed, relatively efficient biotransformations were achieved. Atlower substrate concentration, yields of overall biotransformation were in the 70-85%range. With higher substrate concentration, lower total recovery (55-65%) were obtained.Phosphate buffer at pH 6.3 appeared superior to TrisHCl buffer.Table 6, Effect of increasing substrate concentration on biotransformation of diol 1.Exp No. 11 12 13 14Cell age (days) 18 19 19 18pH 5.25 5.30 5.30 5.3525nD 1.3330 1.3332 1.3331 1.3332Substrate 1/Et0H(mg/ ml) 1010/25 600/30 800/20 1000/10Volume of culture (L) 2.5 1 1 1Substrate concentration (mg/L) 400 600 800 1000Resuspension buffer: type Phosphate Phosphate Phosphate PhosphatepH 6.3 6.3 6.3 6.3Incubation time (h) 168 216 144 240Yield (%)1 0 2.9 22.5 18.24 38.7 28.5 26.9 23.292 4.3 10.5 3.9 4.593 9.2 9.0 7.0 3.99 4 9.4 8.9 7.2 4.0Total recovery (%) 61.6 59.9 67.5 53.959602.1.1.2^Addition of substrate to the growing cell suspension culture inone batchIn the previous experiments, the "spent medium", which was the nutrient growthmedium left after filtration of cells, was just discarded. Based on the considerations thatsuch "spent medium" may contain some "new" enzymes which were generated during cellgrowth and relevant to biotransformation, and also in order to reduce the tediousprocedures for cell resuspension, biotransformation experiments with direct addition ofsubstrate to the growing cells were performed.The procedures for this series of experiments were generally the same as theprocedures outlined in Scheme 31 except that the initial steps 1 to 3 were omitted. The diol1 dissolved in ethanol was added to the growing cell suspension culture (younger cellswith 0-13 days of cell age) in one batch and incubated for shorter time periods (Table 7).When the substrate (50mg) was introduced to a cell suspension (500 ml, 12 daysold) in the PRDCo nutrient medium (Entry 18), only 24% of the starting material wasrecovered unreacted after only 24 h. It was of interest to note that, in addition to the usualproducts 4, 92, 93 and 94, a new product 8 derived from the diol 1 was also isolated in—10% yield. This compound was assigned as the tertiary alcohol 8 based on its MS, 1 11NMR and 13 C NMR data as discussed earlier. Compound 8 has been isolated fromtobacco plants and its synthetic equivalent has also been prepared chemically by reaction ofdiol 1 with singlet oxygen [83}. Comparison of the spectral data of the biotransformationproduct 8 with the published data indicated their identity.Surprisingly, the results indicated that the reaction proceeded at a faster rate whenthe substrate was applied directly to the growing cell suspension in the PRDCo mediumand a better recovery of materials was also observed. Moreover, the metabolites producedby the cell suspension did not cause any problems for our analysis of the results.612.1.1.2.1^Effects of cell age on biotransformation of diol 1With the encouraging results from the application of substrate directly to the cellsuspension, attention was transferred to an investigation of the effects of different cell ageson biotransformation of diol 1. In a series of experiments, the diol 1 was directly incubatedwith the growing cells of various ages. When the diol 1 was administrated during theinoculation (0 day) of the cell suspension, no biotransformation was observed and the cellswere found to stop growing completely. However, when the diol 1 was applied to cellcultures at 5 days, 11 days and 13 days of growth, not only were the incubation timesdramatically reduced to less than 48 h, but also the recovery of material was found to behigher (Table 7).It was revealed that more efficient biotransformation was achieved with youngercells (11-13 days), at a low ratio of substrate to cell suspension (53 mg in 500 mlsuspension culture) and in a relatively short incubation time (Entry 17). The overall yield ofthe 5 products was 72.1% with 13.2% of recovered diol 1.Cell ages (between 5-13 days) did not seem to have any major effect on either therate of reaction or the yields of the products, indicating that enzyme systems were alreadypresent in the young cells. However, using the 5 day old cell culture seemed to be the bestturn around time for the whole process. Therefore, this technique provided an attractivealternative in performing the biotransformation of tobacco cembranoids. Not only was thetedious resuspension procedure being avoided, but also the reaction was found to proceedat a faster rate.Clearly, an additional enzyme system responsible for C-12 hydroxylation wasproduced during the short incubation time with younger cells.6 2Table 7, Effect of cell age on the biotransformation of diol 1 with direct substrateapplication to the cell suspension.Exp No. 15 16 17 18 19Cell age (days) 0 5 11 13 13pH (initial) 5.12 4.90 5.25 5.20 5.2545 (initial) 1.3370 1.3362 1.3347 1.3331 1.3331Substrate 1/EtOH (mg/ ml) 50/5 50/5 53/5 50/5 50/5Volume of culture (L) 0.5 0.5 0.5 0.5 0.5Substrate concentration (mg/L) 100 100 106 100 100Incubation time (h) 98 24 42 24 24Yield (%)^1 83.0 32.4 13.2 24.0 38.08 8.6 14.9 10.4 6.24 27.1 33.4 33.4 25.49 2 - 3.5 4.0 3.7 3.09 3 7.0 9.8 7.5 6.19 4 7.4 10.0 7.5 6.3Total recovery (%) 83.0 86.0 85.3 86.4 85.02.1.1.2.2 Effects of substrate concentration on biotransformation of diol 1The effects of increasing substrate concentrations were also investigated by applyingdifferent levels of substrate directly to the 5 day old cell suspensions. The results aresummarized in Table 8.It is revealed that very young cells, in which the enzyme systems are not so welldeveloped, are still capable of achieving biotransformation provided that low concentrations ofsubstrate are involved (Entry 20). The low yields seen in Entry 22 may also relate to the shortincubation time and high substrate concentration. Under similar conditions, more startingmaterial was recovered as a result of more substrate application to the cell suspensions. At thisstage, the optimum level of substrate for direct application was 100-200 mg per litre of culture,if the concentration was increased to 400 mg per litre of culture, almost 70% of material wasrecovered unreacted after 24 h.Table 8, Effects of substrate concentration on biotransforamtion of diol 1Exp No. 20 21 22Cell age (days) 5 5 5pH (initial) 5.12 5.09 5.1245 (initial) 1.3370 1.3370 1.3371Substrate 1/EtOH (mg/ ml) 50/5 100/5 200/5Volume of culture (L) 0.5 0.5 0.5Substrate concentration (mg/L) 100 200 400Incubation time (h) 24 24 24Yield (%)^1 27.2 52.3 70.28 7.8 4.7 2.14 29.5 15.8 7.29 2 3.3 1.8 1.39 3 7.5 3.1 1.49 4 7.7 3.2 1.4Total recovery (%) 83.0 80.9 83.62.1.1.3 Batchwise additions of substrate to growing cell suspension cultureResults obtained from the experiment using 0 day old cells (Entry 15) suggestedthat the substrate concentration at this level (100 mg/L of culture) could be too high andindeed toxic to the cells, thereby inhibiting the cells to grow. When the cells cease to grow,the production of enzymes responsible for these biotransformations could also bediminished. The toxicity problems encountered prompted the investigation of sequentialadditions of substrate in smaller quantities to the cell suspension with the hope that the cellscould endure the toxicity and ultimately handle larger amounts of substrate. Therefore,batchwise addition of substrate was undertaken. In this procedure, the substrate wasdissolved in ethanol, divided into several batches and added in batches to the cellsuspension over a certain period of time.63In a series of experiments performed, the diol 1 was dissolved in ethanol, dividedinto 5 portions and added twice a day to the cell suspension. After the addition of the lastbatch, the incubation was allowed to continue for a further 24 h. Unfortunately, the resultshowed that no significant improvement in terms of biotransformation yield was achieved(Table 9).Table 9, Biotransformation by batchwise addition of diol 1 to cell suspension cultureExp No. 23 24 25 26 27Cell age (days) 5 5 12 12 12pH (initial) 5.12 4.92 5.12 5.42 5.8125^.^• •nD  (initial) 1.3370 1.3369 1.3351 1.3351 1.3348Volume of culture (L) 0.5 0.5 0.5 0.5 0.5Substrate 1/EtOH (mg/ml) 10/1 x5a 10/1 x5b 10/1 x5b 50/2 x5b 50/2 x5bTotal 1 added (mg) 50 50 50 250 250Concentration (mg/L) 100 100 100 500 500Total incubation time (h) 120 72 72 72 72Yield (%)1 10.8 14.6 17.4 30.7 38.98 9.6 11.6 10.8 7.5 14.34 28.0 29.5 23.2 20.5 22.292 3.2 3.3 2.4 2.2 2.593 5.8 6.6 6.0 4.6 3.594 6.0 6.8 6.2 4.8 3.5Total recovery (%) 63.4 72.4 66.2 70.4 84.9a^The substrate was divided into 5 batches and added to the culture every 24 h.b^The substrate was divided into 5 batches and added to the culture twice a day (10amand 4pm).64652.1.1.4 Semi-continual addition of substrate via a peristaltic pumpBy employing a mechanical peristaltic pump, a series of experiments with controlledsemi-continual addition of substrate to the growing cell suspension were performed. Thesubstrate was added to the cell suspension in much smaller batches and in a more frequentmanner. After the last addition, the incubation was allowed to continue for a further 24 h.The results are in Table 10.Table 10, Biotransformation by semi-continual addition of diol 1 via a peristaltic pumpExp No. 28 29 30 31 32 33 34Cell age (days) 6 6 12 6 6 12 2pH (initial) 5.02 4.75 5.33 4.95 4.95 5.36 5.6025^.^• •nD (initial) 1.3362 1.3361 1.3345 1.3359 1.3362 1.3349 1.3368Volume of culture (L) 0.5 0.5 0.5 0.5 1.0 1.0 0.5Substrate 1/EtOH (mg/m1) 100/12 100/12 100/12 250/12 100/12 100/12 100/12Rate of addition (ml/hr) 0.5/1 0.5/2 0.5/1 0.5/1 0.5/1 0.5/1 0.5/1Time for addition (h) 24 46 24 24 24 24 24Incubation time afterlast addition (h)24 2 24 24 24 24 24Total incubation time (h) 48 48 48 48 48 48 48Yield: (%)^1 33.5 46.5 35.2 16.1 22.8 26.7 13.88 18.6 9.8 5.8 2.1 4.3 5.2 2.04 27.4 19.2 23.6 34.4 33.1 18.3 20.89 2 3.0 2.1 7.0 1.3 1.1 3.1 09 3 5.6 5.7 3.9 5.8 8.3 6.5 2.89 4 5.7 5.9 8.6 2.1 1.6 3.6 0Total recovery (%) 93.8 89.2 84.1 61.8 71.2 63.4 39.4In one particular experiment (Entry 28), diol 1 (100mg) was added to the cellsuspension culture (0.5 L) via a peristaltic pump over 24 h and the incubation was allowed tocontinue for a further 24 h after the last addition. The unreacted starting material was isolated66in 33.5% yield and the products 8, 4, 92, 93 and 94 were obtained in yields of 18.6%,27.4%, 3.0%, 5.6% and 5.7%, respectively. The overall recovery of material was found tobe extremely good in this case (93.8%). It was revealed that the young cells appear to generatelarge amounts of the C-12 "hydroxylase" enzyme since the ratio of C-12 alcohol 8 to epoxide4 was higher (Entry 28 and 29). Very young cells (2 days old, Entry 34), even thoughincapable of producing any significant amounts of allylic alcohols, were still capable ofproducing epoxide 4 and clearly it already possessed the enzyme system for epoxidation.In summary, this technique allowed higher substrate to suspension culture ratiosalthough the overall benefits in terms of biotransformation yields were marginal.2.1.1.5 Analytical method for evaluating cembranoid biotransformationThe above results obtained from the various biotransformation experiments relied onthe actual isolated yields of recovered starting material and products as determined by flashchromatography on silica gel. Since the structures of products and their behaviours towardsthe absorbent are very similar, considerable difficulties were encountered in resolving thesemixtures. In order to analyse the biotransformation results more effectively and quickly, ananalytical method employing GC was developed in our laboratory. In this analysis, theunderivatized products obtained from biotransformation of cembranoids were used directlyeven though there were reports that underivatized cembranoids were not stable at the hightemperature employed with GC column and derivatizations were necessary. [ 14] Theseunderivatized compounds did not seem to undergo decomposition under our experimentalconditions and the retention times were reproducible. Obviously, this analytical system wassuperior to the reported method based on derivatization in which bis(trimethylsily1)-trifluoroacetamide (BSTFA) [ 14]. By careful selection of methyl docosanoate as internalstandard and calibration of the GC detector responses, quantifications of the underivatizedbiotransformation products were carried out. Table 11 listed the retention times and GCconditions.67Table 11, GC conditions and retention timesCompound^Retention Time (min)1^6.1Methyl docosanoate^8.7GC conditionsColumn:^DB-1701(15m x 0.262 mm)Carrier Gas:^Helium (0.68 ml/min)4 11.3 Injection temperature: 250°C8 12.0 Oven temperature: 220°C9 2 12.6 Detector temperature: 300°C9 3 13.3 Detector: FID9 4 14.4 Concentration of internal standard: 0.25 mg/mlWith the development of the analytical GC method on cembranoids in hand, theanalysis of the biotransformation results was performed more efficiently. Entry 35 in Table12 shows the results obtained at different incubation times from an experiment where thediol 1 was added to the suspension culture at a much slower rate (0.5 ml/3 h) via aperistaltic pump and incubated for a longer time (168 h of total incubation time).Table 12, GC results of biotransformation of diol 1Exp No. 35 36Cell age (days) 18 17pH (initial) 6.30 5.6525 (initial)nD 1.3339 1.3332Volume of culture (L) 0.5 0.1Substrate 1/Et0H (mg/ ml) 500/12 15/3Rate of addition (nil/ hr) 0.5/3 -Incubation time (h) 72 96 120 144 168 168GC Yield: (%)^1 61.9 48.1 30.7 15.7 8.9 76.68 0 5.7 6.1 9.0 9.2 1.14 19.1 24.2 33.0 39.4 42.7 2.79 2 1.1 4.7 5.0 6.8 7.093 4.3 6.3 9.0 11.3 13.1 1.79 4 + + 1.1 4.2 4.5 5.4Total recovery (%) 86.4 89.0 84.9 86.4 85.4 87.56 8In order to destroy the enzyme system, the plant cell culture was autoclaved at 1200C for15 minutes and thus used in biotransformation experiment, almost no biotransformation wasobserved and the starting material was recovered in good yield (Entry 36). Therefore, it wasclear that the described biotransformations were being achived by enzymatic processes.2.1.1.6 ConclusionThe above results indicated that diols 1 and 2, two major tobacco cembranoids, can bebiotransformed efficiently into a series of products, mainly epoxide 4 and epoxide 95 alongwith smaller amounts of the allylic alcohols 92, 93 and 94, by the whole cells of TRP4a.The products obtained can be divided into two main categories, epoxide and allylicalcohol. These two distinctive groups of products could be derived from different reactionpathways catalyzed by different enzyme systems.Among the products isolated from the biotransformation experiments, compounds 92and 93 are assigned as a pair of diastereomers with opposite stereochemistry at C-10, the newlycreated chiral centre, based on 1H NMR and 13C NMR studies. Since attempts to obtain therequired crystals of both 92 and 93 failed, X-ray analyses of these compounds are impossibleat present. Attempts at obtaining derivatives of 92 and 93 may be considered in the near futurein order to get the necessary crystalline forms so as to complete the X-ray determinations.Structurally, compound 1 differs from 2 only in the chirality at C-4, however, markeddifferences in reactivities towards the cell derived enzymes were observed. The a-diol 1 reactedwith such enzymes at a much slower rate when compared with its P-epimer 2.The pH of the buffer involved in resuspending the cells prior to addition of substrateplays an important role in the rate of cembranoid biotransforamtion. Reactions involvingresuspension in phosphate buffer at pH 6.3 were found to proceed much faster than thoseinvolving TrisHCl buffer at pH 7.5 and 9.When the substrate concentration was increased to and/or above 600 mg per litre of cellsuspensions at pH 6.3, a significant portion of starting material remained unreacted after 120 h6 9and extended incubation time resulted in poor overall recovery of material. The optimumconditions for carrying out the biotransformation of cembranoids 1 and 2 seemed to be areaction with a substrate concentration below 400 mg per litre of cell suspension at pH 6.3 andwith an incubation time not exceeding 120 h.Application of substrate directly to the growing cell suspension culture provided anattractive alternative in performing cembranoid biotransformation, not only was the tediousresuspension procedure being eliminated, but also the reaction was found to proceed muchfaster. No significant differences in terms of rate of substrate consumption and product yieldswere observed when cultures of different ages (between 5-13 days) were used. The optimumlevel of substrate for direct application is 100-200 mg per litre of culture.The application of total substrate in one single dose can cause a sudden shock to thecells leading to cell injury or indeed cell death thereby reducing the production of the requiredenzymes for the biotransformation. The experiments with batchwise addition of substrate insmaller quantities or semi-continual addition of substrate via a peristaltic pump in even muchsmaller batches and in a more frequent manner over a longer period of time showed that highersubstrate concentration was allowed (500 mg/L) and total recovery was good (as high as93.8%) although the overall benefits in terms of biotransformation yields were marginal.The above studies have provided evidence that the TRP4a cell line, originallypropagated for the production of di- and triterpenes as part of a study relating to Chinese herbalmedicine [78, 801, can tolerate the cembranoid diols 1 and 2 as "foreign" substrates which areunrelated in structure to those normally produced by this T. wilfordii cell line. It is clear thatenzymatic functionalization of the 11,12 double bond is predominant to afford thecorresponding epoxides 4 and 95 as major products. Other enzyme-catalyzed hydroxylationsat C-10, C-12 and C-13 are also noted and it is clear that the enzymatic functionalization has,on the one hand, similarities to chemical transformations but on the other hand, somedifferences.2.1.2 Chemical studies on the cembranoids: preparation of cembranoidanaloguesIn order to pursue one of our objectives of degrading cembranoid diols 1 or 2 intothe low molecular weight compounds responsible for the tobacco aroma and facilitatestructure elucidation of the biotransformation products in our studies, several cembranoidanalogues were prepared from diol 1 by chemical methods. These analogues were eithersubjected to further biotransformation studies or used as standard compounds forcomparison purposes.2.1.2.1 Conversion of diol 1 to norsolandione 78 and methyl ester 103The diol 1 was converted to norsolanadione 78, a target molecule in ourbiotransformation studies, and the methyl ester 103 prepared according to the publishedprocedures (Scheme 32) [34, 35] with slight modifications. Treatment of diol 1 with aceticanhydride and triethylamine in dichloromethane at room temperature gave monoacetate 97in 95% yield. Oxidation of this acetate 97 with osmium tetroxide in pyridine afforded thetriol 98 in 86% yield. Cleavage of 98 with lead tetraacetate in benzene gave the seco-aldehyde 99 in 83% yield. Oxidation of 99 with Jones' reagent in acetone afforded thecorresponding acid which was derivatized as the methyl ester 100 by reaction with etherealdiazomethane in methanol in 80% yield. Subsequent hydrolysis of acetate 100 withpotassium carbonate in methanol gave diol 101 in 86% yield. Oxidation of 101 withpyridinium chlorochromate (PCC) in dichloromethane followed by retro-aldolfragmentation in basic conditions gave norsolanadione 78 (47%) and the methyl ester 103(43%), the latter being a mixture of cis and trans geometrical isomers (2:3) as indicated byGC and 1 H NMR. This mixture was inseparable by conventional chromatography andappeared as one spot on TLC under several solvent systems. The 1H NMR spectrum of103 showed that there were two singlets at 8 6.03 and 6.05 ppm due to the olefmic proton,701 97 98Ac20^ 0s04H. ^Et N PyridineCI Cl 2^86%95%1, Jones'2, CH2 N 280%Pb(OAc)4Benzene83%99100PCCCH2C1 2+78Me0 'O103Scheme 32, Chemical conversion of diol 1 to norsolanadione 7871two singlets at 8 1.89 and 2.13 ppm attributed to the vinyl methyl (H-8) and two almostsuperimposed singlets at 8 3.620 and 3.625 ppm due to the ester methyl group.Norsolanadione 78 was obtained alternatively following the published procedures[34,351 (Scheme 33). Thus, diol 1 was oxidized to tetrol 104 in 76% yield by the reaction0s04Pyridine76%721104 Pb(OAc)486%BenzeneHQ OHPCC^:^•CH 2 C1 2 > l<57% K 2CO3Me0H52%Scheme 33, Alternative route to norsolanadione 78with osmium tetroxide in pyridine for 5 h. Cleavage of this glycol 104 with lead tetraacetate inbenzene at room temperature gave the seco-aldehyde 32 in 86% yield. Oxidation of this allylicalcohol 32 with pyridinium chlorochromate in dichloromethane at room temperature affordedthe enone 106 in 57% yield. Upon reaction with potassium carbonate in methanol, itunderwent a retro-aldol fragmentation to give the norsolanadione 78 in 52% yield. Thiscompound was well characterized and used in our biotransformation studies as a standardcompound for GC calibration.732.1.2.2 Conversion of diol 1 to seco-diketone 44In order to cleave the 4,5 bond, diol 1 was oxidized to enone 43 in 81% yieldusing PCC in dichloromethane. The retro-aldol reaction of enone 43 with potassiumcarbonate in methanol afforded seco-diketone 44 in 85% yield (Scheme 34).1^ 43^ 44Scheme 34, Conversion of diol 1 to seco-diketone 442.1.2.3 Conversion of epoxide 4 to 7 and 107Epoxide 4, which was the major product obtained in the previousbiotransformation experiments using the TRP4a cell line, was subjected to similar chemicalconversion as diol 1. It was first converted to enone 7 using PCC in dichloromethane in69% yield and then to seco-epoxide 107 in 84% yield under basic condition via retro-aldolreaction (Scheme 35).PCCCH 2C1 269%K2CO3Me0H84% 4^ 7^ 107Scheme 35, Conversion of epoxide 4 to 7 and 1077 42.1.2.4^Conversion of seco-aldehyde 32 to triol 110The seco-aldehyde 32 was reduced to the corresponding triol 110, in 69.5% yieldwith sodium borohydride in methanol (Scheme 36).NaBH4CH30 H69.5% 32^110Scheme 36, Conversion of seco-aldehyde 32 to triol 110Among the compounds obtained in these chemical studies, 7, 78, 103, 107 and110 have been used as reference samples in our investigation for the biodegradation ofcembranoids and 32, 43, 44 and 104 were subjected to further biotransformation studieslater on.2.1.3 Biotransformation of diol 1 using cell free extract (CFE) obtainedfrom the TRP4a cell lineAlthough the biotransformation results with the TRP4a cell line showed that thewhole cells were capable of transforming diols 1 and 2 into corresponding epoxides andallylic alcohols, the locations of the enzyme systems within the cells and responsible forsuch oxidative processes were unknown. In addition, no expected ring cleavage products,such as norsolanadione 78, were obtained. This could mean that the TRP4a cell line didnot contain enzymes capable of such transformation, or the enzymes within the cells wereinaccessible, perhaps membrane bound in a manner that the substrate could not reach theactive site. For this reason, CFE techniques were considered since obviously the availableenzymes were now released from the cells and into appropriate buffer systems.2.1.3.1 Preparation of CFEThe general procedures for CFE preparation and biotransformation are outlined inScheme 37. Steps 1 to 4 were performed at 0-4°C to avoid the protein or enzymedenaturation. The homogenization was performed with an IKA Ultra-Turrax Disperser T-25 fitted with an S25N-25F rotor/stator at 24,000 rpm for 30 seconds and the sameprocedure was repeated 3 times. In order to avoid overheating, a 1 minute break wasallowed between each operation. The homogenate was then subjected to centrifugation at10,000 g (8,000 rpm) for 30 minutes in an automatic refrigerated centrifuge. The clearsupernatant was collected as crude CFE.In order to ascertain the quality of the CFE, both soluble protein concentration andperoxidase activity of the crude CFE were determined [86, 87] The soluble proteinconcentration was assayed by the Bio-Rad procedure and expressed as mg/ml. In thisassay, the proteins present in the CFE formed a complex with a dye reagent, Blue G-250,and the absorbance at 595 nm of the resulting complex is measured. The proteinconcentration of the CFE can be calculated from the standard curve which was obtained bydissolving known amounts of bovine serum albumin (BSA) powder in the same buffer toproduce a set of standard solutions and measuring their absorbances at 595 nm, with theassumption that the extinction coefficient of the dye-CFE protein was identical to that ofdye-BSA protein complex.The peroxidase activity of CFE was assayed by the pyrogallol-purpurogallinmethod and expressed as units/ml using the definition that one unit of peroxidase will form75Celite pad1)Sonicationin EtOAc2) FiltrationICell material EtOAc extractsI^Cell suspension culture1)Filtration2) Wash with distilled water3) Homogenization in buffer (pH 6.6)4) CentrifugationCell pellet^Supernatant ( CFE )1)Substrate administrationwith cofactor (H202)2) IncubationWork-upFiltration through Celite76FiltrateEtOAc extractionEtOAc extracts1)wash with water, brine2) dry over Na2SO43) Concentration in vacuo4) Chromatographic separationProductsScheme 37, Preparation of CFE and biotransformation1.0 mg of purpurogallin from pyrogallol in 20 seconds and 200C. The standard curve wasobtained by dissolving known amounts of purpurogallin in ether to produce a set ofstandard solutions and measuring their absorbances at 420 nm.In the following biotransformation studies, the CFE was used in the amount of 25units per 10 mg of the precursor with addition of 2.16 molar equivalents of 0.24%hydrogen peroxide as cofactor.2.1.3.2 Biotransformation of diol 1 using the CFE prepared fromtheTRP4a cell lineIn order to evaluate the effects of cell ages on cembranoid biotransformation, CFEswere prepared from the cell culture of different ages grown in PRDCo medium andbiotransformation experiments using such CFEs were performed. Unfortunately, all theCFEs prepared from 2-18 day old cultures failed to convert the diol 1 into any expectedproducts after 3 h of incubation even in the presence of hydrogen peroxide (2.16 equiv.) asa cofactor. An overall recovery of 85-95% of diol 1 was observed with a negligible amountof epoxide 4 as determined by GC analysis (Table 13).The inability of CFE to transform the diol 1 into any products is in sharp contrastwith the previous results obtained from the whole cell experiments. Presumably, theprocedures for CFE preparation could have destroyed the activities of the enzyme systemsrequired for the expected biotransformation or the CFE does not contain the relevantenzymes and cofactors which are necessary for the biotransformation of cembranoids. Theenzymes could have remained in the pellet after centrifugation.777 8Table 13, Biotransformation of diol lwith CFE prepared from TRP4a grown in PRDCo mediumExpt. No.Age (days)25nDpHVolume of culture (L)37^38^39^40^41^42^43^442^4^7^9^11^14^16^181.3370 1.3368 1.3361 1.3353 1.3347 1.3332 1.3330 1.33315.55^5.10^4.85^5.35^5.60^5.50^5.50^5.601.5^1.0^0.5^0.5^0.5^0.5^0.5^0.5CFE (m1)^92^82^73^86^93^86^96^230peroxidase activity (unit/ml) 3.43^3.53^4.15^3.83^5.51^5.17^5.90^3.94protein concentration (mg/ml)0.98^1.58^3.48^1.21^1.26^0.72^0.90^0.58pH^6.50^6.50 6.80^6.50 6.55^6.55^6.60 6.60Incubation conditions:Substrate 1/EtOH (mg/ml)Volume of CFE (ml)Buffer added (ml)Distilled water(ml)0.24% H202(m1)Incubation time (h)0.51.53.010/2^10/2^10/27.0^6.0^6.535^35^3515^15^151.0^1.0^1.0^recovered substrate86.0^89.5^84.391.2^92.5^91.982.7^87.4^92.910/2^10/2^50/5 50/54.5^4.8^21.0^32.035^35^175^17515^15^75^751.0^1.0^5.0^5.01 (%,by GC)95.8^93.0^89.6^90.295.3^90.3^93.2^91.793.8^92.2^92.1^92.05/14.51571.099.080.178.12.1.4 Biotransformation of diol 1 using the cell homogenate and the pelletprepared from the TRP4a cell line2.1.4.1 Preparation of cell homogenate and resuspended pelletDuring the CFE preparation, the pellet obtained after centrifugation was discardedpreviously. In order to examine the possibility that some enzymes still remain in the pelletafter centrifugation, a series of experiments were performed with the cell homogenate,which was the total "enzyme mixture" obtained by homogenizing the cells in the phosphatebuffer, and with resuspended pellet, that is, with pellet obtained after centrifugation andresuspension in phosphate buffer, as shown in Scheme 38.Work-upas before Additionof buffer Resuspended pelletCell culture1)Filtration2) Wash with distilled water3) Homogenization in buffer (pH 6.6)(24,000rpm, 30sec.x3)Cell homogenate79Centrifugation(10,000g, 30min)1) Substrateadministrationwith cofactors(H202 , FMN,MnC1 2)2) IncubationCell pellet^Supernatant ( CFE )1) Substrateadministrationwith cofactors(H202 , FMN,MnC12)2) Incubation1) Substrateadministrationwith cofactors(H202 , FMN,MnC12)2) IncubationWork-upas beforeWork-upas beforeScheme 38, Preparation of the cell homogenate and resuspendedpellet and biotransformationThe cells were harvested and homogenised as before in the phosphate buffer (0.1M, pH 6.6). The homogenate was then divided into two portions. One portion was useddirectly in the biotransformation experiments. The remaining portion was then centrifugedat 10,000 g (8,000 rpm) for 30 minutes as in the preparation of CFE. The supernatant wascollected as CFE for biotransformation experiments. The pellets were resuspended in thesame buffer and used directly in biotransformation experiments. The peroxidase activityand protein concentration were measured as before for the homogenate, CFE and theresuspended pellet, respectively. Cofactors were also added and based on earlier studies inour laboratory in which peroxidase enzymes were isolated from Catharanthus roseus cellcultures, manganous chloride and flavin mononucleotide (FMN) were chosen for thispurpose.2.1.4.2 Biotransformation of diol 1 using cell homogenate and resuspendedpelletA series of simutaneous biotransformation experiments with diol 1 were thenperformed using CFE, cell homogenate and resuspended pellet (Table 14). It was foundthat the pellet obtained from the 18 day old TRP4a culture with the supplement of hydrogenperoxide, FMN and manganous chloride appeared to be an optimum condition for theformation of the epoxide 4 from diol 1. Thus, when diol 1 (50 mg in 10m1 ethanol) wasadded to the suspension of the pellet in phosphate buffer (prepared from 18 day old cells)containing 2.16 equivalents of hydrogen peroxide, 0.5 equivalents of manganous chlorideand 0.5 equivalents of FMN and incubated at room temperature for 48 h, the epoxide 4was obtained in 40.1% yield and 38.2% of the starting material still remained unreacted(Entry 48R).The results obtained from the resuspended pellet experiments were found to becomparable with those obtained from the whole cell studies, indicating that the enzymeswere indeed present in the pellet portion of the homogenate. Comparing these results withthose obtained in the biotransformation of diol 1 with resuspended pellet in whichhydrogen peroxide is present as sole cofactor (Table 15), it was clear that withoutmanganous chloride and FMN, the yields of epoxide 4 were low, implying that thesecofactors were necessary for the conversion of diol 1 to epoxide 4.80Table 14, Biotransformation of diol 1 with TRP4a cell homogenate, pellet and supernatantExpt. No. 45 46 47 48Cell age (days) 7 12 14 1825nD 1.3335 1.3332 1.3332 1.3342pH 5.15 6.09 5.70 6.30Vol. of culture (L) 2.0 2.0 3.0 2.0Fresh weight (g) 249 503 964 416Buffer added (ml) 200 480 732 480HomogenateExpt. No. 45H^46H 47H 48HVolume (ml) 400 950 1670 880peroxidase activity (unit/nil) 5.28 4.33 3.86 4.50protein concentration (mg/m1) 2.12 1.00 0.80 1.19pH 6.55 6.55 6.50 6.00Incubation conditionsSubstrate 1 /Et0H (mg/m1) 50/10 50/10 50/10 50/10Homogenate used (m1) 23.7 29.0 32.4 28.0Buffer added (pH6.6, ml) 175 175 175 175Distilled water (ml) 75 75 75 75Cofactors^H202 (eq) 2.16 2.16 2.16 2.16FMN (eq) 0.5 0.5 0.5 0.5MnC12 (eq) 0.5 0.5 0.5 0.5Incubation time (h) 24 24 24 24Yield (%)1480.06.278.514.280.20.979.811.981Table 14, Biotransformation of diol 1 with TRP4a cell homogenate, pellet and supernatant(continued)Supernatant (CFE)Expt. No. 45S 46S 47S 48Speroxidase activity (unit/m1) 4.95 4.72 4.72 4.05protein concentration (mg/ml) 1.69 0.67 0.64 1.23pH 6.45 6.45 6.53 6.55Incubation conditionsSubstrate 1/EtOH (mg/ml) 50/10 50/10 50/10 50/10Supernatant used (ml) 25.3 26.5 26.5 31.0Buffer added (pH6.6, ml) 175 175 175 175Distilled water (ml) 75 75 75 75Cofactors^H202 (eq) 2.16 2.16 2.16 2.16FMN (eq) 0.5 0.5 0.5 0.5MnC12 (eq) 0.5 0.5 0.5 0.5Incubation time (h) 24 24 24 24Yield (%)^1 81.6 87.2 68.8 84.84 6.5 4.3 6.9 7.7Resuspended pelletExpt. No. 45R^46R 47R 48Rperoxidase activity (unit/In]) 1.80 2.40 2.50 2.60protein concentration (mg/ml) 1.07 0.51 0.80 0.83pH 6.50 6.00 6.55 6.00Incubation conditionsSubstrate 1/Et0H (mg/ml) 50/10 50/10 50/10 50/10Resuspension used (ml) 25.0 90 100.0 50.0Buffer added (pH6.6, ml) 175 175 175 175Distilled water (ml) 75 75 75 75Cofactors^H202 (eq) 2.16 2.16 2.16 2.16FMN (eq) 0.5 0.5 0.5 0.5MnC12 (eq) 0.5 0.5 0.5 0.5Incubation time (h) 48 48 48 48Yield (%)^1 66.5 75.4 71.6 38.24 19.7 12.2 + 40.18 2Table 15, The effects of manganous chloride and FMN on biotransformation of diol1 with resuspended pelletExpt. No. 49 50 51Cell age (day) 7 12 18Fresh weight of pellet (g) 38 301 84Buffer added (ml) 100 250 200Vol. of resuspension (ml) 134 415 266peroxidase activity (unit/ml) 1.80 2.40 2.60protein concentration (mg/ml) 1.07 0.51 0.83pH 6.50 6.00 6.00Incubation conditions:Substrate 1/EtOH (mg/ml) 50/10 50/10 50/10Resuspended pellet used (ml) 25.0 90 50.0Buffer added (pH6.6, ml) 175 175 175Distilled water (ml) 75 75 75Cofactors^H202 (eq) 2.16 2.16 2.16Incubation time (h) 48 48 48Yield (%):1 77.3 93.9 81.94 11.7 1.9 12.42.1.4.3 ConclusionEven though cell homogenate and CFE prepared from the TRP4a cell line gave verylow yields of biotransformation products, the pellet resuspended in phosphate buffer (pH6.6) afforded reasonable conversion of diol 1 to epoxide 4 when such cofactors ashydrogen peroxide, manganous chloride and FMN were added. The results indicated thatthe enzymes were indeed present in the pellet portion of the homogenate and the cofactors(manganous chloride, hydrogen peroxide and FMN) were necessary for such conversion.Failure to get any ring cleavage products could mean that the system did not contain theenzymes responsible for such biotransformation processes.832.2 Biotransformation of cembranoids using the T-43-T cell line2.2.1 Biotransformation of diol 1 using the T-43-T whole cellsBecause none of the three enzyme systems, ie, whole cells, CFE and resuspendedpellet of TRP4a, can convert diols 1 or 2 into any ring cleavage products, the tobacco cellline T-43-T was then initiated from the seed of Nicotiana sylvestris, a species in whichdiols 1 and 2, and many other cembranoids are encountered. This cell line was used in ourbiotransformation studies with the hope that it might be more effective in biotransformationof the tobacco cembranoids than TRP4a .Initial biotransformation studies with the T-43-T cell line were performed withwhole cells harvested from culture reaching stationary phase and resuspended in phosphatebuffer (pH 6.3) containing sucrose (8%) as those described in the experiments usingTRP4a. However, this method was deemed inappropriate because of the rapid deteriorationof the cell suspension. It was observed that the color of precursor-treated cells changedfrom green to dark brown within 20 minutes and the Evan's Blue stain test showed noviable cells [88].Subsequent biotransformations were then performed by adding the substrate intothe growing cell cultures directly as in the case of TRP4a. Due to the toxicity of substrate toplant cells, both batch-wise addition and semi-continual addition methods were employedagain in order to reduce such toxic shock to cells. The procedures were the same as thoseused in TRP4a experiments. The results are given in Table 16 and Table 17.In this series of experiments, 12 day old cells were used for biotransformation withvarying substrate addition rates and incubation times. Based on recovery of substrate andoverall yield of biotransformation products, longer incubation times were preferable. It wasinteresting to note that epoxide 4, which was the major product in experiments usingTRP4a growing cells, was a minor component in this study. It was possible that theenzymes within the cells of T-43-T cell line performing hydroxylation at C-10 and C-12 to84Table 16, Biotransformation of diol 1 with batch-wise addition of substrate to T-43-T cellsuspensionExpt No. 52 53 54 55 56Cell age (days) 12 12 12 12 1225nD 1.3358 1.3355 1.3355 1.3355 1.3356pH 5.31 5.35 5.35 5.35 5.25Volume of culture (L) 0.5 0.5 0.5 0.5 0.5Substrate 1 (mg) 200a 90b 90b 90c 90dIncubation time (h) 60 60 72 126 158Yield (%)1 62.5 37.3 41.6 30.8 8.98 3.8 14.9 6.0 14.2 19.84 2.9 12.6 12.8 19.8 12.492 0 5.6 1.4 0 6.993 3.6 12.3 7.2 21.4 29.3a The substrate in 25ml of EtOH was divided into 5 batches and added to suspensionculture in 36 h.b The substrate in 12 ml of Me0H (7.5mg/m1) was divided into 20mg, 15mg, 15mg,20mg and 20mg batches and added to suspension culture in 48 h.c The substrate in 12m1 of Me0H was divided into eight 11.25mg batches and added tosuspension culture in 77 h.d The substrate in 12m1 of Me0H was divided into eight 11.25mg batches and added tosuspension culture in 85 h.e Results in b, c and d were taken from P. Nasiri's report [88].afford the C-10 alcohol 93 and the C-12 alcohol 8 were more accessible to achieve thebiotransformations observed.Several experiments were performed in which an alcoholic solution of diol 1 wasadded slowly in a controlled semi-continual manner by means of a peristaltic pump. It wasfelt that this approach may provide a more desirable biotransformation particularly ifsubstrate toxicity was a problem. However, the results were not sufficiently encouraging tojustify further study with this technique.85Table 17, Biotransformation of diol 1 with semi-continual addition of substrate to T-43-Tcell suspension via peristaltic pumpExpt. No. 57 58 59Cell age (days)n25DpH141.33525.68111.33605.50121.33555.30Volume of culture (ml) 350 350 500Substrate 1/EtOH(mg/ml) 35/12 35/6 90/12Addition rate(ml/hr) 0.5 0.25 0.5Incubation time (h) 48 48 48Yield (%)1 54.7 49.0 59.38 1.7 1.8 10.04 0 2.7 15.892 0 0 3.093 0 0 4.894 2.9 0.8 02.2.2^Biotransformation of diol 1 using CFE prepared from the T-43-Tcell lineBecause still no expected ring cleavage products were obtained in the whole cellexperiments using the T-43-T cell line, the strategy of using CFE was considered again.Similar to those experiments using CFEs prepared from the TRP4a cell line, the CFEs wereprepared from the T-43-T cell cultures of various ages (2-11 days old) and subjected tosimilar biotransformation conditions. Again, no biotransformation was observed whenhydrogen peroxide was used as cofactor and only the starting material was recovered in 90-96% yields (Table 18).86Table 18, Biotransformation of diol 1 with CFE prepared from T-43-T cell lineExpt. No. 60 61 62Cell age (days) 2 7 1125nD 1.3373 1.3366 1.3357pH 5.42 5.51 5.39Volume of culture (L) 1.0 1.0 1.0Fresh weight (g) 47 102 63Buffer added(ml) 90 85 95CFE^Volume(m1) 117 160 148Protein concentration (mg/m1) 0.63 1.10 0.88Peroxidase activity (units/m1) 3.16 4.00 4.16pH 6.55 6.53 6.55Incubation^conditionsSubstrate 1/EtOH(mg/ml) 10/2 10/2 20/4Phosphate buffer(pH6.6, ml) 35 35 70Distilled water(ml) 15 15 300.24% H202 (eq) 2.16 2.16 2.16CFE (m1) 8.0 6.3 6.0Incubation time(h) 24 24 24Recovered substrate 1 (%) 95.0 95.7 90.72.2.3 Biotransformation of diol 1 using cell homogenate and resuspendedpellet prepared from the T-43-T cell lineBased on the same consideration as in the case of TRP4a, similar experiments wereperformed using CFE, the cell homogenate and resuspended pellet prepared from thetobacco T-43-T cell line. The results indicated that the epoxide 4 was formed moreefficiently by the reaction of diol 1 with the homogenate and resuspended pellet containing87cofactors (hydrogen peroxide, FMN and manganous chloride). Thus, when diol 1 wassubjected to incubation with the homogenate and resuspended pellet (prepared from the 12day old cell culture) containing 0.5 equivalents of FMN, 0.5 equivalents of manganouschloride and 2.16 equivalents of hydrogen peroxide for 48 h, the epoxide 4 was obtainedin 57.7% and 61.8%, respectively (Table 19).From the results in Table 19, it is clear that centrifugation of cell homogenate at10,000g to afford CFE eliminates the enzymes relevant to biotransformation and lowers thebiotransformation yields, but the enzymes are indeed present in the pellets left aftercentrifugation of the cell homogenate and favorable biotransformation yields are obtained inexperiments using cell homogenate and resuspended pellet.As indicated in Table 19, the results were dramatically different from those obtainedwith whole cells. The epoxide 4, which was obtained only in low yields in the whole cellexperiments, was the major product with yields of 57.7% and 61.8% for the experimentsusing cell homogenate and resuspended pellet.In a similar experiment using cell homogenate (Entry 64, Table 20), the incubationtime was extended to 120 h and the result indicated that the biotransformation yield washigher. The epoxide 4 was obtained in 70.6% yield, that is 75% based on the startingmaterial consumed. This yield exceeded that obtained with the TRP4a cell line. Apparently,the enzyme responsible for epoxidation of diol 1 was readily available in the cellhomogenate, but the enzymes for hydroxylation were probably destroyed during thehomogenization procedure. Also, it was clear that, although the toxicity of diol 1, in termsof inhibition of cell growth, was a problem at higher substrate concentration in whole cellexperiments (Entry 52, Table 16), the enzymes once liberated from the cells, were able totolerate reasonably high concentrations of diol 1 in the cell homogenate experiments andvery respectable biotransformation yields (72%) could be achieved (Entry 65, Table 20).8889Table 19, Biotransformation of diol 1 with cell homogenate and resuspended pelletprepared from T-43-T cell lineExpt No. 63Cell age (days)n25DpH121.33545.47Volume of culture (L) 1.0Fresh weight (g) 158Buffer added (ml) 150Homogenate Supernatant Resuspended pelletProtein concentration (mg/m1) 1.90 0.96 1.68Peroxidase activity (units/in') 1.77 4.11 2.92pH 6.30 6.35 6.55Incubation^conditions:Substrate 1/EtOH (mg/ml) 50/10 50/10 50/10Phosphate buffer (ml) 175 175 175Distilled water (ml) 75 75 75T-43-T Volume (ml) 80 80 60Cofactors0.24% H202 (eq) 2.16 2.16 2.16FMN (eq) 0.5 0.5 0.5MnC12 (eq) 0.5 0.5 0.5Incubation time (h) 48 48 48Yield (%)^1 30.1 70.1 28.24 57.7 18.4 61.8When the CFE was used with addition of the cofactors (FMN and manganouschloride in addition to hydrogen peroxide), 18.4% of epoxide 4 was obtained and 70.1%of diol 1 was still unchanged, indicating that these cofactors (FMN and manganouschloride) did show some effects on the biotransformation of diol 1 to epoxide 4.Table 20, Biotransformation of diol 1 with cell homogenate at a longer incubation timeExpt No. 64 65Cell age (days)n25DpH141.33455.67141.33525.62Volume of culture (L) 0.5 2.5Homogenate:Protein concentration (mg/m1) 1.70 1.82Peroxidase activity (units/m1) 4.39 3.10pH 6.45 6.40Incubation^conditionsSubstrate 1 /Et0H (mg/ml) 50/10 500/100Phosphate buffer (0.1 M, pH6.6, ml) 175 1750Distilled water (ml) 75 750Homogenate (ml) 100 850Cofactors:^0.24% H202 (eq) 2.16 2.16FMN (eq) 0.5 0.5MnC12 (eq) 0.5 0.5Incubation time(h) 120 120Yield (%)1 5.3 6.04 70.6 72.293 0.8 +94 8.5 4.8In control experiments, it was found that if the cell homogenate was autoclaved at1200C for 15 minutes before use in order to deactivate the enzymes, only 9.7% of epoxide4 was obtained when the above conditions were employed. Therefore the results observedin the previous experiments clearly involve enzymatic processes.When diol 1 was incubated with the cell homogenate without cofactors (H202,FMN and MnC12), no significant biotransformation was observed in GC analysis (Table21). Therefore, the cofactors were necessary for such biotransformation.90Table 21, Biotransformation of diol 1 with T-43-T cell homogenate without cofactors orwith autoclaved cell homogenate.Expt No.^ 66Cell age (days) 15n25D^ 1.3347pH 5.95Volume of culture (L)^ 0.5Fresh weight (g) 129Buffer added (ml) 90Homogenate: Protein concentration (mg/ml)^1.62Peroxidase activity (units/m1) 3.30pH^ 6.50Incubation conditions:Substrate 1/Et0H(mg/m1) 5/1 5/1Phosphate buffer (0.1 M, pH6.6, ml) 17.5 17.5Distilled water(ml) 7.5 7.5Homogenate (m1) 10 10(autoclaved)Cofactors:^0.24% H202 (eq) 2.16FMN (eq) 0.5MnCl2 (eq) 0.5Incubation time(h) 48 48Yield (%, GC)^1 74.6 85.58 0 1.94 9.7 5.392 1.0 1.093 1.0 1.894 1.1 1.02.2.4^ConclusionThe results obtained from the biotransformation using the T-43-T cell line show thatthis cell line behaves differently from the TRP4a cell line in some ways. In the case of9 1TRP4a, both whole cells and resuspended pellet give good conversion of diol 1 to epoxide4 and some allylic alcohols, but cell homogenate and CFE gave almost nobiotransformation. In the case of T-43-T, the biotransformation yields using whole cells aremuch lower. Epoxide 4 is obtained in relatively low yield compared with its yield in TRP4aexperiments and considerable amounts of the C-10 alcohol 93 and the C-12 alcohol 8 areobtained.Cell homogenate and resuspended pellet supplemented with such cofactors ashydrogen peroxide, FMN and manganous chloride give best transformation from diol 1 toepoxide 4 (58-72%), even better than TRP4a whole cells which give epoxide 4 in 50%yield. These cofactors are necessary for the bioconversion of diol 1 to epoxide 4.The respective cleavages of the cembrane skeleton are not occurring with diol 1 andplant cell culture derived enzymes of the T-43-T cell line.2.3 Biotransformation of cembranoid analogues using the T -43-T cell lineIn the previous biotransformation experiments, no expected ring cleavage productswere obtained, indicating that diols 1 and 2 were not suitable substrates for such enzymaticprocesses. Therefore, several cembranoid analogues obtained by chemical conversion fromdiol 1 were subjected to biotransformation studies.2.3.1 Biotransformtion of the epoxide 4 using the T -43 -T whole cellsAlthough the TRP4a cell line was capable of converting the diol 1 into a series ofoxidation products (Scheme 27), it was ineffective in transforming the epoxide 4 into otherproducts. When the epoxide 4 was incubated with a TRP4a cell suspension, direct TLCanalysis failed to reveal the presence of any new products and starting epoxide 4 wasrecovered in good yield (-85%). These results suggested that the more polar products 92,93 and 94 obtained in the biotransformation of diol 1 could not be derived from epoxide 492by an enzymatic process and the epoxide 4 was not a suitable substrate for furtherbiotransformation. On the other hand, the whole cells of T-43-T cell line converted theepoxide 4 into a new product , triol 109, in —55% yield after 16 days of incubation timewith recovery of —10% of epoxide 4 (Table 22).93 16OH174^ 109Scheme 39, Biotransformation of epoxide 4 to 109Table 22, Biotransformation of epoxide 4 with T-43-T whole cellsExpt No. 67 68 69Cell age (days) 8 10 1025nD 1.3360 1.3359 1.3360pH 5.30 5.30 5.28Vol. of culture (L) 0.5 1.0 1.5Substrate 4/EtOH (mg/ml) 47/5 100/10 150/15Incubation time (days) 14 12 16Yield (%)4 21 12 13.3109 47 54 52.7Low resolution EI-MS spectrum of the triol 109 showed that the base peak was atm/z 43 and the largest fragment peak was at m/z 320. The weak peak at m/z 302,corresponded to loss of water from the peak at m/z 320. On the other hand, CI-MSspectrum gives its ions at m/z 303(M++H-2H20), 321(M++H-H20),337(M+-H) and356(M++H+NH3). Therefore, the molecular ion was assigned to m/z 338. In accord withthis assignment, a characteristic peak at m/z 279 was found. This ion was due to loss of 59mass units (C3H70), a process similar to loss of 43 mass units from the isopropyl sidechain at C-1 of a typical cembranoid system, thereby indicating that the newly introducedhydroxyl function was attached to the original isopropyl unit at C-15. Placement of thehydroxyl function at the tertiary C-15 position was readily revealed from its 1H NMRspectrum in which the signals due to H-16 and H-17 became two singlets at 8 1.18 and1.11 ppm. These methyl protons were seen as doublets at 8 0.80 and 0.85 ppm in theepoxide 4. In its 13C NMR spectrum, an additional signal for a tertiary carbon possessing ahydroxyl group can be seen at 8 72.2. A comparison of 13 C data with epoxide 4 isprovided in Table 23. All of these results supported that a new hydroxyl group had beenincorporated into the molecule at the non-activated methine position of the isopropyl sidechain, ie, the C-15 position. Therefore, this product was assigned as a 15-hydroxylatedepoxide, (1S, 2E, 4S, 6R, 7E, 11S, 12S)-11, 12-epoxy-2,7-cembradiene-4, 6, 15-triol109.Table 23, 13C NMR Chemical shifts (ppm) of epoxide 4 and triol 109 determined in CDC13C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C104 47.1 127.7 138.4 72.3 52.9 66.1 132.7 135.2 35.6 24.9109 52.0 126.4 132.6 72.3 53.0 66.1 141.2 135.7 36.0 25.0C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-204 61.3 60.1 36.6 28.6 33.1 19.1 20.9 30.0 16.0 16.3109 61.2 59.5 36.6 25.6 72.2 26.9 27.2 29.9 15.8 16.5This result provides further evidence that the tobacco T-43-T cell line behavesdifferently from the TRP4a cell line. Even though T-43-T whole cells do not show verygood ability to transform the starting diol 1 into the epoxide 4 as does TRP4a in whole cell94experiments, they show capability to hydroxylate the non-activated methine position of theisopropyl side chain in the epoxide 4. This enzymatic introduction of hydroxyl function atC-15 to afford the triol 109 is rather remarkable when one considers other "activated"allylic positions in epoxide 4. As seen in our earlier results using the TRP4a cell line, suchallylic hydroxylations did occur but introduction of hydroxyl function at C-15 has not beenobserved before. Unfortunately, still no ring cleavage products were isolated.2.3.2 Biotransformation of the enone 43 using cell homogenate preparedfrom the T-43-T cell lineSince the T-43-T cell homogenate failed to convert starting diol 1 into any ring-cleavage products, diol 1 was then oxidized using PCC to enone 43 in 81% yield andsubjected to biotransformation conditions as before (Entry 65). But unfortunately, noexpected retro-aldol type reaction took place, only the epoxidation of the 11,12 doublebond was observed again and enone 7 was obtained in 65.3 % yield (Table 24). Since thisenone was not a suitable substrate for ring cleavage, no further biotransformationexperiments were performed.1^ 43^ 795Scheme 40, Chemical conversion of diol 1 to enone 43 and biotransformation of 43 to 7Table 24, Biotransformation of enone 43 with T-43-T cell homogenateExpt. No. 70Age (days) 1625nD 1.3334pH 5.74Volume of culture (L) 1.5Volume of homogenate (ml) 825Peroxidase activity (unit/mi.) 2.81Protein concentration (mg/ml) 0.63Incubation conditions:Volume of homogenate (ml) 267Protein/substrate (mg/mg) 3.4Peroxidase/substrate (units/mg) 15Substrate 43 (mg)/EtOH (ml) 50/5Buffer added (ml) 220Distilled water (ml) 950.24% H202 (eq) 4.0FMN (eq) 0.5MnC12 (eq) 0.5Incubation time (h) 120Yield (%)^43 12.87 65.3Table 25, 13C NMR chemical shifts (ppm) of 43 and 7 determined in CDC13C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C10(1) 46.4 127.7 137.5 72.4 52.2 66.2 130.6 136.6 38.8 23.3(43) 47.1 130.2 136.1 72.7 56.3 200.6 127.0 157.9 40.4 23.9(7) 47.9 130.6 136.0 72.4 54.8 200.9 126.4 159.3 38.3 24.7C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20(1) 124.4 133.3 36.8 27.9 33.0 19.3 20.7 30.1 16.1 15.0(43) 122.9 134.8 37.2 28.7 32.6 19.3 20.5 30.6 18.7 15.7(7) 58.6 60.1 33.8 26.2 31.5 19.9 20.3 30.8 19.1 18.196<O 01) 1\13 121514 -^1719^1^ ^"C 169 10207N8 18The spectral data of biotransformation product 7 were identical to those of syntheticproduct 7 obtained by oxidation of epoxide 4 with PCC and consistent with the publisheddata [42]. The chemical shifts of the starting diol 1, enone 43 and epoxide 7, as obtained inthe corresponding 13C NMR spectra are summarized in Table 25.2.3.3 Biotransformation of the seco-diketone 44 and seco-epoxide 107using the cell homogenate9743^ 44^107Scheme 41, Chemical conversion of 43 into 44 and biotransformation of 44 to 107Since the enone 43 did not undergo the desired retro-aldol fragmentation, it wasfurther converted to seco-diketone 44 chemically. With the hope that T-43-T cellhomogenate could perform appropriate conversions on this ring cleavage substrate, seco-diketone 44 was subjected to biotransformation conditions (Table 26). Unfortunately, stillno further cleavage at 7,8 double bond was observed. The only product obtained was seco-epoxide 107 in low yield. Therefore, either in ring closed or ring opened substrates, themost active site was the same, ie, the 11,12 double bond in 1 and the 7,8 double bond in44.The EI-MS spectrum of 107 showed a molecular ion at m/z 320, suggesting thatone oxygen had been incorporated into the substrate molecule. The expected enzymaticattack at the 7,8 double bond, corresponding to 11,12 double bond in the cycliccembranoid skeleton, was readily evident from 1H NMR spectrum. The olefinic protonsignal at 5.06 ppm (t, J=8 Hz, H-7) and the vinyl methyl proton signal at 1.58 ppm (s, H-20) in 44 were now shifted to 2.60 ppm (t, J=6 Hz, H-7) and 1.24 ppm (s, H-20),respectively, in 107.Table 26, Biotransformation of seco-diketone 44 with T-43-T cell homogenateExpt. No. 71 72 73Age (days)n2516 16 16D 1.3340 1.3340 1.3338pH 7.00 5.72 5.74Volume of culture (L) 1.5 1.0 1.0weight of fresh cell (g) 489 283 353Buffer added (ml) 440 250 315Volume of homogenate (ml) 880 520 640pH 6.58 6.40 6.52Peroxidase activity (unit/ml) 4.78 4.45 3.93Protein concentration (mg/ml) 0.81 1.12 1.02Incubation conditions:Volume of homogenate (ml) 315 169 640Protein(g)/substrate(mg) 5.1 3.8 6.5Peroxidase units/mg 30 15 25Substrate 44 (mg)/EtOH(ml) 50/5 50/5 100/10Buffer added (ml) 90 175 175Distilled water(ml) 40 75 750.24% H202(eq) 4.0 4.0 4.0FMN 0.5 0.5 0.5MnC12 0.5 0.5 0.5Incubation time (h) 120 120 120Yield (%)^4 4 56.2 21.0 83.7107 10.3 25.2 4.698In order to confirm the structure and the stereochemistry at C-7 and C-8, 107 wassynthesized from epoxide 4 by oxidation using PCC followed by retro-aldol reaction withpotassium carbonate. Since the 1 H NMR spectrum of biotransformation product 107 wasidentical to that of synthesized compound 107, this establishes 107 as (3E, 7S, 8S, 11S,12E)-4, 8 -dimethy1-7 ,8-epoxy-11 -is opropy1-3 , 7, 12-pentadecadiene-2, 14-dione.994^ 7^ 107Scheme 42, Chemical conversion of epoxide 4 to 1072.3.4 Biotransformation of the seco-aldehyde 32 using the cell homogenateUpon incubation of the seco-aldehyde 32 with the buffered solution containing thehomogenate and the cofactors (4.0 equivalents of hydrogen peroxide, 0.5 equivalents ofFMN and 0.5 equivalents of manganous chloride) at room temperature for 48 h (Table 27),a more polar spot was observed by TLC analysis. When the reaction was allowed toproceed for a further 72 h, TLC indicated that most of the starting material was consumedwith the appearance of two major new spots. Subsequent chromatography andspectroscopic studies suggested that the more polar product was the primary alcohol 110, areduced product, and the less polar one the cyclized ether 111 (Scheme 43).GC analysis indicated that both compounds 110 and 111 exhibited the sameretention time and it was conceivable that the tetrahydrofuran derivative 111 could bederived from the primary alcohol 110 through a cyclization involving nucleophilic attack ofthe hydroxyl group onto the allylic alcohol system in a S N2'-like manner. Fortunately,these two products were well separated on TLC and could be purified by column32 110 111110 19 OH9 1083 11[1/4^843 12 2 1 0i97 \ ,s0H5 6r-N0 108 -^172 0^1> -<161813 121415174 ^20^11, ".< 62^141'rl^15H ,,100chromatography. The actual isolated yields for 110 and 111 after chromatographicseparation were 29% and 32%, respectively.Scheme 43, Biotransformation of seco-aldehyde 32 into 110 and 111Among the two experiments performed, the results were dramatically different interms of yields of biotransformation products. The only significant difference in thereaction parameters used was the higher level of homogenate volume employed (Entry 75in Table 27). Apparently, the higher concentration of "reductase" enzymes present in thelatter experiment was sufficient to perform the reduction of 32 to 110 and the latter, inturn, could undergo cyclization, as noted above, to give the novel cyclic ether 111.The CI-MS spectrum of 110 indicated that the molecular peak was at m/z 341((M+Hr), even though the most prominent ion observed in the EI-MS spectrum was atm/z 304, due to loss of two molecules of water from 110.The 1H NMR spectrum of 110 is very similar to that of starting material 32 exceptthat the aldehyde signal at 8 9.78 ppm (-CH2CHO) in 32 was replaced by a triplet at 8 3.65ppm (-CH2CLI2OH).In order to aid our structural determination, this primary alcohol 110 was preparedby reduction of the seco-aldehyde 32 with 1.0 equivalent of sodium borohydride inmethanol. Spectroscopic data of the synthetic sample 110 were found to be consistent withproduct 110 isolated from the biotransformation experiment.Table 27, Biotransformation of seco-aldehyde 32 with T-43-T cell homogenateExpt. No. 74 75Age (days)n25DpH151.33515.51161.33455.72Volume of culture (L) 0.5 1.0weight of fresh cell (g) 123 299Buffer added (ml) 100 250Volume of homogenate (ml) 210 540Peroxidase activity (unit/m1) 4.00 3.55Protein concentration (mg/ml) 1.75 1.57pH 6.40 6.45Incubation conditions:Volume of homogenate (ml) 90 422Protein(mg)/substrate(mg) 3.2 6.6Peroxidase (units)/substrata(mg) 7.2 15.0Substrate 32 (mg)/EtOH(ml) 50/10 100/20Buffer added (ml) 175 350Distilled water(ml) 75 1500.24% H202(eq) 2.4 4.0FMN 0.5 0.5Mn02 0.5 0.5Incubation time (h) 120 120Yield (%)^3 2 73.1 2.3110 10.4 29.3111 31.9The CI-MS spectrum of 111 showed that the molecular ion at m/z 323 ([M+H]+).In comparing the 1H NMR spectrum of 111 with that of seco-aldehyde 32, the signal at 84.70 ppm (dt ,H-6) in 32 was missing and vinyl methyl proton signal at 8 1.71 ppm (s, H-20) in 32 was now shifted to high field at 8 1.29 ppm (s, H-20) in the spectrum of 111.101The olefinic proton signal at 8 5.25 ppm (doublet, J=8 Hz, H-5) and the proton signal at 84.78 ppm (multiplet, H-6) in aldehyde 32 were replaced by two new sets of signals at 85.48 ppm (doublet, J=16 Hz, H-5) and 8 5.31 ppm (multiplet, H-6) in the spectrum of111. Finally, in the 13 C NMR spectrum, the C-4 carbon signal at 8 135.7 ppm wasreplaced by a new C-4 carbon signal at 8 82 ppm. Table 28 provides a summary of 13CNMR data for the compounds 32, 110 and 111. Therefore, it was clear that the allylicsystem was attacked and the structure was assigned as the tetrahydrofuran derivative 111with the stereochemistry at C-4 unsolved. In accord with this assignment, a peak at m/z 85can been seen in its MS and this could be due to the cleavage of the 4,5 bond in 111.The results (Entry 75) showed that no oxidation product was isolated in significantamounts and the aldehyde functionality was reduced selectively in the presence of themethyl ketone. This was in sharp contrast with the previous results. Therefore, the cellhomogenate of the tobacco cell line T-43-T was capable of performing both chemicaloxidation and reduction processes. These contrasting enzymatic properties of the tobaccoplant cells could be utilized in other areas of organic synthesis.Table 28, 13C NMR chemical shifts (ppm) of 32, 110 and 111 determined in CDC13C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C103 2 201.9 41.8 31.4 135.7 128.5 66.1 26.2 73.2 139.9 128.7110 62.4 35.7 29.7 137.5 128.6 66.2 27.0 73.1 139.9 127.9111 67.4 37.7 45.8 82.0 138.7 129.2 26.2 72.2 140.3 122.4C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-203 2 48.6 47.5 42.1 209.0 30.0 16.7 26.9 32.0 20.6 19.1110 48.6 47.6 42.1 210.0 30.3 16.5 26.2 32.0 20.6 19.1111 48.7 48.6 42.1 209.5 30.0 25.7 32.0 32.1 19.1 20.7102OH^HO112 ^113 1142.3.5 Biotransformation of the tetrol 104 using the cell homogenateSeveral biotransformation experiments using tetrol 104 as a substrate wereperformed with the T-43-T cell homogenate (Table 29). In this series of experiments,resuspension of cells in buffer was omitted and the homogenate was prepared by directhomogenization of the suspension culture with an Ultra-Turrax Disperser T-25 at 24,000rpm. No cofactors were added in the biotransformation process.When tetrol 104 (50mg/5m1Et0H) was incubated with such a cell homogenate, nobiotransformation was observed and precursor was recovered in 75% yield. Because of thesolubility problem encountered in the experiment using 104 as a substrate, a large amountof ethanol (50mg/50m1 EtOH) was used in subsequent studies. In such a case, a less polarproduct was isolated in approximately 20% yield along with recovery of 50-70% of theprecursor (Entry 77 and 78). The structure of the new product was assigned as ether 112.Scheme 45, Biotransformation of tetrol 104103The mass spectrum of product 112 shows a molecular ion at m/z 338 which is 2mass units less than that of substrate 104. The 13C NMR spectrum indicates that there areone double bond and one carbonyl group in the molecule. The structure for 112 wasconfirmed by direct comparison of its spectral data with the published result.[42] Thisproduct could be formed by oxidation at C-6 to an enone followed by Michael addition ofthe C-11 hydroxyl group to the conjugated enone system. In one experiment (Entry 80),very low yields of the known cyclic ethers 113 and 114 were obtained. Their identitieswere established by comparison the mass spectrometric data with published data.[42, 82]Table 29, Biotransformation of tetrol 104 with T-43-T cell homogenateExpt. No. 76 77 78 79 80Age (days) 20 16 19 18 1625nD 1.3335 1.3338 1.3334 1.3334 1.3339pH 6.85 6.35 5.80 5.60 4.40Volume of culture (L) 0.4 0.4 0.4 1.2 0.8Peroxidase activity (unit/m1) 3.26 4.44 4.94 2.33 2.44Protein concentration (mg/ml) 0.67 0.63 0.64 0.81 0.89Incubation conditions:Substrate 104/EtOH (mg/nil) 50/5 50/50 50/50 150/150 100/200Incubation time (h) 120 120 120* 96 120Yield (%)^104 75 59 52 63 58112 0 21 23 22 23113 0 0 0 0 211 el 0 0 0 0 2* Air was bubbled (500m1/L/min) into cell homogenate during the entire period ofincubation.Since no expected ring cleavage products were obtained in this series of experiments,tetrol 104 seemed to be not a suitable substrate for our purpose. No further optimizationconditions were considered and further studies with this substrate were discontinued.1041052.3.6 ConclusionFive cembranoid analogues obtained either by chemical manipulation or previousbiotransformation have been evaluated as substrates in biotransformation experiments with"growing" cells of the T-43-T cell cultures and with the cell homogenates derived fromthese cultures.Regioselective hydroxylation at C-15, a non-activated methine position in thecembranoid epoxide 4, by whole cells of tobacco T-43-T cell line reveals an interestingnovel enzymatic bioconversion and provides a possible method to hydroxylate the non-activated carbon centres in other substrates. This result could be further explored in otherareas of natural products chemistry where oxidation of a non-activated carbon atom isrequired.In the biotransformation experiments performed using the cell homogenate, enone43 affords only the corresponding epoxide 7 in low yield and the expected ring cleavageproducts via retro-aldol reaction are not observed.The seco-diketone 44 undergoes regioselective attack, in a manner reminiscent ofthe parent cembranoid skeleton, to afford an epoxide 107.When seco-aldehyde 32 is exposed to enzymatic conditions, only reduction of thealdehyde function is noted and the resulting primary alcohol 110 undergoes a precedentedcyclization onto the allylic alcohol component in a SN2'-like manner to give ether 111.It is rather remarkable to note the completely different modes of enzymatic attackwhen various cembranoid analogues are exposed to the enzymes of N. sylvestris cellcultures. It is clear that this tobacco cell line is capable of performing both oxidation andreduction, namely selective epoxidation of double bond, hydroxylation at allylic position,hydroxylation at non-activated methine position and selective reduction of an aldehydefunction to the corresponding alcohol. These contrasting enzymatic properties of thetobacco plant cells could be utilized in other areas of organic synthesis.1062.4 Overall conclusionsAlthough the results presented in this thesis do not realize the goal of establishingan efficient route from tobacco cembranoids, mainly two isomeric diols 1 and 2, to nor-solanadione 78 which is a compound of potential use in the area of aroma and fragrancechemicals, with plant cell culture technology, biotransformation experiments using plantcell cultures derived from Tripterygium wilfordii and Nicotiana sylvestris do show theircapabilities to biotransform the tobacco cembranoids and their analogues into oxidation orreduction products. These studies involving biotransformation of tobacco cembranoidswith plant cell cultures afford novel products and provide information relevant to possiblebiosynthetic pathways involved in the tobacco plant. As proposed in the literature,epoxidation at the 11,12 double bond to give epoxides 4 and 95 was the most favoredprocess and the yields of such processes were generally in the range of 40-75%. Besides,hydroxylated products 8, 92, 93 and 94 resulting from hydroxylation at the C-12, C-10and C-13 positions were obtained in relatively low yields. The stereochemistry at C-10position in the two diastereomeric allylic alcohol 92 and 93 remains to be solved.By using the tobacco cell line T-43-T, hydroxylation at a non-activated position waspossible. In addition, selective reduction of an aldehyde function in the presence of ketonefunction was observed.2.5 Further Research DirectionsSince no expected ring cleavage products were obtained in studies using plant cellcultures, efforts have been directed to the biotransformation studies using microorganisms.Biotransformations with both bacteria and fungi are currently under investigation in ourlaboratory.1 0 73 EXPERIMENTALMelting points were determined using a Reichert melting point apparatus and were notcorrected. Infrared spectra were recorded on Perkin Elmer 710 spectrometers using chloroformsolutions in sodium chloride cells (0.1 mm path length). Optical rotations were recorded on aPerkin-Elmer 141 polarimeter at ambient temperature using a quartz cell of 10 cm pathlength withthe solvent and concentration (in g/100m1) given in parentheses. 1H NMR spectra were recordedon Bruker WH-400, Bruker AC 200 or Varian XL-300 spectrometers. Chemical shift values werereported in ppm relative to tetramethylsilane as an internal standard. 13C NMR spectra wererecorded on a Varian XL-300 spectrometer at 75.3 MHz or a Bruker AC-200 spectrometer at 50.2MHz. Mass spectra were recorded on AEI-MS-902 (low resolution ) and Kratos-MS-50 (highresolution) spectrometers. Elemental analyses were performed by Microanalysis Laboratory,University of British Columbia. GC analyses were performed on Hewlett-Packard 5890A GasChromatography connected to a Hewelett-Packard 3388A integrator using DB-1701 column (J &W Scientific: 15m x 0.262 mm) and Flame Ionization Detector (Carrier gas: helium, Injectiontemperature: 250°C, Oven temperature: 220°C, Internal Standard: methyl docosanoate).All the cell cultures were grown in a rotary shakers at 26°C and 135 rpm withoutillumination. The medium pH was measured on Electrometer Model 265 while the refractive indexwas measured using an Officine Galileo refractometer at 25°C. The cell homogenization wasaccomplished with an IKA Ultra-Turrax Disperser T-25 fitted with an S25N-25F rotar/stator(Janike and Kunkel GmbH and Co. KG) and centrifugation was performed using Sorvallsuperspeed RC2-B automatic refrigerated centrifuge. UV absorbances in protein and peroxidaseactivity assay were recorded on Bausch and Lomb Spectronic 20 spectrophotometer. Semi-continual addition of precursor was accomplished via a peristaltic pump (Cole-Parmer Model7401-20 with Master Flex pump drive).Column chromatography was performed using silica gel (230-400 mesh) with air ornitrogen gas pressure to obtain a suitable flow rate, while thin layer chromatography was1 0 8performed using commercial aluminum-backed silica gel plates (Merck, art. 5554). Visualizationwas accomplished by spraying with a solution of vanillin in concentrated sulfuric acid (0.5%)followed by heating in an oven at 120°C for 1 minute.(1S, 2E, 4S, 6R, 7E, 11E)-2,7,11-cembratriene-4,6-diol 1 and (1S, 2E, 4R, 6R, 7E,11E)-2,7,11-cembratriene-4,6-diol 2 were provided by the Swedish Tobacco Company,Stockholm. All the cell suspension cultures were maintained and supplied by the BiologicalServices of the Department of Chemistry , University of British Columbia.3.1 Chemical conversions of diol 1 to cembranoid analogues3.1.1 Conversion of (1S, 2E, 4S, 6R, 7E, 11E)-2,7,11-cembratriene-4,6-diol 1to (1S, 2E, 4S, 6R, 7E, 11E)-6-acetoxy-2,7,11-cembratriene-4-ol 97 and (1S,2E, 4S, 6R, 7E, 11S, 12S)-6-acetoxy-2,7-cembradiene-4,11,12-triol 98 [ 35 ]1^97^ 98To a stirred solution of diol 1 (300 mg, 0.98 mmol) and triethyl amine (2 ml) indichloromethane (50 ml), acetic anhydride (0.5 ml) was added. After being stirred at roomtemperature for 18 h, water (10 ml) was added and the reaction mixture was allowed to stir foranother 2 h. The reaction mixture was then extracted with ethyl acetate (3x100 ml). The extractswere washed with water, then with brine and dried over anhydrous sodium sulphate.Concentration in vacuo and chromatography on silica gel using ethyl acetate as eluent gave theacetate 97 (330 mg, 95%).Osmium tetroxide (250 mg, 0.98 mmol) was then added to the solution of 97 (330 mg)in pyridine (50 ml). After being stirred at room temperature for 4 h, an aqueous solution of981 0 9sodium bisulfate (500 mg in 50 ml of water) was added and the reaction mixture was allowed tostir for an additional 2 h. The reaction mixture was then diluted with ethyl acetate (50 ml) andwater (30 ml). The aqueous layer was extracted with ethyl acetate (2x300 ml). The combinedorganic extract was washed with water and brine and then dried over anhydrous sodiumsulphate. Concentration in vacuo and chromatography on silica gel using ethyl acetate andhexanes (1:1) as eluents gave triol 98 (313 mg, 86%). The physical properties are as follows:mp: 59-600C; [a]D : +170 (c =0.45, EtOH); IR (CHC13) Vmax : 3590, 3450, 1720 and 1250cm-1; 1 H NMR (CDC13) 8: 0.82(d, J=6.5 Hz, 3H)/0.89(d, J=6.5 Hz, 3H)(H-16/H-17), 1.05(s,3H, H-20), 1.31(s, 311, H-18), 1.68(d, J=1.0 Hz, 3H, H-19), 2.04(s, 3H, -OCOCH3),3.28(m, 1H, H-11), 5.4-5.9(m, overlapped, 4H, H-2, H-3, H-6 and H-7); LRMS m/z(%):322(W-60, 0.2), 304(5), 286(1), 261(3), 243(2), 227(3), 206(3), 139(12), 121(26), 109(23),95(29), 81(34), 71(37), 55(26), 43(100); HRMS calcd. for C 20113403 (W-60): 322.2509,found: 322.25143.1.2^Conversion of 98 to (4E, 6R, 8S, 9E, 11S)-6-acetoxy-4, 8-dimethy1-8-hydroxy-11-isopropy1-14-oxo-4, 9-pentadecadienal 99 [ 35 ]Powdered lead tetra-acetate (45 mg, 0.21 mmol) was added to a solution of triol 98 (70mg, 0.18 mmol) in benzene (10 ml). After being stirred at room temperature for 10 mins, thereaction mixture was diluted with ethyl acetate (20 ml), saturated sodium thiosulfate solution (5ml) was added and the resulting solution was allowed to stir for an additional 10 min. Thereaction mixture was then separated into two layers and the aqueous layer was extracted with ethylacetate (3x50 ml). The combined extracts were washed with water and brine and then dried over1 1 0anhydrous sodium sulphate. Concentration in vacuo and chromatography on silica gel using ethylacetate and hexanes (1:1) as eluents gave seco-aldehyde 99 (56 mg, 83%). The physicalproperties are as follows: oil; [a]p: -4.8 0 (c =0.27, EtOH); IR (CHC13) Vmax: 3460, 2740,1720 and 1250 cm -1 ; 111 NMR (CDC13) 8: 0.84(d, J=6.7Hz, 3H)/0.88(d, J=6.7Hz, 3H)(H-17/H-18), 1.28(s, 3H, H-19), 1.75(d, J=1.3 Hz, 3H, H-20), 1.77(dd, J=6 and 15Hz, 1H, H-7a), 1.99(dd, J=6.8 and 15 Hz, 1H, H-7b), 2.01(s, 3H, -000CL13), 2.13(s, 3H„H-15),5.20(d, J=9Hz, 1H, H-5), 5.36(dd, J=8 and 15.5 Hz, 1H, H-10), 5.49(d, J=15.6 Hz, 1H, H-9), 5.66(ddd, J=6, 6.8 and 9.2Hz, 1H, H-6), 9.76(t, J=1.4 Hz, 1H, H-1); LRMS m/z (%): 362(M-F-18, 0.1), 337 (0.1), 320 (0.4), 302 (6), 277 (0.6), 259 (3), 201 (3), 179 (8), 161 (18), 135(20), 121 (50), 109 (40), 95 (50), 81 (45), 71 (42), 55 (33), 43 (100); HRMS calcd. forC22H3404 (M -F-18): 362.4657, found: 362.4650.3.1.3^Conversion of 99 to methyl (4E, 6R, 8S, 9E, 11S)-6-acetoxy-4, 8-dimethy1-8-hydroxy-11-isopropy1-14-oxo-4, 9-pentadecadienoate 100 [ 35 1AcO=AcO ,OH^0 ^0, z^\, / >"‹\^>i<HN Me0 099^ 100Jones' reagent (chromium trioxide in sulphuric acid, 2.7 M) was added dropwise to asolution of 99 (300 mg, 0.79 mmol) in acetone (50 ml) at -100C until the orange color persisted.The excess Jones' reagent was destroyed by dropwise addition of isopropanol and the reactionmixture was then diluted with ethyl acetate (250 ml). The resulting solution was separated intotwo layers and the aqueous layer was extracted with ethyl acetate (2x200 ml). The combinedorganic extracts were washed with water and brine and then dried over anhydrous sodiumsulphate. Concentration in vacuo gave the crude product. The crude product was dissolved inAc0.OH0%rMe0^ 0100> <HO PH0, z\N7Me0 0101>w<1 1 1methanol (50 ml) and ethereal solution of diazomethane was then added dropwise until thereaction mixture attained a permanent yellow color. Excess diazomethane was removed bypassing a stream of nitrogen through the solution. The resulting solution was concentrated invacuo to give the crude mixture of the methyl ester. Chromatography on silica gel using hexanesand ethyl acetate (3:1) as eluents gave ester 100 (260 mg, 80%). The physical properties are asfollows: oil; [alp: -4.20 (c =0.81, EtOH); IR(CHC13) Vmax : 3680, 3550, 1720 cm-1 ; 1 H NMR(CDC13) 8: 0.84(d, J=6.6 Hz, 3H)/0.88(d, J=6.6Hz, 3H)(H17/H18), 1.27(s, 3H, H-19),1.75(d, J=1.2Hz, 3H, H-20), 2.00(s, 3H, OCOCH3), 2.13(s, 3H, H-15), 3.67(s, 3H, OCH3),5.21(dd, J=1.3 and 9.2Hz, H-5), 5.38(dd, J=8.8 and 15.6Hz, 1H, 11-10), 5.49(d, J=15.7Hz,H-9); LRMS m/z(%): 350(M±-60, 0.1), 332(4), 289(2), 197(8), 154(26), 121(22), 109(11),95(32), 79(19), 17(17), 55(17), 43(100); HRMS calcd. for C21113404 (M±-60): 350.2458,found: 350.2456.3.1.4^Conversion of 100 to methyl (4E, 6R, 8S, 9E, 11S)-6,8-dihydroxy-4, 8-dimethyl-ll-isopropyl-14-oxo-4, 9-pentadecadienoate 101 [ 35 1Powdered potassium carbonate (50 mg) was added to the solution of 100 (240 mg, 0.585mmol) in methanol (50 ml). After being stirred at room temperature for 2 h, the reaction mixturewas diluted with ethyl acetate (250 ml) and washed with water until neutral pH. The organic layerwas then washed with brine (100 ml) and dried over anhydrous sodium sulphate. Concentrationin vacuo and chromatography on silica gel using hexanes and ethyl acetate (3:1) as eluents gave101 (185 mg, 86%). The physical properties are as follows: oil; [ta]D : +260 (c =0.57, CHC13);OH0Mee()^Me0 0101 102> <781 1 2IR (CHC13): Vmax 3680, 3600, 3480, 1730, 1710 cm -1 ; 111 NMR (CDC13) 8: 0.83 (d,J=6.5Hz, 3H)/0.88 (d, J=6.5Hz, 311) (H-17/H-18), 1.41 (s, 3H, H-19), 1.70 (d, J=1.0Hz, 3H,H-20), 2.12 (s, 3H, H-15), 3.65 (s, 3H, OMe), 4.78 (ddd, J=2.5, 8.2 and 10.0Hz, 111, H-6),5.24 (dd, J=1.1 and 8.2Hz, 111, H-5), 5.34 (dd, J=8.6 and 15.3 Hz, 111, H-10), 5.50 (d,J=15.3Hz, 1H, H9); LRMS m/z(%): 350 (M+-18, 0.2), 335 (0.2), 332 (2), 289 (1), 231 (1),223 (3), 289 (1), 231 (1), 223 (1), 194 (11), 154 (15), 136 (25), 121 (23), 109 (12), 93 (41), 79(21), 69 (19), 55 (20), 43 (100); HRMS calcd. for C21113404 (M+-18): 350.2458, found:350.2467.3.1.5 Conversion of 101 to methyl 4-methyl-6-oxo-heptene-4-oate 103 and (3E,5S)-5-isopropyl-3-nonene-2,8-dione, norsolanadione, 78 [ 34 ]Pyridinium chlorochromate (250 mg, 0.55 mmol) was added to the solution of 101 (180mg, 0.49 mmol) in dichloromethane (30 ml). After being stirred at room temperature for 2 h, thesolution was diluted with ether (30 ml) and filtered through a thin layer of silica gel.Concentration in vacuo gave the crude enone 102 (158 mg). The crude enone 102 (158 mg) wasdissolved in methanol (20 ml) and powdered potassium carbonate (200 mg) was then added.After being stirred at room temperature for 2 h, the solution was diluted with ethyl acetate (100ml), washed with water, then brine and dried over anhydrous sodium sulphate. Concentration invacuo and chromatography on silica gel using hexanes and diethyl ether (2:1) as eluents gaveinitially methyl 4-methyl-6-oxo-4-hepteneoate 103 (36 mg, 43%) as a mixture of cis- and trans-geometrical isomers (2:3), followed by (3E, 5S)-5-isopropyl-3-nonene-2,8-dione 78 (45 mg,47%). The physical properties of 103 are as follows: oil; 1 11 NMR (CDC13) 8: 1.89 & 2.13 (s,11 1 3H-8), 2.45 (s, H-7), 3.620 & 3.625 (s, -OMe), 6.03 & 6.05 (s, H-5); LRMS m/z(%): 170 (Mt,14.9), 155 (3.9), 141 (2.6), 138 (62.4), 127 (21.7), 111 (18.3), 96 (83.1), 85 (26.3), 67(18.7), 59 (9.8), 55 (14.6), 43 (100); HRMS calcd. for C91-11403: 170.0943, found: 170.0940.The physical properties of 78 are as follows: oil; [a] D : -2.00 (c =0.67, CHC13); IR (CHC13)Vmax : 1675, 1715 cm-1 ; 1H NMR (CDC13) 8: 0.81 (d, J=6.5Hz, 3H)/0.86(d, J=6.5Hz, 3H)(H-11/H-12), 2.04 (s, 3H, H-9), 2.18 (s, 3H, H-1), 5.94 (d, J=16Hz, 1H, H-3), 6.46 (dd, J= 9and 16Hz, 1H, H-4); LRMS m/z(%): 178 (M+-18, 2.7), 163 (5.6), 153 (8.5), 145 (10.6), 135(31.3), 126 (36.9), 121 (30.9), 111 (22.6), 97 (60.2), 93 (36.5), 84 (44.5), 43 (100); HRMScalcd. for C12H2002: 196.1464, found: 196.1458.3.1.6^Conversion of diol 1 to (1S, 2E, 4S, 6R, 7E, 11S, 12S)-2,7-cembradiene-4,6,11,12-tetrol 104 [ 35 ]Osmium tetroxide (420 mg, 1.64 mmol) was added to a solution of diol 1 (500 mg, 1.63mmol) in pyridine (25 ml). After being stirred at room temperature for 3 h, sodium bisulfite (800mg) in water (20 ml) was added to the reaction mixture and the resulting solution was allowed tostir for another 3 h. The reaction mixture was then diluted with ethyl acetate (50 ml) and washedwith water (100 ml). The aqueous layer was further extracted with ethyl acetate (2x200 ml) andthe organic extracts were combined and washed with brine, then dried over anhydrous sodiumsulphate. Concentration in vacuo and chromatography on silica gel using ethyl acetate as eluentafforded tetrol 104 (420 mg, 76%) as a white foam. The physical properties of 104 are asfollows: mp: 133-40C; [a]p: +6.50 (c =1.3, EtOH); IR (CHC13) Vmax : 3682, 3404 cm-1 ; 1HNMR (CDC13) 8: 0.85(d, 3H, J=6.5Hz)/0.90(d, 3H, J=6.5Hz) (H-16/H-17), 1.09(s, 3H, H-HO/ ',coH / 0104^ 32HO ;OH> <1 1 420), 1.28(s, 3H, H-18), 1.70(d, J=1Hz, 3H, H-19), 1.84(dd, J=2.5 and 14Hz, 1H, H-5),2.10(d, J=7.5 and 14Hz, 1H, H-5'), 2.32(bs, 1H, -OH), 2.77(bs, 1H, -OH), 3.37(m, 1H, H-11), 3.58(bs, -OH), 4.62(ddd, J=2.5, 7.4 and 8.8Hz, 1H, H-6), 5.5(d, J=15.4Hz, 1H, H-3),5.64(dd, J=8.5 and 15.4Hz, 1H, H-2), 5.70(dd, J=1.1 and 8.8Hz, 1H, H-7); LRMS m/z(%) :322 (M+-18, 1.2), 304 (9), 289 (1.2), 261 (5.4), 243 (3.1), 227 (4.6), 136 (28.3), 121 (39.8),109 (41.6), 95 (46.5), 81 (53.9), 71 (54.1), 55 (55.0), 43 (100); HRMS calcd. for 020113403(M+-18): 322.2508, found: 322.2513; Elemental analysis calcd. for C20113604: C 70.55, H10.66, found: C 70.40, H 10.46.3.1.7^Conversion of tetrol 104 to (4E, 6R, 8S, 9E, 11S)-4,8-dimethy1-6,8-dihydroxy-11-isopropy1-14-oxo-4,9-pentadecadienal 32 [ 33 ]Powdered lead tetra-acetate (460 mg, 1.03 mmol) was added to the solution of tetrol 104(350 mg, 1.03 mmol) in benzene (50 ml). After being stirred at room temperature for 10 mins, thereaction mixture was diluted with ethyl acetate (100 ml), then saturated sodium thiosulfatesolution (50 ml) was added and the resulting solution was allowed to stir for an additional 10mins. The reaction mixture was then separated into two layers and the aqueous layer wasextracted with ethyl acetate (200 ml). The combined organic extract was washed with water andbrine and then dried over anhydrous sodium sulphate. Concentration in vacuo andchromatography on silica gel using hexanes and ethyl acetate (2:1) as eluents afforded 32 (300mg, 86%). The physical properties of 32 are as follows: mp: 51-20C; [a]D : 54.1 0 (c =0.44,CHC13); IR (CHC13) Vmax: 1720 cm -1 ; 1 H NMR (CDC13) 8: 0.89 (d, J=6.5Hz, 3H)/ 0.93 (d,)f)N>11.< 0  ^> - <()/HO OH 0^,OH1 1 5J=6.5Hz, 3H)(H-17/H-18), 1.40 (s, 3H, H-19), 1.70 (d, J=1.0Hz, 3H, H-20), 2.10 (s, 3H, H-15), 4.80 (ddd, J=2.5, 7.4 and 8.8Hz, 1H, H-6), 5.22 (dd, J=1.1 and 8.8Hz, 1H, H-5), 5.30(dd, J=8.5 and 15.4Hz, 1H, H-10), 5.72 (d, J=15.4Hz, 1H, H-9), 9.80 (s, 1H, H-1); LRMSm/z(%): 338 (Mt 0.1), 302 (2.5), 284 (0.6), 277 (0.5), 259 (1.5), 241 (1.1), 223 (0.7), 136(58.1), 121 (80.5), 109 (47.3), 97 (53.8), 93 (100), 81 (62.4), 71 (40.7), 55 (23.4), 43 (59.9);HRMS calcd. for C20113404: 338.2457, found: 338.2450; Elemental analysis calcd. forC20H3404: C 70.97, H 10.13, found: C 70.79, H 10.06.3.1.8^Conversion of 32 to (4E, 8S, 9E, 11S)-4, 8-dimethy1-8-hydroxy-11-isopropy1-6,14-dioxo-4,9-pentadecadienal 106 and nor-solanadione 78H 0^ H 032 106^78Pyridinium chlorochromate (350 mg, 0.77 mmol) was added to the solution of seco-aldehyde 32 (250 mg, 0.74 mmol) in dichloromethane (50 ml). After being stirred at roomtemperature for 1 hr, the solution was diluted with ether (200 ml) and the resulting mixture wasthen filtered through a layer of silica gel. Concentration in vacuo and chromatography on silica gelusing hexanes and ethyl acetate (2:1) as eluents gave enone 106 (142 mg, 57%). Enone 106(140 mg, 0.42 mmol) was then dissolved in methanol and potassium carbonate (50 mg) wasadded. After being stirred at room temperature for 1 hr, the reaction mixture was diluted withethyl acetate and washed with water, then brine and dried over anhydrous sodium sulphate.Concentration in vacuo and chromatography on silica gel using hexanes and ethyl acetate (95:5) aseluents afforded nor-solanadione 78 (43 mg, 52%), which is identical to that obtained previously.1 1 63.1.9^Conversion of diol 1 to (1S, 2E, 4S, 7E, 11E)-2,7,11-cembratriene-4-ol-6-one 43 [5, 39 ]43To a solution of pyridine (0.24 ml, 3 0 mmol) in dichloromethane (10 ml), chromiumtrioxide (100 mg, 1 0 mmol) was added at 0°C. After being stirred at 0°C for 30 minutes, diol 1(100 mg, 0.327 mmol) in dichloromethane (2 ml) was added and the mixture was stirred for 2 hat 0°C. Water (10 ml) was then added and the mixture was extracted with ethyl acetate (3x50 ml).The extract was washed with water and brine and then dried over anhydrous sodium sulphate.Concentration in vacuo and chromatography on silica gel using ethyl acetate and hexanes (1:3) aseluents gave enone 43 (80 mg, 80.5%). The physical properties of 43 are as follows: mp 77-8°C; [a]p: +930 (c =0.62, CHC13); IR(CHC13) V max : 3400, 1680 cm-1 ; 1 H NMR (CDC13) 8:0.80 (d, J=8.0 Hz, 3H)/0.86 (d, J=8.0 Hz, 311) (H-16/H-17), 1.35 (s, 311, H-18), 1.57 (s, 3H,H-20), 2.08 (d, J=1.0 Hz, 3H, H-19), 2.2-2.4 (m), 2.54(d, J=12 Hz, 111, H-5a), 2.73 (d, J=12Hz, 111, H-5b), 2.84(s, OH), 4.95(m, 1H, H-11), 5.55-5.35 (m, 2H, H-2 and H-3), 6.05 (s,1H, H-7); LRMS m/z(%) : 304(5.8), 286(4.5), 261(7.0), 243(10.0), 203(11.5), 191(3.9),163(34.2), 123(53.7), 109(48.9), 98(60.8), 71(51.8), 55(52.3), 43(100); HRMS calcd. forC20113202: 304.2402, found: 304.2399; Elemental analysis calcd. for C20113202: C 78.89, H10.59, found: C 79.00, H 10.73.3.1.10^Conversion of 43 to (3E, 7E, 11S, 12E)-11-isopropy1-4,8-dimethyl-3,7,12- pentadecatriene-2,14-dione 44 [41]HOHOOH,-^ .• OHHO/1101 1 74 3^ 4 4Enone 43 (55 mg, 0.181 mmol) was dissolved in a mixture of aqueous sodiumcarbonate solution (1.0%, 10 ml) and ethanol (5 ml) and stirred at room temperature for 24 h. Thereaction mixture was worked up by extraction with ethyl acetate (3x20 ml ). The extract waswashed with water and brine and then dried over anhydrous sodium sulphate. Concentration invacuo. and chromatography on silica gel using ethyl acetate and hexanes (1:3) as eluents gaveseco-diketone 44 (46.8 mg, 85.1%). The physical properties are as follows: oil; [a]p: 9.5 0 (c0.4, CHC13); IR(CHC13) Vmax: 1690, 1675, cm -1 ; 1H NMR (CDC13) 5: 0.85 (d, J=8.0 Hz,3H)/0.90 (d, J=8.0 Hz, 3H) (H-16/11-17), 1.58(s, 3H, H-20), 2.10(d, J=1 Hz, 3H, H-19),2.15(s, 3H, H-15), 2.25(s, 3H, H-1), 5.06(t, J=8 Hz, 1H, H-7), 6.02(d, J=6 Hz, 1H, H-13),6.07(s, 1H, H-3), 6.59(dd, J=12 and 16 Hz, 1H, H-12); LRMS m/z(%): 304(1.0), 289(0.7),261(3.6), 243(4.9), 219(1.2), 203(9.4), 191(2.5), 149(22.7), 123(48.1), 109(58.4), 98(95.9),81(100), 71(49.2), 55(24.9), 43(31.9); HRMS calcd. for C20H3202: 304.2402, found:304.2402.3.1.11 Conversion of 32 to (4E, 6R, 8S, 9E, 11S)-4,8-dimethy1-11-isopropyl-14-oxo-4,9-pentadecadiene-1,6,8-triol 110Sodium borohydride (5.0 mg, 0.13mmol) was added to a solution of 32 (50 mg, 0.148mmol) in methanol (5 ml) at 00C. After being stirred for 5 min, the reaction mixture was diluted1 1 8with ethyl acetate (50 ml) and the resulting solution was washed with water, then with brine anddried over anhydrous sodium sulphate. Concentration in vacuo and chromatography on silica gelusing ethyl acetate and hexanes (1:2) as eluents gave initially the recovered 32 (11 mg, 22.0%)followed by triol 110 (35 mg, 69.5%). The physical properties of 110 are as follows: oil;[a]D :33.60 (c =0.3, CHC13); IR Vmax (CHC13): 3600, 1710 cm -1 ; 1H NMR (CDC13) 8: 0.82(d, J=8.0 Hz)/0.86 (d, J=8.0 Hz) (6H, H-18/H-17), 1.40(s,3H, H-19), 1.70(d, J=1 Hz, 3H, H-20), 2.12(s, 3H, H-15), 3.65(t, J=6 Hz, 2H, H-2/H-3), 4.79(dt, J=4 and 10 Hz, 1H, H-6),5.25(d, 12 Hz, 114, H-5), 5.35(dd, J=12 and 16 Hz, 1H, H-10), 5.50(d, J=16 Hz, 1H, H-9);LRMS m/z(%) :304(M+-36, 39.6), 289(9.8), 261(12.1), 246(6.4), 231(7.6), 197(30.1),136(49.8), 121(92.0), 111(89.1), 93(100), 85(51.6), 71(23.6), 43(5.1); HRMS calcd. forC20113202 (M+-36): 304.2402; found: 304.2403.3.1.12^Conversion of epoxide 4 to (1S, 2E, 4S, 7E, 11S, 12S)-11, 12-epoxy-4-hydroxy-2, 7-cembradien-6-one 7 [42]I I <4 7To a stirred cool solution of pyridinium chlorochromate (35.6mg, 0.165 mmol) indichloromethane (15 ml) was added a solution of epoxide 4 (50 mg, 0.155 mmol) indichloromethane (1 ml). The mixture was stirred at 0°C for 2.5 h. Water (40 ml) was then addedand the mixture was stirred for a further 5 min. Extraction with ethyl acetate (3x50m1) followedby solvent removal in vacuo and chromatography on silica gel (25g) using ether and hexanes(2:1) as eluents gave enone 7 (34.2 mg, 69%) and recovered epoxide 4 (8.9 mg, 18%). Thephysical properties of 7 are as follows: mp: 66-67°C; [a] D : +85° (c =0.60, CHC13); IR (CHC13)Vmax : 3610, 3470, 1665 cm-1 ; 1 H NMR (CDC13) 8: 0.81 (d, J=6.5 Hz, 3H)/0.84 (d, J=6.51 1 9Hz, 3H) (H-16/H-17), 1.27 (s, 3H, H-20), 1.35 (s, 3H, H-18), 2.20 (d, J=1.2 Hz, 3H, H-19), 2.49 (d, J=12.6 Hz, 1H, H-5a), 2.62 (dd, J=4.5 and 9.0 Hz, 1H, H-11), 2.74(d, J=12.6Hz, 1H, H-5b), 3.60 (br s, OH), 5.42 (d, J=15.5 Hz, 111, H-3), 5.49 (dd, J=8.0 and 15.5 Hz,1H, H-2), 6.08 (br s, 1H, H-7); LRMS m/z(%): 320(10.2), 305(2.8), 302(9.0), 287(3.3),277(13.0), 259(11.3), 241(4.7), 219(16.5), 201(10.4), 194(47.5), 137(98.1), 123(70.5),109(100), 97(78.3), 81(66.6), 71(25.5), 55(11.7), 43(13.8); HRMS calcd. for 020113203:320.2352; found: 320.2361.3.1.13^Convertion of 7 to (3E, 7S, 8S, 11S, 12E)-4, 8-dimethyl-7, 8-epoxy-11-isopropyl-3, 7, 12-pentadecadiene-2, 14-dione 107I , K 7 107Enone 7 (30 mg, 0.094mmol) was dissolved in methanol (10m1) and water (1 ml).Powdered potassium carbonate (75mg, 0.54mmol) was added and the mixture was stirred atroom temperature for 2 h. Water (40 ml) was then added and the mixture was extracted with ethylacetate (3x50 ml). The extract was washed with water until neutral pH, then with brine and driedover anhydrous sodium sulphate. Concentration in vacuo and chromatography on silica gel usingethyl acetate and hexanes (1:1) as eluents gave seco-diketone 107 (25.2 mg, 84%). The physicalproperties of 107 are as follows: oil; [a]D: +18.90 (c =0.28, CHC13); IR Vmax (CHC13): 1685,1675 cm-1 ; 1 H NMR (CDC13) 8: 0.81 (d, J=6.5 Hz, 311)/0.84 (d, J=6.5 Hz, 3H) (H-17/H-18),1.24 (s, 311, H-20), 2.12 (s, 311, H-19), 2.18 (s, 3H, 11-15), 2.22 (s, 311, H-1), 2.60 (t,J=6Hz, 111, H-7), 5.59(s, 1H, H-3), 6.02(d, J=10Hz, 1H, H-13), 6.50(dd, J=15 and 10 Hz,1H, H-12); LRMS m/z(%): 320(M+, 0.8), 305(0.2), 302(0.3), 295(0.4), 277(1.5), 262(2.4),1 2 0194(2.6), 137(14.3), 123(20.3), 109(19.2), 95(21.8), 81(28.1), 69(16.0), 55(22.8), 43(100);HRMS calcd. for C20113203 (M±-18): 320.2351, found: 320.2345.3.2 Propagation of the plant cell cultures3.2.1 Propagation of TRP4a cell cultureTRP4a cell line was derived from a leaf explant of Tripterygium wilfordii.[ 801 Thesuspensions were maintained in PRDCo medium which was the PRL-4 medium of Gamborg andEveleigh [89] supplemented with 2,4-dichlorophenoxyacetic acid (D) (2 mg/L) and coconut milk(100 ml/L). Maintenance of this stock culture was carried out by subculturing of a 10% (v/v)inoculum at 14-17 days intervals into a fresh PRDCo medium and agitating on a rotary shaker at26°C and 135 rpm without illumination. The growth profiles were monitored by pH andrefractive index of the spent medium.3.2.2 Propagation of T-43-T cell cultureTobacco cell line T-43-T was initially derived from seeds of Nicotiana sylvestris whichwere obtained from the Swedish Tobacco Company. The leaf derived tobacco calli were culturedon solidified agar (8 g/L) with Murashige-Skoog (MS) medium [ 90] containing sucrose (30g/L)and 2,4-D (1 mg/L) and were kept at 25°C with a cycle of 8 h darkness and 16 h light. Calli weresubcultured every 3-4 weeks. The cell suspension cultures were inoculated at a rate of 1.8 g drycell mass/L of liquid MS medium. All suspension cultures were grown at 26°C and 135 rpmwithout illumination in 1 L Erlenmeyer flasks with 500 ml liquid Murashige-Skoog mediumcontaining sucrose (30g/L) and 2,4-D (1 mg/L). Cultures were maintained by transferring 15%inoculum to fresh MS medium at 20-21 day intervals and agitating on a rotary shaker at 26°C and135 rpm without illumination. The growth profiles were monitored by pH and refractive index ofthe spent medium.1213.3 Biotransformations using TRP4a cell line3.3.1^Biotransformations using TRP4a whole cells3.3.1.1^Preparation of buffers for resuspending cells.TrisHC1 (0.05M, pH 7.5):Trizma® (6.06 g, 0.05 mole) was dissolved in distilled water (800 ml) and then sucrose(80 g) was added. The volume of the solution was made up to 1 L with distilled water, while thepH of the solution was adjusted to 7.5 by adding 5% hydrochloric acid.Phosphate (0.1 M, pH 6.3):K2HPO4 (4.1 g, 0.0235 mole), KH2PO4 (10.44 g, 0.0765 mole) and sucrose (80 g)were dissolved in 800 ml of distilled water and the volume of the solution was made up to 1 Lwith distilled water.3.3.1.2^General procedure for Experiments 1-14: addition of substrate to thecells resuspended in bufferThe buffer solutions and all the apparatus used were autoclaved at 120 0C for 15 minutesbefore use. All culture manipulations including cell resuspension and biotransformation in thissection were performed under aseptic conditions. The suspension culture was filtered throughMiracloth and resuspended in the same volume of buffer. The substrate (diol 1 or 2) dissolved inethanol was then added to the resuspension culture and incubated at 260C and 135 rpm on arotary shaker for an appropriate time. The biotransformation mixture was worked up by ethylacetate extraction as described below. Chromatography on silica gel afforded pure products.3.3.1.2.1^Typical procedure for biotransformation of diol 1: addition of diol 1to the cells resuspended in buffer (Entry 6, Table-4)The cells from 4 flasks of suspension culture of the TRP4a cell line (4x500 ml, 16 daysold) were filtered through a Buchner funnel equipped with Miracloth, washed with phosphatebuffer (400 ml) and subjected to suction to remove excess water. The resulting cells (800 g) were1 2 2then transferred to a 4 L. beaker containing 2000 ml of phosphate buffer (pH 6.3). After agitatingwith a spatula, the resuspended cells were poured into four 1 L Erlenmeyer flasks to which diol 1(4x100 mg) dissolved in ethanol (4x10 ml) was added. The mixture was then incubated at 26°Cand 135 rpm on a rotary shaker. The control experiment was set up in the same way except thatno diol 1 was added .After 144 h of incubation time, the biotransformation mixture in each flask was filteredthrough Miracloth and the filtrate was extracted with ethyl acetate (3x200 ml). The combinedextracts were washed with water (200 ml) and brine (200 ml) and then dried over anhydroussodium sulphate. Concentration of combined extracts from each flask in vacuo afforded a crudemixture of products (318 mg) from the broth.Ethyl acetate (300 ml) was added to the combined cell material and the resultingsuspension was homogenised with an IKA Ultra-Turrax disperser T-25 at 20,000 rpm for 5 min.The resulting homogenate was then filtered through Miracloth, the filtrate was washed with water(200 ml), then brine (200 ml) and dried over anhydrous sodium sulphate. Concentration in vacuoafforded a crude mixture of products (105 mg) from the cell mass.The combined extract (423 mg) was chromatographed on silica gel (200 g) using ethylacetate as eluent and separated into three portions (A: 35 mg, B: 239 mg, C: 89 mg). The portionA was purified again on silica gel (25 g) using ethyl acetate and hexanes (2:1) as eluents to giverecovered diol 1 (20 mg, 5%), while portion B was purified again on silica gel (100 g) usingacetone and hexanes (1:2) as eluents to give epoxide 4 (140 mg, 35%) and allylic alcohol 92 (64mg, 14%). Finally, portion C was purified again on silica gel using acetone and hexanes (1:1) aseluents to give C-10 alcohol 93 (20 mg, 5%) and C-13 alcohol 94 (48 mg, 12%). The physicalproperties of the isolated products are as follows:HO^ HOpH ^pHHO^ HO924 93^94OHHO123(1S, 2E, 4S, 6R, 7E, 11S, 12S)-11,12-epoxy-2,7-cembradiene-4,6-diol 4 [ 361: mp 99 - 1000C.[a]D : +113.90 (c =0.62, CHC13); IR (CHC13): Vmax 3430 cm -1 ; 1 11 NMR (CDC13) 8: 0.8 (d,J=7Hz, 3H)/0.85 (d, J=7Hz, 3H) (H-16/H-17), 1.20 (s, 3H, H-20), 1.37 (s, 3H, H-18), 1.78(s, 311, H-19), 1.75 (dd, J=6Hz, 14.2 Hz, 1H, H-5), 2.20 (dd, J=6.land 14.2Hz, 1H, 11-5),2.88 (dd, J=2Hz and 8Hz, 11-1, H-11), 4.48 (dt, J=2Hz and 8Hz, 1H, H-6), 5.30 (m,overlapped, 2H, H-2 and H-3), 5.40 (d, J=8Hz, 111, H-7); LRMS m/z(%): 322([M]+, 0.1),304(0.9), 286(0.8), 261(3.8), 243(3.9), 203(5.1), 177(5.8), 163(10.7), 136(21.4), 123(28.9),109(33.8), 95(43.2), 81(52.8), 69(41.6), 55(35.5), 43(100); HRMS calcd. for C20H3403:322.2508, found: 322.2510; Anal. calcd. for C20H3403: C 74.49, H 10.63; found: C 74.31, H10.46(1S, 2E, 4S, 6R, 7E, 1041, 11E)-2,7,11-cembratriene-4,6,10-triol 92: mp: 41-42 0C; [a]p:+51.30 (c =0.60, CHC13); IR Vmax (CHC13): 3625, 3420 cm -1 ; 1H NMR (CDC13) 8 : 0.8 (d,J=7Hz, 3H)/0.85 (d, J=7Hz, 3H) (H-16/H-17), 1.25 (s, 3H, H-20), 1.58 (s, 3H, 11-18), 1.60(s, 3H, H-19), 4.48 (t, J=7Hz, 111, H6), 4.54 (dt, J=2Hz and 8Hz, 1H, H10), 5.16 (d, J=8Hz,111, H-11), 5.30 (d, J=8Hz, 111, 11-7), 5.40 (d, J=15Hz, 1H, H-3), 5.50 (dd, J=8 and 15Hz,1H, 1-1-2); LRMS m/z(%): 304([M-18] -1- , 0.4), 286(4.5), 268(2.4), 243(2.6), 223(10.7),203(6.4), 177(6.0), 165(19.1), 136(21.4), 123(24.7), 109(25.7), 97(51.5), 81(62.8), 69(57.0),55(41.9), 43(100); HRMS calcd. for C20H3202 [M-18]+: 304.2402, found: 304.2406; Anal.calcd. for C20H3403: C 74.49, H 10.63; found: C 74.50, H 10.581 2 4(1S, 2E, 4S, 6R, 7E, 1042, 11E)-2,7,11-cembratriene-4,6,10-triol 93: mp: 63-64°C;[a]D :+74.5° (c =0.51, CHC13); IR (CHC13) Vmax: 3620, 3410 cm -1 ; 1H NMR (CDC13) 8:0.80 (d, J=7Hz, 3H)/0.85 (d, J=7Hz, 3H) (H-16/H-17), 1.37 (s, 3H, H-18), 1.69 (s, 6H, H-18 and H-19), 2.55 (dd, J=7Hz and 14Hz, 1H, H-9), 4.43 (dt, J=2Hz and 8Hz, 1H, H-6), 4.54(m, 1H, H-10), 5.22 (d, J=8Hz, 1H, H-11), 5.30-5.37 (m, overlapped, 2H, H-2 and H-3), 5.43(d, J=8Hz, 111, H-7); LRMS m/z(%): 322(M+, 0.1), 304( 0.4), 286(1.1), 262(2.7), 243(1.8),223(8.1), 205(5.7), 177(4.7), 165(10.9), 123(19.5), 112(41.4), 97(32.4), 81(35.7), 69(29.6),59(100), 55(43.0), 43(53.2); HRMS calcd. for C20113403 : 322.2508, found: 322.2498; Anal.calcd. for C20H3403: C 74.49, H 10.63; found: C 74.40, H 10.39(15, 2E, 4S, 6R, 7E, 11E, 13S)-2,7,11-cembratriene-4,6,13-triol 94: mp: 66-67°C; [a] D :+25.6° (c 0.18, CHC13); IR Vmax (CHC13): 3620, 3420 cm -1 ; 1 11 NMR (CDC13) 8 : 0.8 (d,J=7Hz, 3H)/0.85 (d, J=7Hz, 3H) (H-16/H-17), 1.35 (s, 3H, 11-18), 1.54 (s, 3H, H-19), 1.66(s, 311, H-20), 3.98(dd,J=8Hz and 14Hz, 1H, H-11), 4.45 (t, 8Hz, 1H, H-6), 5.20 (d, J=8Hz,1H, 11-3), 5.30 (m, overlapped, 2H, H-7 and H-11), 5.40 (dd, J=8 and 15Hz, 1H, H-2); LRMSm/z(%): 304([M-18]+, 0.5), 260(2.8), 243(1.1), 227(4.2), 203(6.4), 177(6.0), 165(4.0),140(34.6), 123(20.2), 109(21.9), 95(29.5), 81(34.9), 69(35.6), 55(49.3), 43(100); HRMScalcd. for C201-13202 (M+-18): 304.2403, found: 304.2401; Anal. calcd. for C20113403: C74.49, H 10.63; found: C 74.38, H 10.363.3.1.2.2^Typical procedure for biotransformation of diol 2: addition of diol 2to the cells resuspended in buffer (Entry 8, Table-5)Diol 2 (50 mg) was dissolved in ethanol (5 ml) and added to an Erlenmeyer flaskcontaining 19 day old cells (211 g, wet weight) resuspended in Tris HC1 buffer (500 ml, pH 7.5).The mixture was incubated in exactly the same way as in experiment using diol 1. Work-up after144 h as before afforded crude product mixture from broth (25 mg) and from cells (45 mg).1 2 5Chromatography of combined extracts (70 mg) on silica gel (25 g) using ethyl acetate as eluentgave epoxide 95 (26.3 mg, 53%) followed by a mixture (12.4 mg) which could not be purifiedby conventional silica gel chromatography and was not studied further. The physical propertiesfor product 95 are as follows:95(1S, 2E, 4R, 6R, 7E, 11S, 12S)-11, 12-epoxy-2, 7-cembradiene-4, 6-diol 95[36]: oil, [a]D :+66.40 (c =1.1, CHC13); IR V max (CHC13): 3400, 1390 and 1375 cm -1 ; 1H NMR (CDC13) 8:0.81 (3H, d, J=6Hz)/0.86 (3H, d, J=6Hz), 1.20 (3H, s), 1.38 (3H, s), 1.72 (3H, d, J=1.3Hz),2.83 (1H, dd, J=2 and 7.5 Hz), 4.85 (1H, dt, J=2 and 9Hz), 5.20 (1H, dd, J=7.5 and 16 Hz),5.35 (1H, d, J=8Hz), 5.48( 1H, d, J=16Hz); MS[m/z(%)]: 322(1), 304(7), 286(6), 261(9),243(7), 233(3), 205(4), 163(18), 150(17), 136(54), 121(41), 107(43), 95(51), 81(70), 69(57),55(58), 43(100); HRMS calcd. for C20H3202, 304.2402[M-18]±, found304.23893.3.1.3 General procedures for Experiments 15-35: addition of substrate to thegrowing cell suspension cultureIn all instances, the diol 1 was dissolved in ethanol and added to the growing cells in oneof the following ways: In experiments 15 to 22, the diol 1 was added to the cell suspensionculture in one batch while in experiments 23 to 27, the alcoholic solution of diol 1 was dividedinto several portions and added to the cell suspension culture batchwise over a certain period oftime. The mixture thus formed was incubated on a rotary shaker at 135rpm and 260C. Inexperiments 28 to 35, the substrate was added to the cell suspension culture in a bottom stirredbenchtop bioreactor semi-continually via a peristaltic pump and incubated at room temperature. All> <HO81 2 6other parameters (pH, age of culture, etc.) are indicated in the Tables. The work-up procedureswere the same as those used in Experiments 1-14.3.3.1.3.1 Typical procedure for biotransformation with one batch addition ofdiol 1 to the growing cell suspension culture (Entry 18, Table-7)Diol 1 (50 mg) was dissolved in ethanol (5 ml) and added directly to a Erlenmeyer flaskcontaining cell suspension culture (500 ml, 13 days old). The resulting mixture was incubated ona rotary shaker for 24 h. The biotransformation mixture was worked up as before to afford crudeextracts (67 mg). Chromatography on silica gel (25 g) using ethyl acetate as eluent afforded therecovered diol 1 (12 mg, 24%), tertiary alcohol 8 (5.2 mg, 10%), epoxide 4 (16.5 mg, 33%), C-10 alcohol 92 (2 mg, 4%), C-10 alcohol 93 (3.7 mg, 8%) and C-13 alcohol 94 (3.9 mg, 8%).The physical properties for tertiary alcohol 8 are as follows:HO^,OH(1S, 2E, 4S, 6R, 7E, 10E)-2,7,10-cembratriene-4,6,12-triol 8[451: mp: 134-1350C; [a]D : +420(c =0.18, CHC13); IR Vmax (CHC13): 3602, 3460 cm-1 ; 1H NMR (CDC13) 8: 0.8 (d, J=7Hz,311)10.85 (d, J=7Hz, 3H) (H-16/H-17), 1.21 (s, 3H, 11-20), 1.29 (s, 3H, H-18), 1.66 (s, 3H,11-19), 1.75 (dd, J=6Hz and 14.2 Hz, 1H, 11-5), 2.20 (dd, J=6.land 14.2Hz, 1H, 11-5'), 2.70(dd, J=0.8Hz and 7Hz, 2H, H-9a and H-9b), 4.80 (t, J=7Hz, 111, H-6), 5.40 (d, J=15.5Hz,1H, H-3), 5.42 (d, J=16Hz, H-11), 5.54 (dd, J=8.5 and 15.5Hz, 1H, H-2), 5.54 (d, J=8.2Hz,1H, 11-7), 5.64 (dt, J=6.9 and 16Hz, 1H, 11-10); LRMS m/z(%): 304(W-18, 1.6), 289(1.2),261(5.8), 243(6.1), 223(8.5), 203(8.3), 189(3), 177(9.7), 161(12.5), 151(13.8), 136(38.7),123(45.6), 109(57.5), 95(55.8), 79(60), 69(59.3), 55(60.6), 43(100); HRMS calcd. for1 2 7C2043202: 304.2402 (M+-18), found: 304.2398; Anal. calcd. for C20113403: C 74.49, H10.63; found: C 74.35, H 10.48^3.3.1.3.2^Typical procedure for biotransformation with batch-wise addition ofdiol 1 to the growing cell suspension culture (Entry 23, Table-9)Diol 1 (50 mg) was dissolved in ethanol (5 ml) and added in 5 equal batches at 24 hintervals over 96 h to an Erlenmeyer flask containing suspension culture (500 ml, 5 days old).The resulting cell suspension was incubated on a rotary shaker. After addition of the last batch,the incubation was allowed to continue for a further 24 h (total incubation time, 120 h). Work-upas before afforded a crude extract (60 mg). Chromatography on silica gel (25 g) using ethylacetate as eluent afforded recovered diol 1 (5.4 mg, 10.8%), tertiary alcohol 8 (4.8 mg, 9.6%),epoxide 4 (14.0 mg, 28.0%), C-10 alcohol 92 (1.6 mg, 3.2%), C-10 alcohol 93 (2.9 mg,5.8%) and C-13 alcohol 94 (3.0 mg, 6.0%), respectively.^3.3.1.3.3^Typical procedure for biotransformation with semi-continual additionof diol 1 to the growing cell suspension culture via a peristaltic pump (Entry 32,Table-10)The suspension culture (2x500 ml, 6 days old) was transferred into a 1 L benchtopbioreactor which was bottom stirred with a magnetic stir bar, aerated through a sintered glass diskat 200 mVL/min, and kept at room temperature. Diol 1 (100 mg) was dissolved in ethanol (12 ml)and added into the above suspension culture through a timer-controlled peristaltic pump over aperiod of 24 h at a rate of 0.5 ml/h. After the last addition, the incubation was allowed to continuefor a further 24 h. Work-up as before afforded crude extract (132 mg). Chromatography on silicagel (50 g) using ethyl acetate as eluent afforded recovered diol 1 (22.8 mg, 22.8%), tertiaryalcohol 8 (4.3 mg, 4.3%), epoxide 4 (33.1 mg, 33.1%), C-10 alcohol 92 (1.1 mg, 1.1%), C-10alcohol 93 (8.3 mg, 8.3%) and C-13 alcohol 94 (1.6 mg, 1.6%), respectively.1283.3.2 Biotransformations using CFE3.3.2.1 Preparation of phosphate buffer (0.1 M, pH 6.6)KH2PO4 (13.6 g, 0.10 mol) was dissolved in 1 L distilled water as solution A andNa2HPO4 (14.2 g, 0.10 mol) was dissolved in 1 L distilled water as solution B. Solution A(-600 ml) and solution B (-350 ml) was mixed and the final pH was adjusted to 6.6 with additionof solution B.3.3.2.2 Preparation of CFEAll procedures were performed at 0-4°C.TRP4a cell suspension culture was harvested by filtering through a Buchner funnelequipped with Miracloth and the filtrate was collected for pH and refractive index measurements.The cells were washed with equal amount of distilled water (wt/vol) and allowed to suction dry.The fresh weight was determined.Phosphate buffer (0.1 M, pH 6.6, 140 ml/100 g fresh weight) was added to the cells andthe resulting suspension was then homogenised with an IKA Ultra-Turrax Disperser T-25 at24,000 rpm for 30 sec. The same procedure was repeated three times. In order to avoidoverheating the suspension, a 1 minute break was allowed between each operation.The homogenate thus obtained was then subjected to centrifugation at 10,000g (8,000rpm) for 30 min. The clear supernatant was collected as crude CFE and used in biotransformationexperiments. The peroxidase activity and soluble protein concentration were assayed according tothe following procedures.3.3.2.3 Measurement of protein concentration: Bio -Rad protein assay [86]One part of the dye reagent (Bio-Rad Protein Assay Dye Reagent Concentrate) was dilutedwith four parts of distilled water. The diluted dye reagent solution (5 ml) was then added to a test-1 2 9tube containing CFE (0.1 ml) and the solution was mixed thoroughly. After using a referencesample (prepared by mixing phosphate buffer (0.1 ml, 0.1M, pH 6.6) and the diluted dyesolution (5 ml)) to adjust the reading of the UV spectrometer at 595 nm to zero, the absorbance ofthe CFE was then measured at the same wavelength. The protein concentration can be calculatedfrom the standard curve which was produced by dissolving known amounts of bovine serumalbumin (BSA) powder in the same buffer to produce a set of standard solutions (0.1 mg/ml to1.0mg/m1), adding aliquots (0.1 ml) of these solutions to the diluted dye (5 ml) and andmeasuring absorbances at 595nm.3.3.2.4 Measurement of peroxidase activity: pyrogallol -purpurogallin assay [ 87 ]CFE (1 ml) was added to a 50 ml Erlenmeyer flask containing 5% aqueous pyrogallolsolution (2 ml), 0.1 M phosphate buffer (2 ml, pH 6.6), freshly-prepared 0.5% hydrogenperoxide solution (1 ml) and distilled water (14 ml) at 20°C. This mixture was allowed to standfor 20 seconds at 20°C, then 2M sulphuric acid (1 ml) was added to quench the reaction and thesolution was then extracted with ether (2x25 ml). After the reading of the UV spectrometer wasadjusted to zero at 420 nm by a reference sample which was an ether extract (2x25 ml) from amixture of 5% pyrogallol solution (2 ml), 0.1M phosphate buffer (3 ml, pH 6.6), freshly-prepared 0.5% hydrogen peroxide solution (1 ml) and distilled water (14 ml), the absorbance ofthe organic extract from the CFE reaction was then recorded at the same wavelength. The standardcurve can be obtained by measuring absorbance at 420 nm of a set of standard solutions preparedby dissolving purpurogallin (0.5 to 3.5 mg) in ether (50 ml).3.3.2.5 General procedures for biotransformations using CFE (Entry 37 -44)The CFE solution (25 units of peroxidase per 10 mg of precursor) was added to anErlenmeyer flask containing precursor (10 mg) dissolved in ethanol (2 ml), distilled water (15m1),phosphate buffer (35m1, 0.1 M, pH 6.6), and 0.24% hydrogen peroxide (1 ml, 2.16 equiv.).1 3 0After being stirred at room temperature for an appropriate interval, 25 ml of ethyl acetate wasadded and the mixture was allowed to stir for another 5 min. The resulting mixture was thenfiltered through Celite and the filtrate was extracted with ethyl acetate (3x50 ml). The Celite padwas sonicated with ethyl acetate (50 ml) for 30 min and then filtered. The combined organicextracts were washed with water, then brine and dried over anhydrous sodium sulphate.Concentration in vacuo afforded the crude mixture of products which were then chromatographedon silica gel column.3.3.2.6^Typical procedure for biotransformation using CFE (Entry 44, Table-13)The CFE solution (32 ml, containing 125 units of peroxidase, 18.6 mg of protein)prepared from 18 day old TRP4a cell suspension was added to an Erlenmeyer flask containingprecursor (50 mg) dissolved in ethanol (10 ml), distilled water (75m1), phosphate buffer (175m1,0.1 M, pH 6.6), and 0.24% hydrogen peroxide (5 ml, 2.16 equiv.). After being stirred at roomtemperature for an appropriate interval, small amounts of reaction mixture (5% by volume) waswithdrawn and extracted with ethyl acetate to provide a sample for TLC and GC analysis. After 3h of incubation, ethyl acetate (125 ml) was added and the mixture was allowed to stir for another5 min. The resulting mixture was then filtered through Celite and the filtrate was extracted withethyl acetate (3x250 ml). The Celite was sonicated with ethyl acetate (150 ml) for 30 minutes andthen filtered. The combined organic extracts were washed with water and brine and then driedover anhydrous sodium sulphate. Concentration in vacuo afforded the crude extract (76 mg).Chromatography on silica gel (25 g) using ethyl acetate as eluent gave the recovered diol 1 (37.8mg, 88.9%).3.3.3^Biotransformations using cell homogenate, resuspended pellet andsupernatant (CFE) with cofactors added3.3.3.1 Preparation of cell homogenate, resuspended pellet and supernatant1 3 1The procedures were similar to those mentioned in the preparation of CFE and wereperformed at 0-4°C.The homogenate obtained as before was divided into two portions. One portion of thehomogenate was used directly in the biotransformation experiment. The remaining portion of thehomogenate was then centrifuged at 10,000 g (8,000 rpm) for 30 min. The supernatant wascollected as CFE for biotransformation. The pellet was resuspended in the same phosphate buffer(pH 6.6, 40 m1/10 g wet weight) and also used in biotransformation experiments. The peroxidaseactivity and protein concentration were measured as before for the homogenate, CFE and theresuspended pellet, respectively.^3.3.3.2^General procedures for biotransformations using cell homogenate,resuspended pellet and supernatant (CFE)The CFE solution, cell homogenate or resuspended pellet was added to an Erlenmeyerflask containing precursor (50 mg) dissolved in ethanol (10 ml), distilled water (75 ml),phosphate buffer (175 ml, 0.1 M, pH 6.6), 0.24% hydrogen peroxide (2.16 equiv.), FMN (0.5equiv.) and manganous chloride (0.5 equiv.). After being stirred at room temperature for anappropriate interval, ethyl acetate (125 ml) was added and the mixture was allowed to stir foranother 5 min. The resulting mixture was then filtered through Celite and the filtrate was extractedwith ethyl acetate (3x250 ml). The Celite pad was sonicated with ethyl acetate (150 ml) for 30minutes and then filtered. The combined organic extracts were washed with water, then brine anddried over anhydrous sodium sulphate. Concentration in vacuo afforded the crude mixture ofproducts which were then chromatographed on silica gel column.^3.3.3.3^Typical procedure for biotransformation of diol 1 using cellhomogenate (Entry 46H, Table-14)The cell homogenate (29 ml, containing 125 units of peroxidase and 29 mg of protein)prepared from 12 day old cell suspension culture was added to an Erlenmeyer flask containing1 3 2diol 1 (50 mg) dissolved in ethanol (10 ml), distilled water (75 ml), phosphate buffer (175 ml,0.1 M, pH 6.6), 0.24% hydrogen peroxide (2.16 equiv.), FMN (0.5 equiv.) and manganouschloride (0.5 equiv.). After being stirred at room temperature for 24 h, the reaction mixture wasworked up as before through ethyl acetate extraction etc. to afford combined crude extracts (89mg). Chromatography on silica gel (25 g) using ethyl acetate as eluent gave recovered diol 1(39.3 mg, 78.5%) followed by epoxide 4 (7.1 mg, 14.2%).3.3.3.4 Typical procedure for biotransformation of diol 1 using pelletresuspended in buffer (Entry 48R, Table-14)The pellet resuspended in phosphate buffer (50 ml, containing 125 units of peroxidase and42 mg of protein) prepared from 18 day old cell suspension culture was added to an Erlenmeyerflask containing diol 1 (50 mg) dissolved in EtOH (10 ml), distilled water (75 ml), phosphatebuffer (175 ml, 0.1 M, pH 6.6), 0.24% hydrogen peroxide (2.16 equiv.), FMN (0.5 equiv.) andmanganous chloride (0.5 equiv.). After being stirred at room temperature for 48 h, the reactionmixture was worked up as before through ethyl acetate extraction etc. to afford combined crudeextracts (85 mg). Chromatography on silica gel (25 g) using ethyl acetate as eluent gave recovereddiol 1 (19.1 mg, 38%) followed by epoxide 4 (20.1 mg, 40%).3.4 Biotransformations using the T-43-T cell line3.4.1 Typical procedure for biotransformation with batchwise addition of diol 1to the growing cell suspension culture (Entry 52, Table-16)Diol 1 (200 mg) was dissolved in ethanol (25 ml), divided into 5 portions and then addedto the cell suspension culture at time intervals of 0, 12, 7, 5 and 12 h. After the last addition, theincubation was allowed to continue for 24 h. Workup as before, with ethyl acetate extraction etc.afforded a combined crude extracts from broth and cells (278 mg). Chromatography on silica gel(85 g) using ethyl acetate as eluent gave successively the recovered diol 1 (125 mg, 62.5%), 8(7.6 mg, 3.8%), epoxide 4 (5.8 mg, 2.9%) and 93 (7.2 mg, 3.6%).1 3 33.4.2 Typical procedure for biotransformation with addition of diol 1 to T-43-Tgrowing cells via peristaltic pump (Entry 58, Table-17)The suspension culture (350 ml, 11 days old) was transferred into a 500 ml benchtopbioreactor which was bottom stirred, aerated through a sintered glass disk at 200 ml/L/min andkept at room temperature. Diol 1 (35 mg) was dissolved in ethanol (6 ml) and added into the cellsuspension at a rate of 0.25 ml/h over a 24 h period. After the last addition, the incubation wasallowed to continue for a further 24 h. Workup with ethyl acetate extraction etc.as  before affordeda combined crude extract (79.6 mg). Chromatography on silica gel (25 g) using ethyl acetate aseluent gave successively the recovered diol 1 (17.4 mg, 49.0%), 8 (0.6 mg, 1.8%), epoxide 4(1.0 mg, 2.7%) and 94 (0.3 mg, 0.8%).3.4.3 Typical procedure for biotransformation of diol 1 using cell homogenate(Entry 64, Table-20)The cell homogenate (100 ml, containing 439 units of peroxidase and 170mg of protein)prepared from cell suspension culture (14 days old) was added to an Erlenmeyer flask containingdiol 1 (50 mg/10m1 EtOH), distilled water (75 ml), phosphate buffer (175 ml, 0.1 M, pH 6.6),0.24% hydrogen peroxide (2.16 equiv.), FMN (0.5 equiv.) and manganous chloride (0.5equiv.). After being stirred at room temperature for 120 h, ethyl acetate (125 ml) was added andthe mixture was allowed to stir for another 5 min. The resulting mixture was then filtered throughCelite and the filtrate was extracted with ethyl acetate (3x250 ml). The Celite pad was sonicatedwith ethyl acetate (150 ml) for 30 min and then filtered. The combined organic extracts werewashed with water (400 ml), then brine (400 ml) and dried over anhydrous sodium sulphate.Concentration in vacuo afforded the crude mixture of products (89 mg). Chromatography onsilica gel (25 g) using ethyl acetate as eluent gave recovered diol 1 (2.7 mg, 5%), epoxide 4 (35.5mg, 71%), C-10 alcohol 92 (0.4 mg, 1%) and C-10 alcohol 93 (4.3 mg, 9%).1 3 43.4.4 Typical procedure for biotransformation of diol 1 using CFE preparedfrom T-43-TCFE (80 ml, containing 329 units of peroxidase and 77 mg of protein) prepared from cellsuspension culture (12 days old) was added to an Erlenmeyer flask containing diol 1 (50 mg/10m1EtOH), distilled water (75 ml), phosphate buffer (175 ml, 0.1 M, pH 6.6), 0.24% hydrogenperoxide (2.16 equiv.), FMN (0.5 equiv.) and manganous chloride (0.5 equiv.). After beingstirred at room temperature for 48 h, the reaction mixture was worked up as before and a crudemixture of products (59 mg) was obtained. Chromatography on silica gel (25 g) using ethylacetate as eluent gave recovered diol 1 (35.0 mg, 70%) and epoxide 4 (39.2 mg, 18%).3.4.5 Typical procedure for biotransformation of diol 1 using pelletresuspended in buffer(Entry 63, Table-19)The resuspended pellet (80 ml, containing 175 units of peroxidase and 100 mg of protein)prepared from cell suspension culture (12 days old) was added to an Erlenmeyer flask containingdiol 1 (50 mg/10 ml EtOH), distilled water (75 ml), phosphate buffer (175 ml, 0.1 M, pH 6.6),0.24% hydrogen peroxide (2.16 equiv.), FMN (0.5 equiv.) and manganous chloride (0.5equiv.). After being stirred at room temperature for 48 h, the reaction mixture was worked up asbefore and a crude mixture of products (88 mg) was obtained. Chromatography on silica gel (25g) using ethyl acetate as eluent gave recovered diol 1 (14.1 mg, 28.2%) and epoxide 4 (30.9 mg,61.8%).3.5 Biotransformation of cembranoid analogues using T-43-T cell line3.5.1^Biotransformation of epoxide 4 using T-43-T whole cells (Entry 69,Table-22) 16OH1741 3 5Epoxide 4 (150 mg) was dissolved in absolute ethanol (15 ml) and divided into threeequal portions. Each portion was added to an Erlenmeyer flask containing the growing cellsuspension culture of T-43-T (500 ml, 10 days old). The resulting suspensions were incubated at135 rpm and 26°C on a rotary shaker. After shaking for 16 days, the cell suspensions werefiltered through Miracloth and the filtrate was extracted with ethyl acetate (3x500 ml). Thecombined organic extracts were washed with water, brine and dried over anhydrous sodiumsulphate. Concentration in vacuo afforded the crude broth extract (145 mg).Ethyl acetate (500 ml) was added to the cell material and the resulting suspension washomogenised at 24,000 rpm for 5 min. The homogenate was filtered through Miracloth and thefiltrate was washed water (300 ml), brine (300 ml) and dried over anhydrous sodium sulphate.Concentration in vacuo gave the crude cell extract (124 mg).The crude extracts from broth and cell were combined (269 mg). Chromatography onsilica gel (75 g) using ethyl acetate as eluent first gave the recovered epoxide 4 (20 mg, 13.3%)and followed by (1S, 2E, 4S, 6R, 7E, 11S, 12S)-11,12-epoxy-2,7-cembradiene-4,6,15-triol109 (83 mg, 52.7%). The physical properties of 109 are as follows: mp 74-5°C; [a]p: 93.6° (c=0.22, CHC13); IR Vmax (CHC13): 3600, 3400 cm -1 ; 111 NMR (CDC13) 8: 1.11(s, 3H, H-17),1.18(s, 3H, H-16), 1.21(s, 311, H-20), 1.38(s, 3H, H-18), 1.77(s, 3H, H-19), 2.86(dd, J=1and 5Hz, 1H, H-11), 4.48(dt, J=1 and 5Hz, 1H, H-6), 5.4(d, J=10Hz, 1H, H-7), 5.48(m,overlapped, 2H, H-2/H-3); LRMS m/z(%): 320(W-18, 0.2), 302(0.2), 287(0.5), 279(3.7),262(2.5), 149(15.5), 121(11.8), 87(50.3), 58(30.2), 43(100); HRMS calcd. for C20113203(W-18): 320.2352, found: 320.2341; Anal. calcd. for C20113404: C 70.97, H 10.12, found: C70.77, H 9.96.3.5.2^Biotransformation of (1S, 2E, 4S, 7E, 11E)-4-hydroxy-2,7,11-cembratriene-6-one 43 using T-43-T cell homogenate (Entry 70, Table-24) 13643^ 7Enone 43 (50 mg) was dissolved in ethanol (5 ml) and added to a 1 L Erlenmeyer flaskcontaining T-43-T cell homogenate (267 ml, containing 750 units of peroxidase and 168 mg ofprotein), phosphate buffer (200m1, pH 6.6), distilled water (95m1). After addition of cofactors(4.0 equiv. of hydrogen peroxide, 0.5 equiv. of FMN and 0.5 equiv. of MnC12), the mixture wasincubated at room temperature with stirring for 120 h. Work up as before afforded a crudeproduct mixture (119mg). Chromatography on silica gel (25g) using ethyl acetate and hexanes(1:3) as eluents gave recovered enone 43 (6.4 mg, 12.8%) followed by (1S, 2E, 4S, 7E, 11S,12S)-11,12-epoxy-4-hydroxy-2,7-cembradiene-6-one 7 (32.7 mg, 65.3%). The physicalproperties (MS, 1H and 13C NMR) of 7 are identical to those of chemical product 7 obtained byoxidation of epoxide 4 with PCC.3.5.3^Biotransformation of (3E, 7E, 11S, 12E)-11-isopropy1-4,8-dimethy1-3,7,12-pentadecatriene-2,14-dione 44 using T-43-T cell homogenate (Entry 72,Table-26)44 107Seco-diketone 44 (50 mg) was dissolved in ethanol (10 ml) and added to a 1 LErlenmeyer flask containing T-43-T cell homogenate (169 ml, containing 752 units of peroxidaseand 189mg of protein) prepared from cell suspension culture (16 days old). To this mixture,phosphate buffer (175 ml, pH 6.6), distilled water (75 ml), hydrogen peroxide (4.0 equiv.),197 ‘ 0, OH5 6/-8N9102 04 02 1 "_ 11 413 1215117161832HO 1 9 HOOH ^OH''' 9 10820^1 >< +3/HO/17<6181 3 7FMN (0.5 equiv.) and manganous chloride (0.5 equiv.) were added and the mixture wasincubated at room temperature with stirring for 120 h. The reaction mixture was worked up asbefore to afford crude product mixture (129 mg). Chromatography on silica gel (30g) using ethylacetate and hexanes (1:3) as eluents gave recovered 44 (10.5mg, 21.0%) and (3E, 7S, 8S, 11S,12E)-4,8-dimethy1-7,8-epoxy-11-isopropy1-3,7,12-pentadecatriene-2,14-dione 107 (12.6 mg,25.2%) which was identical to the chemical product 107 as described before.3.5.4^Biotransformation of (4E, 6R, 8S, 9E, 11S)-48-dimethy1-68-dihydroxy-11-isopropyl-14-oxo-4,9-pentadecadienal 32 using T-43-T cell homogenate(Entry 75, Table-27)Seco-aldehyde 32 (100 mg) was dissolved in ethanol (10 ml) and added to a 1 LErlenmeyer flask containing T-43-T cell homogenate (422 ml, containing 1498 units ofperoxidase and 662 mg of protein) prepared from cell suspension culture (16 days old). To thismixture, phosphate buffer (350m1, pH 6.6), distilled water (150 ml), hydrogen peroxide (4.0equiv.), FMN (0.5 equiv.) and manganous chloride (0.5 equiv.) were added and the mixture wasincubated at room temperature with stirring for 120 h. The reaction mixture was worked up asbefore to afford crude product mixture (285 mg). Chromatography on silica gel (70 g) using ethylacetate and hexanes (1:1) as eluents gave (44, 5E, 8S, 9E, 11S)-4, 8-dimethyl-1, 4-epoxy-8-hydroxy-11-isopropy1-5, 9-pentadecadiene-14-one 111 (31.9 mg, 32%), recovered 32 (2.3 mg,2%) and (4E, 6R, 8S, 9E, 11S )-4,8-dimethy1-11-isopropy1-14-oxo-4,9-pentadecadiene-1,6,8-triol 110 (29.3 mg, 29%). The spectral data of 110 were identical with those of the chemicalproduct 110 obtained by reduction of 32 with sodium borohydride. The physical properties of„OH^HO113 1141121 3 8another product 111 are as follows: oil; [a] D : - 13.00 (c =0.96, CHC13); IR Vmax (CHC13):1710 cm-1 ; 1H NMR (CDC13) 8: 0.82 (d, J=8.0 Hz)/0.86 (d, J=8.0 Hz) (H-18/H-17), 1.25(s,3H, H-19), 1.29(s, 3H, H-20), 2.12(s, 3H, H-15), 3.85(m, 2H, H-1), 5.31(ddd, J=2, 7 and 16Hz, H-6), 5.48(d, J=16 Hz, H-5), 5.60(m, overlapped, 2H, H-9/H-10); LRMS m/z(%):304(M+, 21.0), 289(7.0), 286(2.8), 261(9.9), 246(4.6), 203(15.2), 177(22.4), 197(10.9),111(10), 93(46.1), 85(50.8), 71(20.3); HRMS calcd. for 020143202 (M±-18): 304.2402;found: 304.24033.5.5^Biotransformation of (1S, 2E, 4S, 6R, 7E, 11S, 12S)-2,7-cembradiene-4,6,11,12-tetrol 104 with T-43-T cell homogenate (Entry 80, Table-29)Tetrol 104 (100 mg) was dissolved in ethanol (200 ml) and divided into two equalportions. Each portion was added to a 1 L Erlenmeyer flask containing T-43-T cell homogenate(400 ml, containing 976 units of peroxidase and 357 mg of protein) prepared from cellsuspension culture (16 days old) by direct homogenization. The mixture was incubated at room1 3 9temperature with stirring using a magnetic stir bar for 120 h. The reaction mixture was worked upas before to afford crude product mixture (197 mg). Chromatography on silica gel (50 g) usingethyl acetate as eluent gave (1S, 2E, 4S, 8R, 11S, 12S)-8,11-epoxy-6-oxo-2-cembrene-4,12-diol112 (23.3 mg, 23%) followed by (1S, 2E, 4S, 6E, 8R, 11S, 12S)-8,12-epoxy-2,6-cembradiene-4,11-diol 113 (1.6 mg, 2%), (1S, 2E, 4S, 6E, 8R, 11S, 12S)-8,11-epoxy-2,6-cembradiene-4,12-diol 114 (1.5 mg, 2%) and recovered 104 (58.4 mg, 54%). The physicalproperties of (1S, 2E, 4S, 8R, 11S, 12S)-8, 11-epoxy-6-oxo-2-cembren-4,12-diol 112 are asfollows[42]: mp 175-6°C; [a]p: +38° (c =0.18, CHC13); IR Vmax (CHC13): 3530, 1700 cm-1 ;1 H NMR (CDC13) 8: 0.88 (d, 3H, J=6.5Hz)/0.89(d, 3H, J=6.5Hz)(H-16/H-17), 0.93(s, 3H,H-20), 1.19(s, 3H, H-19), 1.21 (s, 3H, H-18), 2.59(d, J=15Hz, H-7'), 2.61(d, J=18Hz, 1H,H-5'), 2.91(d, J=15Hz, 1H, H-7), 2.97(d, J=18Hz, 1H, H-5), 3.75(m, 1H, H-11), 5.29(d,J=15Hz, 1H, H-3), 5.44(dd, J=9.2 and 15.4Hz, 1H, H-2); LRMS [m/z(%)]: 338(0.5), 320(6),302(2), 281(4), 223(3), 205(2),^194(33),^180(13),^141(30),^135(51),^127(31),^121(30),109(22), 93(26), 81(37), 71(30), 55(20), 43(100); HRMS calcd. for C20H3404: 338.2457;found: 338.2461. The structures for 115 and 116 was established by comparison of massspectrometric data with those described in the literature. [82]5FF^ F3^ 2 1^ 0 PPmFigure-5, 1 H NMR spectrum of dial 1 in CDC1 3 (400 MHz )F787I^3^' 2 1 0 P PinFigure-6, 1 1-1 NMR spectrum of nor-solanadione 78 in CDCI 3 (400 MHz )53^2^ 1^0 ppmFigure-7, 1 H NMR spectrum of triol 92 in CDC13 (400 MHz )45933 2 1Figure- 8 , 1 H NMR spectrum of triol 93 in CDC1 3 (400 MHz )45943 2^ 1Figure- 9, 1 H NMR spectrum of triol 94 in CDC13 (400 MHz )110III^r•-•^ TV^WI'^ f^I5^4 3 2 1^0 ppmFigure-10, I H NMR spectrum of seco-alcohol 110 in CDC1 3 (400 MHz )CCtoz1.4OIDD1tO109-t I T T^'6.0^5.0PPMf 1 TIlire4.0^3.0^2.0^1.011.0^10.0^9.0^8.0 7.0 0.0Figure-11, 1 H NMR spectrum of epoxide 109 in CDC1 3 (200 MHz )147REFRENCES1^J.G. Gary, S. D. Kung, S. G. Wildman, S. J. Sheen, Nature (London), 1974, 252,226.2^K. Grob, J. A. Voellmin, J. Chromatog. Sci., 1970, 8, 218.3^I. Wahlberg, C. R. Enzell, Nature Product Reports, 1987, 237.4^C. R. Enzell, I. Wahlberg, A. J. Aasen, Fortschritte. d. Chem. Org. Naturst,1977, 34, 1.5^C.R. Enzell,I. Wahlberg, Recent Adv. Tob. Sci., 1980, 6, 64.6^D. L. Roberts, R. L. Rowland, J. Org. Chem., 1962, 27, 3989.7^D. L. Roberts, R. L. Rowland, J. Org. Chem., 1963, 28, 1165.8^J. P. Springer, J. Clardy, R. H. Cox, H. G. Culter, R. J. Cole, Tetra. Lett.,1975, 2737.9^A. J. Aasen, N. Junker, C. R. Enzell, J. E. Berg, A. M. Pilotti, Tetra. Lett.,1975, 2607.10^I. Wahlberg, I. Wallin, C. Narbonne, T. Nishida, C. R. Enzell, J. E. Berg, Acta.Chem. Scand., 1982, B36, 147.11^A. Colledge, W. W. Reid, R. Russel, Chem. Ind.(London), 1975, 13, 570.12^A. W. Johnson, R. F. Severson, J. Hudson, G. R. Carner, R. F. Arrendale,Tob.Sci., 1985, 29, 67.13^C. Keene, G. J. Wagner, Plant Physiol., 1985, 79, 1026.14^R. F. Severson, R. F. Arrendale, 0. T. Chorty, A. W. Johnson, D. M. Jackson, G. R.Gwynn, J. F. Chaplin, M. G. Stepheneson, J. Agric. Food Chem., 1984, 32, 566.15^V. Heemann, U. Brummer, C. Paulsen, F. Seehoffer, Phytochem., 1983, 22, 133.16^L. Crombie, D. McNamara, D. F. Firth, S. Smith, P. C. Bevan, Phytochem., 1988,27, 1685.17^I. Wahlberg, K. Karlsson, D. J. Austin, N. Junker, J. Roeraade, C. R. Enzell,Phytochem. , 1977, 16, 1217.18^(a) H. G. Cutler, R. J. Cole, Plant Cell Physiol., 1974, 15, 19.(b) H. G. Cutler, W. W. Reid, J. Deletang, Plant Cell Physlol., 1977, 18, 711.19^Y. Saito, H. Takayawa, S. Konishi, D. Yoshida, S. Mizusaki, Carcinogenesis, 1985,6, 1189.20^A. W. Johnson, R. F. Severson, J. Agric. Entomol., 1984, 1, 23.14821^D. M. Jackson, R. F. Severson, A. W. Johnson, J. F. Chaplin, M. G. Stepheson,Environ. Entomol,. 1984, 13, 1023.22^I. Cruichshank, D. Perrin, M. Mandryk, Phytopath. Z., 1977, 90, 243.23^W. J. Guilford, R. M. Coates, J. Amer. Chem. Soc., 1982, 104, 3506.24^P. Manito, "Biosynthesis of Natural Products", Ellis Horwood Ltd., 1973, p.213.25^(a) M. A. Tius, Chem. Rev., 1988, 88, 719.(b) P. C. Astles, E. J. Thomas, Synlett., 1989, 1, 42.26^J. A. Marshall, E. D. Robinson, J. Lebveton, Tetra. Lett., 1988, 29, 3547.27^J. A. Marshall, E. D. Robinson, Tetra. Lett., 1989, 30, 1055.28^J. A. Marshall, E. D. Robinson, R. D. Adam, Tetra. Lett., 1988, 29, 4913.29^J. A. Marshall, T. M. Jenson, B. S. DeHoff, J. Org. Chem., 1987, 52, 3860.30^K. Omura, D. Swern, Tetrahedron, 1978, 34, 1651.31^E. W. Collington, A. I. Mayers, J. Org. Chem., 1971, 36, 3044.32^I. Wahlberg, K. Nordfors, C. Vogt, T. Nashida, C. R. Enzell, Acta. Chem. Scand.,1983, B37, 653.33^V. Sinnwell, V. Heemann, A. M. Bylov, W. Hass, C. Kahre, F. Seehofer, Z.Naturforsch., 1984, 39C, 1024.34^G. W. Kinzer, T. F. Page, R. R. Johnson, J. Org . Chem., 1966, 31, 1797.35^I. Wahlberg, R. Arndt, T. Nishida, C. R. Enzell, Acta. Chem. Scand., 1986, B40,123.36^D. Behr, I. Wahlberg, T. Nishida, C. R. Enzell, J. E. Berg, A. M. Pilotti, Acta Chem.Scand., 1980, B34, 195.37^A. J. Aasen, A. Pilotti, C. R. Enzell, J. E. Berg, A. M. Pilotti, Acta. Chem. Scand.,1976, B33, 999.38^D. Behr, I. Wahlberg, A. J. Aasen, T. Nishida, C. R. Enzell, T. E. Berg, A. M. Pilotti,Acta. Chem. Scand., 1978, B32, 221.39^M.J.Begley, L. Crombie, D. McNamara, D. F. Firth, S. Smith, P. C. Bevan,Phytochem., 1988, 27, 1695.40^I. Wahlberg, A. M. Eklund, C. Vogt, C. R. Enzell, J. E. Berg, Acta. Chem. Scand.,1986, B40, 855.41^(a) A. Zane, Phytochem. , 1973, 12, 731.(b) U. Bruemmer, C. Paulsen, G. Spremberg, F. Seehofer, V. Heemann, V. Sinnwell,Z. Naturforsch.,1981, 36C, 1077.1 4 942^I. Wahlberg, I. Forsblom, C. Vogt, A-M. Eklund, T. Nishida, C. R. Enzell, J. E. Berg,J. Org. Chem., 1985, 50, 4527.43^R. L. Rowland, D. L. Roberts, J. Org. Chem., 1963, 28, 1165.44^J. L. Courtney, S. McDonald, Tetra. Lett., 1967, 459.45^(a) R. A. Lloyd, C. W. Miller, D. L. Roberts, J. A. Giles, J. P. Dickerson, N. H.Nelson, C. E. Rix, P. H. Ayers, Tob. Sci., 1976, 20, 40.(b) I. Wahlberg, R. Arndt, I. Wallin, C. Vogt, T. Nishida, C. R. Enzell, Acta. Chem.Scand., 1984, B38, 21.46^I. Wahlberg, D. Behr, A. M. Eklund, T. Nishida, C. R. Enzell, J. E. Berg, Acta.Chem. Scand., 1982, B36, 37.47^I. Wahlberg, D. Behr, A. M. Eklund, T. Nishida, C. R. Enzell, J. E. Berg, ActaChem. Scand., 1982, B36, 443.48^E. Demole, Int. Congr. Essent. Oils, 6th, San Francisco, California, 1974, 154.49^E. Demole, P. Enggist, Hely. Chim. Acta, 1975, 58, 1602.50^Y. Takagi, T. Chuman, T. Fujimori, H. Kaneko, T. Fukuzumi, M. Noguchi, Agric.Biol. Chem., 1978, 42, 327.51^I. Wahlberg, D. Behr, A. M. Eklund, T. Nashida, C. R. Enzell, Acta. Chem.Scand., 1980, B34, 675.52^E. Demole, C. Demole, Hely. Chim. Acta., 1975, 58, 1867.53^R. R. Johnson, J. A. Nicholson, J. Org . Chem., 1965, 30, 2918.54^D. L. Roberts, W. A. Rohde, Tob. Sci., 1972, 16, 107.55^I. Wahlberg, A -M. Eklund, C. R. Enzell, Acta. Chem. Scand., 1990, 44, 504.56^F. DiCosmo, P. J. Facchini, M. M. Kraml, Chemistry in Britain, 1989, 1001.57^T. Suga, T. Hiram, Phytochem., 1990, 29, 2393.58^W. G. W. Kurz, F. Constable, J. P. Kutney, "Plant Tissue Culture 1982",Proc. 5th. Intl. Cong. Plant Tissue & Culture (A. Fujuwara Ed.) , P.361.59^(a) T. Ikeda, T. Matsumoto, M. Noguchi, Phytochem., 1976, 15, 568.(b) T. Matsumoto, Agric. Biol. Chem., 1981, 45, 1627.60^(a) Y. Fujita, Y. Hara, C. Suga, T. Morimoto, Plant Cell Rep., 1981, 1, 61.(b) Y. Fujita, S. Takahashi, Y. Yamada, Agric. Biol, Chem., 1985, 49, 1755.61^H. Bohm, "Plant Tissue Culture 1982", Proc. 5th. Intl. Cong. Plant Tissue& Culture (A. Fujuwara Ed.) , P.325.62^E. Reinhard, W. Kreis, U. Barthlen, U. Helmbold, Biotechnol. Bioeng.,1989, 34,502.1 5 063^U. Tuominen, L. Toivonen, V. Kauppinen, P. Markkanen, L. Bjork, Biotechnol.Bioeng., 1989, 33, 589.64^M. Misawa, T. Endo, A. Goodbody, J. Vukovic, C. Chapple, L. Choi. J. P. Kutney,Phytochem., 1988, 27, 355.65^J. P. Kutney, C. A. Boulet, L. Choi, W. Gustowski, M. McHugh, J. Nakano, T.Nikaido, H. Tsukamoto, G. M. Hewitt, R. Suen, Heterocycles, 1988, 27, 621.66^T. J. McMurry, J. T. Groves, in "Cytochrome P-450, Structure, Mechanismand Biochemistry", (Ed. P. R. 0. Montellano), P. 1, Plenum Press, 1986.67^F. P. Guengerich, T. L. MacDonald, Acc. Chem. Res., 1984, 17, 9.68^F. P. Guengerish, G. A. Dannan, S. T. Wright, M. V. Martin, L. S. Kaminsky,Biochemistry, 1982, 21, 6019.69^T. L. MacDonald, L. T. Burka, S. T. Wright, F. P. Guengerich, Biochem. Biophys.Res. Commun., 1982, 104, 620.70^S. W. Cummings, F. P. Guengerich, R. A. Prough, Drug Metab. Dispos.,1982, 10, 459.71^E. H. Oliw, F. P. Guengerich, J. A. Oates, J. Biol. Chem., 1982, 257, 3771.72^R. E. Miller, F. P. Guengerich, Biochemistry, 1982, 21, 1090.73^F. P. Guengerich, T. W. Strickland, Mol. Pharmacol., 1977, 13, 993.74^(a) T. Hirata, T. Aoki, Y. Hirano, T. Ito, T. Suga, Bull. Chem. Soc. Jpn.,1981,54, 3527.(b) T. Suga, Y. S. Lee, T. Hirata, Bull. Chem. Soc. Jpn.,1983, 56, 784.75^T. Hirata, S. Izumi, T. Ekida, T. Suga, Bull. Chem. Soc. Jpn., 1987, 60, 289.76^G. L. Lappin, J. D. Stride, J. Tampion, Phytochem., 1987, 26, 995.77^J. P. Kutney, Pure Appl. Chem., 1984, 56, 1011.78^J. P. Kutney, G. M. Hewitt, T. Kurihara, P. J. Salisbury, R. D. Sindelar, K. L. Stuart,P. M. Townsley, W. T. Chalmers, G. G. Jacoli, Can. J. Chem. 1981, 59, 2677.79^(a) T. Jarvis, Ph. D. Dissertation, Dept. of Chem. , U.B.C., 1991.(b) J. Palaty, M. Sc. Thesis, Dept. of Chem., U. B. C., 1990.80^J. P. Kutney, L. Choi, R. Duffin, G. Hewitt, N. Kawamura, T. Kurihara, P. Salisberg,R. Sindelar, K. L. Stuart, P. M. Townsley, W. T. Chalmers, F. Webster, G. G. Jacoli,Planta Med., 1983, 48, 158.81^M. Robert, Ph. D. Dissertation, Dept. of Chem., UBC, 1989.82^C. R. Enzell, I. Wahlberg, Mass Spectrom. Rev., 1984, 3, 395.15183^I. Wahlberg, I. Wallin, C. Narbonne, T. Nishida, C. R. Enzell, J. E. Berg, Acta.Chem. Scand., 1987, B36, 147.84^C. R. Enzell, private communication with Dr. Kutney.85^A. Chu, Y. Okada, J. P. Kutney, unpublished results.86^M. M. Bradford, Anal. Biochem., 1976, 72, 248.87^B. Chance, A. C. Maehly, in "Methods in Enzymology", Vol. 2, (S. P. Colowick,N. 0. Kaplan, Eds.), Academic Press, New York, 1955, P. 773.88^P. Nasiri, J. P. Kutney, unpublished results.89^0. L. Gamborg, D. E. Eveleigh, Can. J. Biochem., 1968, 46, 417.90^T. Murashige, F. Skoog, Physiol Plant, 1962, 15, 473.

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0061784/manifest

Comment

Related Items