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

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Biotransformation Studies on Tobacco Cembranoids using Plant Cell Cultures By Kai Li  B.Sc. Jiangsu Teachers' College, China, 1982 M.Sc., Suzhou University, China, 1985 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA November 1991 © K. Li, 1991  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of Chemistry The University of British Columbia Vancouver, Canada Date: 30 November. 1991  Abstract This thesis deals with the biotransformation studies on tobacco cembranoids using the plant cell culture lines coded as TRP4a and T-43-T which are derived from Tripterygium wilfordii , an important Chinese herbal plant, and Nicotiana sylvestris, respectively.  The studies are divided into three parts: 1) Biotransformation of the diols 1 and 2 using the 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 in phosphate (pH 6.3) or TrisHC1 (pH 7.5) buffers for varying time periods affords five products 4, 8, 92, 93 and 94. The epoxide 4 is the major product occurring in about 50% yield. The  allylic alcohols 92 and 93 are assigned as a pair of diastereoisomers with different chiralities at C-10 but their absolute configurations at this centre have not been established. Similarly, when the diol 2 is incubated with the growing cells of TRP4a, the epoxide 95 is obtained as a major product in about 50% yield. Therefore, it is confirmed that the 11,12 double bond in the diols 1 or 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 have been investigated. No significant biotransformations are achieved when the cell free extract (CFE) and the cell homogenate prepared from the TRP4a cells are involved and the substrate diol 1 is recovered in each case. However, the pellet fractions obtained during the centrifugation in the preparation of CFE, when resuspended in phosphate buffer (pH 6.6) and with addition of hydrogen peroxide, FMN and manganous chloride as cofactors, are capable of transforming the diol 1 into the epoxide 4 in about 40% yield. Biotransformation studies using the T-43-T cell line derived from Nicotiana sylvestris, a tobacco 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 the  allylic 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, the C-10 and C-12 alcohols, 93 and 8 respectively, are obtained in relatively higher yields (2029%). However, in contrast to the data obtained with TRP4a, when such cofactors as hydrogen peroxide, FMN and manganese chloride are added, both the T-43-T cell homogenate and the pellets resuspended in phosphate buffer (pH 6.6) afford good yields of the epoxide 4 (72% and 62%, respectively) in biotransformation experiments with the diol 1. Biotransformation of cembranoid analogues using the T-43-T cell line was also investigated. The growing cells can transform the epoxide 4 into the triol 109, a product formed by 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 is observed in the enone 43 and the seco-diketone 44 and epoxides 7 and 107 are obtained respectively. 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 of such cofactors as hydrogen peroxide, manganous chloride and FMN. The tetrol 104, when exposed to cell homogenate, is oxidized at C-6 and the latter intermediate undergoes a spontaneous intra-molecular Michael addition to give the ether 112 in approximately 20% yield. In addition, the ethers 113 and 114 are obtained in very small amounts. In conclusion, the T-43-T cell line is capable of performing both oxidation and reduction reactions, namely selective epoxidation of the 11,12 double bond, hydroxylation at the allylic positions (C-10 and C-12), hydroxylation at a non-activated methine position (C-15) and selective reduction of aldehyde to the corresponding alcohol.  iii  43  ().\ --\  ...  z  (),,/^/ \ 78  94  95  110  ^  107  111^  114  iv  112  ^  109  113  Table of Contents  Title page ^ II Abstract ^ Table of Contents ^ V List of Schemes ^ VIII List of Figures ^ IX List of Tables ^ IX List of abbreviation ^ XI Acknowledgements ^ XIII 1 1 INTRODUCTION ^ 1.1 Tobacco cembranoids ^ 1 1.1.1^Structures and Nomenclatures ^ 3 1.1.2^Biological considerations ^ 4 1.1.3^Biogenesis ^ 5 1.1.4^Synthesis of cembranoids ^ 7 1.2 Biogenetic considerations for cembranoid transformations ^ 10 1.2.1^Oxidation of the 11,12 double bond ^ 11 1.2.2^Oxidation of the 7,8 double bond ^ 15 1.2.3^Oxidation of the hydroxyl group at C-6 ^ 16 1.2.4^Acid-induced reactions ^ 20 22 1.2.5 Degraded cembranoids ^ 1.3 Plant tissue cultures ^ 30 1.3.1^Applications of plant tissue cultures in biotransformation studies ^ 30 1.3.2^Oxidative reactions in biotransformation studies using plant cell cultures ^ 37 1.3.3^Reductive reactions in biotransformation studies using plant cell cultures ^ 42 1.3.4^Plant cell lines developed and available in our laboratory ^ 43 1.4 Objectives of the project ^ 44 2 RESULTS AND DISCUSSION ^ 46 2.1 Biotransformation of cembranoids using the TRP4a cell line ^ 46 2.1.1^Biotransformation of the diols 1 and 2 using the whole cells ^ 54 2.1.2^Chemical studies on the cembranoids: preparation of cembranoid analogues ^ 70 2.1.3^Biotransformation of the diol 1 using cell free extract (CFE) ^ 74 2.1.4^Biotransformation of the diol 1 using the cell homogenate and the pellet ^ 78 2.2 Biotransformation of cembranoids using the T-43-T cell line ^ 84 2.2.1^Biotransformation of the diol 1 using the whole cells ^ 84  ^  2.2.2 Biotransformation of the diol 1 using the CFE prepared from the T-43-T cell line ^ 86 2.2.3^Biotransformation of the diol 1 using the cell homogenate and the resuspended pellet prepared from the T-43-T cell line ^ 87 2.3 Biotransformation of cembranoid analogues using the T-43-T cell line ^ 92 2.3.1^Biotransformtion of the epoxide 4 using the T-43-T whole cells ^ 92 2.3.2^Biotransformation of the enone 43 using the cell homogenate ^ 95 2.3.3^Biotransformation of the seco-diketone 44 and seco-epoxide 107 using the cell homogenate ^ 97 2.3.4^Biotransformation of the seco-aldehyde 32 using the cell homogenate ^ 99 2.3.5^Biotransformation of the tetrol 104 using the cell homogenate ^ 103 2.4 Overall conclusions ^ 106 2.5 Further Research directions ^ 106 3 EXPERIMENTAL ^ 107 3.1 Chemical conversions of diol 1 to cembranoid analogues ^ 108 3.1.1^Conversion of diol 1 into (1S, 2E, 4S, 6R, 7E, 11E)-6-acetoxy-2,7,11cembratriene-4-ol 97 and (1S, 2E, 4S, 6R, 7E, 11S, 12S)-6-acetoxy-2,7cembradiene-4,11,12-triol 98 ^ 108 3.1.2^Conversion of triol 98 to (4E, 6R, 8S, 9E, 11S)-6-acetoxy-4, 8-dimethy1-8hydroxy-11-isopropy1-14-oxo-4, 9-pentadecadienal 99 ^ 109 3.1.3^Conversion of 99 to methyl (4E, 6R, 8S, 9E, 11S)-6-acetoxy-4, 8-dimethy18-hydroxy-11-isopropy1-14-oxo-4, 9-pentadecadienoate 100 ^ 110 3.1.4^Conversion of 100 to methyl (4E, 6R, 8S, 9E, 11S)-6,8-dihydroxy-4, 8dimethy1-11-isopropy1-14-oxo-4, 9-pentadecadienoate 101 ^ 111 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 ^ 112 3.1.6^Conversion of diol 1 to (1S, 2E, 4S, 6R, 7E, 11S, 12S)-2,7-cembradiene4,6,11,12-tetrol 104 ^ 113 3.1.7^Conversion of tetrol 104 to (4E, 6R, 8S, 9E, 11S)-4,8-dimethy1-6,8dihydroxy-11-isopropy1-14-oxo-4,9-pentadecadienal 32 ^ 114 3.1.8^Conversion of 32 to (4E, 8S, 9E, 11S)-4,8-dimethy1-8-hydroxy-11isopropy1-6,14-dioxo-4,9-pentadecadienal 106 and nor-solanadione 78 ^ 115 3.1.9^Conversion of diol 1 to (15, 2E, 4S, 7E, 11E)-2,7,11-cembratriene4-o1-6-one 43 ^ 116 3.1.10 Conversion of 43 to (3E, 7E, 11S, 12E)-11-isopropy1-4,8-dimethy1-3,7,12116 pentadecatriene-2,14-dione 44 ^ vi  ^  3.1.11 Conversion of 32 to (4E, 6R, 8S, 9E, 11S)-4,8-dimethy1-11-isopropy1-14oxo-4,9-pentadecadiene-1,6,8-triol 110 ^ 117 3.1.12 Conversion of epoxide 4 to (1S, 2E, 4S, 7E, 11S, 12S)-11,12-epoxy-4hydroxy-2,7-cembradien-6-one 7 ^ 118 3.1.13 Conversion of 7 to (3E, 7S, 8S, 11S, 12E)-4, 8-dimethy1-7,8-epoxy11-isopropy1-3, 7, 12-pentadecadiene-2, 14-dione 107 ^ 119 3.2^Propagation of the plant cell cultures ^ 120 3.2.1^Propagation of the TRP4a cell culture ^ 120 3.2.2^Propagation of the T-43-T cell culture ^ 120 3.3 Biotransformation using the TRP4a cell line ^ 121 3.3.1^Biotransformation using the whole cells ^ 121 3.3.2^Biotransformation using the CFE ^ 128 3.3.3^Biotransformation using the cell homogenate, resuspended pellet and supernatant (CFE) with cofactors ^ 130 3.4 Biotransformation using the T-43-T cell line ^ 132 3.4.1^Typical procedure for biotransformation with batchwise addition of the diol 1 to the growing cell suspension culture ^ 132 3.4.2^Typical procedure for biotransformation with addition of the diol 1 to the growing cells via peristaltic pump ^ 133 3.4.3^Typical procedure for biotransformation of the diol 1 using the cell homogenate ^ 133 3.4.4^Typical procedure for biotransformation of the diol 1 using the CFE ^ 134 3.4.5^Typical procedure for biotransformation of the diol 1 using the resuspended pellet ^ 134 3.5 Biotransformation of cembranoid analogues using the T-43-T cell line ^ 134 3.5.1^Biotransformation of the epoxide 4 using the whole cells ^ 134 3.5.2^Biotransformation of (1S, 2E, 4S, 7E, 11E)-4-hydroxy-2,7,11cembratriene-6-one 43 using the cell homogenate ^ 135 3.5.3^Biotransformation of (3E, 7E, 11S, 12E)-11-isopropy1-4,8-dimethy1-3,7,12pentadecatriene-2,14-dione 44 using the cell homogenate ^ 136 3.5.4^Biotransformation of (4E, 6R, 8S, 9E, 11S)-48-dimethy1-68-dihydroxy-11isopropy1-14-oxo-4,9-pentadecadienal 32 using the cell homogenate ^ 137 3.5.5^Biotransformation of (15, 2E, 4S, 6R, 7E, 11S, 12S)-2,7-cembradiene138 4,6,11,12-tetrol 104 using the cell homogenate ^ 140 APPENDIX ^ 147 REFERENCES ^ vii  List of Schemes  Scheme 1, Proposed biosynthetic pathways for the diols 1 and 2 ^ 6 Scheme 2, Synthesis of a-diol 1 ^ 8 Scheme 3, Proposed biogenetic pathways leading to the triols 8 and 9 ^ 12 Scheme 4, Products obtained from photooxygenation of the diol 1 followed by reduction with triethylphosphite ^ 13 Scheme 5, Epoxidation of the 11,12 double bond in the diol 1 ^ 13 Scheme 6, Formation and acid-induced reaction of the epoxide 4 ^ 14 Scheme 7, Epoxidation of the 7,8 double bond in the diol 1 ^ 15 Scheme 8, Proposed biogenetic pathways related to the oxidation at C-6 ^ 17 Scheme 9, Products obtained from photooxygenation of the ketol 43 followed by reduction with triethyl phosphite ^ 18 Scheme 10, Products obtained from acid-induced rearrangement of the epoxide 7 ^ 19 Scheme 11, Proposed pathways via acid-induced rearrangements of the diol 1 ^ 20 Scheme 12, Products obtained in acid-induced rearrangements of the diol 1 ^ 21 Scheme 13, Formation of the (8S, 11R)-8,11-epoxy-diol 57 ^ 22 Scheme 14, Formation of the C19 compound 61 ^ 24 Scheme 15, Formation of the C18 compound 63 ^ 24 Scheme 16, Formation of the C15 compounds ^ 26 Scheme 17, Formation of the C14 compounds ^ 27 Scheme 18, Formation of solanone 73 and the other C13 compounds ^ 28 Scheme 19, Formation of nor-solanadione 78 and the other C12 compounds ^ 29 Scheme 20, The development of a plant cell suspension culture ^ 32 Scheme 21, The preparation of cell free extract (CFE) ^ 35 Scheme 22, Production of 3',4'-anhydrovinblastine 91 by CFE from C. roseus ^ 37 Scheme 23, Some examples of allylic hydroxylation using plant cell cultures ^ 41 Scheme 24, Some examples of reduction using plant cell cultures ^ 43 Scheme 25, Oxidative coupling leading to lignan 118 ^ 44 Scheme 26, Proposed objective for cembranoid biotransformation ^ 45 Scheme 27, Biotransformation of the diol 1 using TRP4a cell line ^ 47 Scheme 28, The mass spectrometric fragmentation pattern of the diol 1^ 51 Scheme 29, The mass spectrometric fragmentation pattern of the triol 92^ 52 Scheme 30, Biotransformation of the diol 2 using TRP4a cell line ^ 54 Scheme 31, Procedures for biotransformation using re-suspension culture of TRP4a ^ 55 Scheme 32, Chemical conversion of the diol 1 to norsolanadione 78 ^ 71 VIII  Scheme 33, Alternative way to norsolanadione 78 ^ 72 Scheme 34, Conversion of the diol 1 to seco-diketone 44 ^ 73 Scheme 35, Conversion of the epoxide 4 to 7 and 107 ^ 73 Scheme 36, Conversion of the seco-aldehyde 32 to alcohol 110 ^ 74 Scheme 37, Preparation of CFE and biotransformation using CFE ^ 76 Scheme 38, Preparation of cell homogenate and resuspended pellet and biotransformation ^ 79 Scheme 39, Biotransformation of the epoxide 4 to 109 ^ 93 Scheme 40, Chemical conversion of the diol 1 to enone 43 and biotransformation of 43 to 7 ^ 95 Scheme 41, Chemical conversion of 43 into 44 and biotransformation of 44 to 107 ^ 97 Scheme 42, Chemical conversion of the epoxide 4 to 107 ^ 99 Scheme 43, Biotransformation of the seco-aldehyde 32 into 110 and 111 ^ 100 Scheme 44, Biotransformation of the tetrol 104 ^ 103 List of Figures  Figure 1, Some cembranoids isolated from tobacco ^ Figure 2, Fundamental carbon skeletons of cembranoid 10, labdanoid 11 and carotenoid 12 ^ Figure 3, Degradation patterns of cembranoids ^ Figure 4, Structure of iron protoporphyrin IX and catalytic cycle of cytochrome P-450 ^ Figure 5, 1 H NMR spectrum of the diol 1 in CDC13 (400 MHz) ^ Figure 6, 1 H NMR spectrum of nor-sorlanadione 78 in CDC13 (400 MHz) ^ Figure 7, 1 H NMR spectrum of the triol 92 in CDC13 (400 MHz) ^ Figure 8, 1 H NMR spectrum of the trio! 93 in CDC13 (400 MHz) ^ Figure 9, 1 H NMR spectrum of the triol 94 in CDC13 (400 MHz) ^ Figure 10, 1 H NMR spectrum of the seco-alcohol 110 in CDC13 (400 MHz) ^ Figure 11, 1 H NMR spectrum of the epoxide 109 in CDC13 (200 MHz) ^  2 2 23 38 140 141 142 143 144 145 146  List of Tables  Table 1, Concentrations of the diols 1 and 2 in the leaf surface gum of different 4 N. tabacum varieties ^ Table 2, Concentrations of diols 1 and 2 in N. tobacum organs ^ 5 Table 3, 13 C NMR chemical shifts (ppm) of cembranoids in CDC13 ^ 48 ix  57 Table 4, Effect of pH on biotransformation of the diol 1 ^ Table 5, Effect of pH on biotransformation of the diol 2 ^ 57 Table 6, Effect of increasing substrate concentration on biotransformation of the diol 1 ^ 59 Table 7, Effect of cell age on the biotransformation of the diol 1 with direct substrate ^ 62 Table 8, Effects of substrate concentration on biotransforamtion of the diol 1 ^ 63 Table 9, Biotransformation by batchwise addition of the diol 1 to cell suspension culture ^ 64 Table 10, Biotransformation by semi-continual addition of diol 1 via a peristaltic pump ^ 65 Table 11, GC conditions and retention times ^ 67 Table 12, GC results of biotransformation of diol 1 ^ 67 Table 13, Biotransformation of diol 1 with the CFE prepared from TRP4a cell line ^ 78 Table 14, Biotransformation of diol 1 with the cell homogenate, pellet and supernatant 81 prepared from TRP4a cell line ^ Table 15, The effects of manganese chloride and FMN on biotransformation of diol 1 ^ with resuspended pellet ^ 83 Table 16, Biotransformation with batch-wise addition of substrate to T-43-T whole cells ^ 85 Table 17, Biotransformation of the diol 1 with semi-continual addition of substrate to T-43-T whole cells via peristaltic pump ^ 86 Table 18, Biotransformation of the diol 1 with the CFE prepared from the T-43-T cell line ^ 87 Table 19, Biotransformation of the diol 1 with the cell homogenate and resuspended pellet prepared from T-43-T cell line ^ 89 Table 20, Biotransformation of diol 1 with cell homogenate at a longer incubation time ^ 90 Table 21, Biotransformation with the T-43-T cell homogenate without co-factors. ^ 91 Table 22, Further biotransformation of the epoxide 4 with the T-43-T whole cells ^ 93 Table 23, 13 C NMR Chemical shifts (CDC13) of the epoxide 4 and the triol 109 ^ 94 Table 24, Biotransformation of the enone 43 with the T-43-T cell homogenate ^ 96 Table 25, 13 C NMR chemical shifts (ppm) in CDC13 ^ 96 Table 26, Biotransformation of the seco-diketone 44 with the T-43-T cell homogenate ^ 98 Table 27, Biotransformation of the seco-aldehyde 32 with the T-43-T cell homogenate ^ 101 102 Table 28, 13 C NMR chemical shifts (ppm) in CDC13 ^ Table 29, Biotransformation of the tetrol 104 with the T-43-T cell homogenate ^ 104  List of abbreviations  ^Specific optical rotation recorded at ambient temperature (23 0C) using sodium D-line ^ Ac acetyl [alD  APT^attached proton test br^broad c^concentration (g/100m1) CFE^cell free extracts CI-MS^mass spectrum with chemical ionization cm-1^wave number 8^chemical shift relative to TMS d^doublet dd^doublet of doublets dt^doublet of triplets EI-MS^mass spectrum with electron impact eq.^equivalent GC^gas-liquid chromatography h^hours HRMS^high resolution mass spectroscopy Hz^hertz IR^infrared J^coupling constant L^litre LRMS^low resolution mass spectroscopy M^molar M+^molecular ion mCPBA^meta-chloroperoxybenzoic acid xi  mg^milligrams MHz^megahertz ml^milliliter mmol^millimole mp^melting point MS^mass spectroscopy MS medium^Murashige and Skoog's nutrient medium m/z^mass to charge ratio  nr25,^refractive index recorded at 25°C using sodium D-line nm^nanometer NMR^nuclear magnetic resonance PCC^pyridinium chlorochromate ppm^parts per million PRDCo^PRL-4 medium[ 89] supplemented with 2,4-dichlorophenoxyacetic acid (D) (2 mg/L) and coconut milk (100 ml/L) rpm^rotation per minute s^singlet t^triplet TBHP^t-butyl hydroperoxide TLC^thin layer chromatography TMS^tetramethylsilane Trizma®^tris(hydroxymethyl)aminomethane TRP4a^plant cell line derived from Tripterygium wilfordii T-43-T^plant cell line derived from Nicotiana sylvestris  xii  ACKNOWLEDGEMENTS I wish to express my appreciation to Professor James P. Kutney for his valuable  advice, 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 cell lines, to Mr. Francisco Kuri-Brena and Mr. Caries Cirera for their suggestions, discussion and 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 British Columbia and the Swedish Tobacco Company, Stockholm, Sweden is gratefully acknowledged. Finally, I am deeply indebted to my wife for her support and encouragement.  1 1 INTRODUCTION 1.1 Tobacco cembranoids Apart from cotton and food-providing plants, tobacco is one of the most important cultivated plants in the world. Even though there are more than sixty different species, most of the commercial tobacco products are prepared from one species only, Nicotiana tabacum which is a hybrid between N. sylvestris and N. tomentostformis .[ 1 ] N. tabacum has not been found growing in a wild environment but only as a cultivated species. Due to their economic importance both in producing and consuming countries, together with the health factors associated with their uses, 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 known pharmacologically active compound, about 2500 chemical constituents have been isolated and identified so far from tobacco and its smoke.[ 2,3 ] Among them are isoprenoids, one of the major classes of tobacco chemical constituents. The content of isoprenoids has been examined thoroughly and the results show that mono-, sesqui-, di- and triterpenoids, carotenoids or even higher isoprenoids have been found. 3, 4 [  ]  As far as tobacco aromas are concerned, carotenoids, labdanoids and cembranoids are the three major sources in the isoprenoid family.[ 3,4] It is a well known fact that the typical aroma of the tobacco leaf is created during the post-harvest treatment, which involves air-, fire-, or suncuring and aging. These processes lead to very substantial chemical changes and thus account for the generation of flavorants. Among the three major sources, the 14-membered macrocyclic cembranoids, which belong to the diterpenoid class, have received considerable attention because these compounds are prone to undergo biodegradation and give rise to a variety of volatile, low molecular weight compounds, many of which are important flavour substances thereby providing significant contributions to the aromas of commercial tobaccos.1 51  2 HO  1  2  4  5 HO so  6 OH II I  HO 7  8  9  Figure 1, Some cembranoids isolated from tobacco  10  11  Figure 2, Fundamental carbon skeletons of cembranoid 10, labdanoid 11 and carotenoid 12  3 1.1.1 Structures and Nomenclatures  The discovery of cembranoids in tobacco dates back to the early 1960's.[ 6 1 Since then, more than fifty cembranoids have been reported as constituents of tobacco. (See Figure 1 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 the major components and, in fact, the first examples of macrocyclic diterpenes containing the 14-membered ring system.[ 7] These two diols were first isolated in 1962. Their relative stereochemistry and absolute configurations were solved by X-ray analysis [ 8 ] , ozonolytic degradations [ 9 ] and chemical correlations [ 10]. HOHO OH ^ OH  18  III  1  10  2  Cembrane nomenclature, which is based on the structure of cembrane 10, is used throughout this thesis. Thus, the name for 1 is (1S, 2E, 4S, 6R, 7E, 11E)-2,7,11cembratriene-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)-2,7,11thunbergatriene-4,6-diol and a-4,8,13-duvantriene-1,3-diol based on the thunbergane and duvane nomenclature for its parent saturated compound 10. The former name derives from Pinus thunbergii, a species name for pine, the latter is a Serbian word for tobacco. In  Chemical 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 these names can be found in the literature. The diol 2 differs from the diol 1 only in the stereochemistry at C-4 and is referred to, in abbreviated form, as "0 diol". -  4 1.1.2 Biological considerations  Depending on the genetic background, tobacco plants produce cembranoids, labdanoids, or both types of cyclic diterpenoids. For example, Oriental tobaccos usually produce both cembranoids and labdanoids while Virginia and Burley tobaccos synthesize cembranoids exclusively.[ 11 ] These genetic differences relate back to the fact that Nicotiana tabacum is a hybrid between N. sylvestris (female) and N. tomentosiformis (male). These  species synthesize cembranoids and labdanoids, respectively.[Lill It is found that the cembranoids are synthesized in the glandular heads of the trichome on the surface of the leaf and in the flower [12, 131 and are present in the gummy exudates. They can be isolated in 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 account for 0.5-10% of the fresh weight of the leaves, depending on variety [ 11 ] and the cembranoid content in the leaf exudate may be as high as 75%.[ 15] It is reported that the diols 1 and 2 constitute 0.01% and 0.005%, respectively, of the dry burley leaf.[ 6] As a mater of fact, the abundance of these cembranoids depends on the genetic background as well as the edaphic conditions, even the leaf positions. [15, 16] post-harvest treatment also can affect its compositions.[ 17] Table 1, The concentrations of diols 1 and 2 in the leaf surface gum of different N. tabacum varieties [ 151 N. tabacum variety  (1 + 2)%  Ti 1112 Virgin Bright NC 2326 Vinica Burley B5 Amarelinho Kurztag  0.5 5 7 8 8 25 75  5  Table 2, Concentrations of the diols 1 and 2 in N. tabacum organs 161 [  Plant organ  Young up haves Old lower leaves Immature petals Mature petals Mature calyces Fertilized calyces  Fresh weight (g)  Weight of 1 & 2 (mg)  %  50.1 47.1 3.7 10.5 3.0 2.5  73 20 18 61 70 63  0.15 0.04 0.49 0.58 2.33 2.52  Ratio (1 : 2)  43:57 39:61 76:24 75:25 76:24 60:40  While the physiological functions of the tobacco cembranoids still have not been established, 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 can [  ,  stimulate oviposition activity of tobacco budworm 21 and inhibit germination of fungal [  ]  spores [ 22 ]. These properties could play an important role in the development of pestresistant high-flavor tobaccos. 1.1.3^Biogenesis Little is known about the biogenesis of the tobacco cembranoids compared to that of tobacco alkaloids. This could be due to the fact that the tobacco cembranoids are synthesized 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 using  isotopically labelled precursors 16 these diols seem to arise from geranylgeraniol 13, an [  ],  accepted precursor in terpenoid synthesis. The mechanism by which geranylgeraniol 13 is coverted into the cyclopropane ent-casbene 14 remains unclear although the stereochemistry of the process has been studied and a possible mechanism proposed.[ 231 It is considered that in the formation of the cyclopropane ring, the pro-S hydrogen at C-1 of  6  Acetate  Mevalonic acid  1\^9 7\ 5 16 14 12^10 8^6^4  OH  13^  14  4  I  III  III  1  16  2 Scheme 1, Proposed biosynthetic pathways for diols 1 and 2  7 geranylgeranyl pyrophosphate is eliminated and the methyl groups on the cyclopropane ring are formed by suprafacial attack on the re re face of the 14,15 double bond of the -  geranylgeranyl pyrophosphate. Ent-casbene 14 thus formed is then converted to cembrene 15 (also a constituent of tobacco), via 5-hydroxylation, solvolysis and reduction. An stereospecific enzymatic hydroxylation of cembrene 15 at the allylic C-6 position gives 2,4,7-cembratetraene-6-ol 16 and hydration of the 4,5 double bond leads to the final products, 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- 14 g-acetate, [2- 14 C)-mevalonate, all(E)-[2- 14 C]-geranylgeraniol and [3- 14 C]-cembrene can be incorporated into cembranoids .[ 16] 1.1.4 Synthesis of cembranoids Recently, significant advances have been made in the synthesis of cembranoids and several new approaches have been reported.[ 25 ] One of the synthetic routes to the diol 1 developed by Marshall et al. [26, 27, 28] is via the 17-membered ether 23, which is readily prepared 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 alcohol 18 is obtained.[ 29 ] Swern oxidation [ 30] using oxalyl chloride and dimethyl sulfoxide leads to aldehyde 19. Addition of propargylmagnesium bromide followed by alcohol protection with 2,3-dihydropyran (DHP) affords the alkyne 20. The latter, upon desilylation with tert-butylammonium fluoride in THF, affords an alcohol which is converted to the allylic chloride 21 by treatment with methanesulfonyl chloride (MsC1) and lithium chloride (LiC1) according to the Collington-Mayers' procedure.[ 31 ] Treatment of the lithiated acetylide with  8 1) Se0 2 /t-BuO0H CH 2 C1 2 , 25%  OH^OTBS  2) TBSCI/DMAP 3) K2 CO 3 /Me0H 85% 18  17  (C0C1) 2 /DMS0 Et 3 N/CH 2 C1 2 92%  THPO OTBS 1)BrMgCH2C--= CH Et 2 0, 96% -  OTBS  -  2) DHP, CH 2 Cl 2 , 96% 0 19  1) Bu 4 NF, THF 98% 2) LiCI, MsCI 84% THPO Cl  n-BuLi,(CH 2 0) n THF, 83% 22  21 RO  1) n-BuLi,TMEDA OH^90% 2) PPTS, Me0H 4 ..,..<^85% 3) TBSC1,DMF, Im  EtMgBr THF-HMPA 84% 0 ^V  THPO  N 24 R=THP 25 R=TBS  23  Scheme 2, Synthesis of a diol 1 -  9  TBSO BSO OH  (COC1)2 DMSO, CH 2 C1 2  26 25  Me 2 CuLi Et 2O 90% TBSO 1) (iBu) 2 A1H THF, 96% 2) (Ph 3 P) 3 R h Cl C 6 H 6, EtOH H2, 69%  27  1) VO(acac)2, t-BtOOH, 88% 2) CH 3 S 0 2 C1,C 5 11 5 N 92% 3) Bu 4 NF,THF, 92%  Na, NH3 THF,75 %  29^ Scheme 2, Synthesis of a - diol 1 (continued)  1  10  paraformaldehyde gives the chloroalcohol 22 which cyclizes to the ether 23 upon low temperature (0°C) addition of 1 equivalent of ethylmagnesium bromide in tetrahydrofuran and hexamethylphosphorylamide (THF-HMPA) followed by reflux. The THP ether 23 readily undergoes a [2,31-Wittig rearrangement with nbutyllithium 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 by silylation with tert-butyldimethylsilyl chloride (TBSC1) converts 24 into 25, the latter being a better substrate than 24 for further reactions. Swern oxidation of 25 gives the ketone 26. Treatment of 26 with lithium dimethyl copper yields the enone 27 which is then reduced with di-isobutyl aluminium hydride and finally hydrogenated selectively with Wilkinson's catalyst to afford alcohol 28 in 69% yield. Epoxidation of the allylic alcohol 28 with vanadyl acetylacetonate and tert-butyl hydroperoxide affords a single epoxide which is then converted to mesylate 29 through treatment with methanesulfonyl chloride in pyridine followed by desilylation. Reductive elimination with sodium ammonia affords the final product, a-diol 1. The (3-diol 2 can also be synthesized in a similar manner. [27]  1.2 Biogenetic considerations for cembranoid transformation  From the results obtained during structure elucidation of compounds isolated from tobacco and "biomimetic" interconversions, it was recognized that the diols 1 and 2 are the key intermediates in the biogenesis of the majority of the other tobacco cembranoids. The metabolic transformations are postulated to be initiated by 1)  oxidation of the 11,12 double bond  2)  oxidation of the 7,8 double bond  11  3)  oxidation of the hydroxyl group at C-6  4)  acid-induced rearrangements  1.2.1 Oxidation of the 11,12 double bond For the sake of clarity, only the 4S epimer, diol 1, which is the more abundant epimer 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 route leading to the formation of a number of tobacco constituents . By the first of the two principal pathways (Scheme 3), the diol 1 is converted into the 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 to the formation of hydroperoxides. The most direct evidence is that these two hydroperoxides have been isolated from the flowers of Greek tobacco.[ 32] They are obvious 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, as suggested by the isolation of the seco-aldehyde 32.[ 33 ] This compound could undergo oxidation to give the seco acid 33, which has been known as a component of tobacco.[ 341 -  In agreement with this, the diol 1 reacts smoothly with singlet oxygen at the 11, 12double bond, giving the two 4,6,11-triols, 9, 34, and the two 4,6,12-triols, 8, 35, in the ratio of 63:1:31:5 after reduction of the initially generated hydroperoxides using triethyl phosphite (Scheme 4).[ 35 ] This sensitized photooxygenation proceeds by a preferential attack of singlet oxygen on the trisubstituted 11,12 double bond both regio- and stereospecifically, leaving the trisubstituted 7,8 double bond, which is less active due to the deactivating effect of the hydroxyl group at C-6, unattacked. Epoxidation of the 11,12 double bond in the diol 1 is the second principal pathway and leads to the formation of the (11S, 12S)-11,12-epoxide 4, which is an abundant  12  1  I  I  1 8  9  Scheme 3, Proposed biogenetic pathways leading to triols 8 and 9  13  a  1  b  +  +  ^ 35 34^ 5% 1% Scheme 4, Products obtained from photooxygenation of the diol 1 followed by reduction with triethylphosphite  39  4  Scheme 5, Epoxidation of the 11,12 double bond in the diol 1  14 tobacco cembranoid (Scheme 5).[ 10] This reaction can be accomplished by oxidation of the diol 1 with m-chloroperoxybenzoic acid (m-CPBA) and shows a regioselectivity reminiscent of that exhibited by its reaction with singlet oxygen [ 36 ]. The epoxide 4 undergoes an acidinduced rearrangement to form 8, 11- and 8, 12-epoxy-diols 36 and 37 which have been isolated from Greek tobacco.[ 37, 38 Subsequent dehydration leads to the formation of ]  compounds 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 by biomimetic experiments.[ 36] Thus, treatment of the epoxide 4 with dilute hydrochloric acid in dioxane-water affords four major products 36, 37, 38 and 39 (Scheme 6).  mCPBA III  1  4  a III  39  36  III  III<  HO  37  Scheme 6, Formation and acid-induced reaction of epoxide 4  38  15 The generation of 36 is explicable by the anti-addition of water to the 11,12epoxide group followed by an attack of the newly formed 11-hydroxyl group on the 7,8 double 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 would be involved in the reaction leading to the 8,12-epoxy-diol 37. Elimination of water from 36 leads to the alcohols 38 and 39. 1.2.2 Oxidation of the 7,8 double bond  Oxidation of the 7,8 double bond in the diol 1 is less common than that of the 11,12 bond and only two compounds in which the vulnerable 11,12 double bond is retained have so far been isolated from tobacco in minute quantities. These two compounds have been identified as the (7S, 8S)-7,8-epoxide 41 and the (7R, 8R)-7,8-epoxides 42 by X-ray analysis and synthesis.[ 401 Thus, treatment of the diol 1 with t-butyl hydroperoxide and a catalytic amount of vanadyl acetylacetonate results in a regiospecific epoxidation of the 7,8 double bond and affords 41 and 42 in a 76:24 ratio (Scheme 7).  ...< ButOOH VO(acac) 2  1  41 76%  42 24%  Scheme 7, Epoxidation of the 7,8 double bond in the diol 1 1.2.3 Oxidation of the hydroxyl group at C 6 -  Oxidation of the hydroxyl group at C-6 converts the diol 1 into the ketol 43, and the latter could serve as a precursor of the seco-diketone 44 via a retro-aldol type of reaction.  16  Both 43 and 44 have been isolated from dark-fired tobacco (Scheme 8).[ 41 ] Oxidation of the 11,12 double bond in the ketol 43 provides a route for the biogenesis of 6-oxo-compounds 7, 30 and 45, which could be obtained alternatively by oxidation of the hydroxyl groups at C-6 in epoxide 4, (4, 6, 12)-triol 8 and (4, 6, 11)-triol 9, respectively. The epoxide 7 can be converted to the 8,11-epoxy-diol 46 via an acid induced rearrangement and the diol 46, in turn, may serve as the precursor of the dehydration products 47 and 48. These three compounds are the only 8, 11-epoxy-compounds containing 6-oxo group so far isolated from tobacco [42], Experimental results support the viability of the biogenetic pathways outlined in Scheme 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 cyclization of 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 also explored 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 be identical to 46 in addition to two minor ones 47 and 48. The three remaining products were formulated as 51, 52 and 53. The generation of these products may be accounted for as shown in Scheme 10. An initial anti-addition of water to the 11,12-epoxy group in 7 yields the triol 31 which undergoes protonation of the oxo-group at C-6, migration of the 7,8 double bond, and attack of the 11-hydroxyl group on C-8 to give two diols, 46 and 51. These two diols are plausible precursors of 47, 48 and 52. Since the product 53 is an (8R, 12S)-8,12epoxy-diol, it is likely to be formed by a mechanism that involves a C-12 carbocation as an intermediate. [42]  17  44^  1  48  III  46 III  43  47  Scheme 8, Proposed biogenetic pathways related to the oxidation at C-6  1 8  +  I 43 4% (84,11R), 49  I II  +  3n  Scheme 9, Products obtained from photooxygenation of the ketol 43 followed by reduction with triethyl phosphite  III  19  III  —  H+  Q  C, pH  III  HO  I  I  III  53  46  1  1 47  ^  48  ^  52  Scheme 10, Products obtained from acid-induced rearrangement of 7  20 1.2.4^Acid-induced reactions  An 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. These compounds have long been known as tobacco constituents (Scheme 11).[ 43, 441 The 4, 8-diol 55 is susceptible to dehydration and oxidation of the 11,12 double bond. 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.[461  1  2  5R  Scheme 11, Proposed pathways via acid-induced rearrangements of the diol 1  21  The results of acid-induced reaction of the diol 1 using dilute sulfuric acid in dioxane-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 is subjected to competing allylic rearrangement, epimerization and dehydrative fragmentation reactions. Compound 55 can also be obtained in 20% yield when passing the diol 1 through a chromatographic column packed with acid-washed alumina.rn  WI  +  10%  15%  55  59  +  24%  3%  1  44  +  12%  5%  2  54  Scheme 12, Products obtained in acid-induced rearrangements of the diol 1  22  The (8S, 11R)-8,11-epoxy-diol 57 in Scheme 11 is the only cembranoid of this stereochemistry that has been encountered so far.[ 461 The synthesis of 57 is shown in Scheme 13. Thus treatment of the (4S, 8S)-diol 55 with m-chloroperoxybenzoic acid affords the corresponding (11S,12S)-11,12-epoxide 59. When exposed to a trace of aqueous hydrochloric acid in chloroform, the epoxide 59 undergoes a facile SN2 type of epoxide ring opening at the secondary carbon C-11 by attack of the hydroxyl group at C-8 to give the (8S, 11R)-8,11-epoxy-diol 57 .[46} OH^ o^ mCPB A  OH^ o^ HCI CHCI 3  0 OH  HO"  55^  59  ^  57  Scheme 13, Formation of (8S, 11R) 8,11 epoxy diol 57 -  -  -  1.2.5 Degraded cembranoids  Detailed studies have revealed that, in addition to cembranoids, tobacco contains a substantial amount of volatile compounds which may be classified as degraded cembranoids. About sixty of such compounds have been isolated from tobacco.[ 3 } The characteristic features of these compounds are their irregular isoprenoid skeleton containing an isopropyl group and consisting of 8 to 19 carbon atoms. With a few exceptions they are carboacyclic. 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 of the bonds in the parent cembranoid skeleton as shown in Figure 3 even though the mode of biogenesis has not yet been verified by labelling studies.[3, 4, 5 1 The key metabolites thus  23  formed have 12, 13, 14, 15, 18 and 19 carbon atoms. They may undergo subsequent chemical alternations involving loss of carbon atoms to give further degraded products. Since the concentration of degraded cembranoids in the aroma fractions obtained from cured leaves is high (it has been estimated that they constitute about 10% of the total volatile material of Burley tobacco [ 48]), it can be concluded that these bond ruptures are favored reactions. It should be emphasized, however, that although some degraded cembranoids are generated during the curing processes of the tobacco leaves, many of them are present in green leaves and fresh flowers, although at a lower concentration.[ 17] This indicates that these degradation reactions also occur in the growing tobacco plants and they are not mere artefacts during the post-harvest treatments. C18 C13 C14 C12 18  16  C18, C15  15 13  17  12 10  1  C19  C12 C13 20 C14 C15 Figure 3, Degradation patterns of cembranoids The cleavage of the 12,20 bond leads to the C19 key metabolite 62, a compound isolated from tobacco flowers.1 47 1 It might occur by the biogenetic pathway shown in Scheme 14, although not validated experimentally. The latter process involves the triol 60  24  and the corresponding epoxide 61 as intermediates. The latter undergoes an acid-induced rearrangement, 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 representative encountered to date.[49 1 Its biosynthesis would involve rupture of the 5,6 and 7,8 bonds in the parent cembranoid. This process has previously been suggested to take place by a retroaldol type fragmentation from seco-aldehyde 54 as shown in Scheme 15.[ 4 ] — HO  1 HO  0^ 62  (-0 H+ CH2-ei 61  Scheme 14, Formation of the C19 compound 61  0  1, (45) 2, (4R)  54  63  Scheme 15, Formation of the C18 compound 63  25  The biogenesis of C15 compounds is explicable by breakage of the 7,8 and 11,12 bonds in parent cembranoids. As shown in Scheme 16, the C15 key metabolites 66, an aldehyde not yet encountered in tobacco, may arise via the 7,11- and/or 7,12dihydroperoxides 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 isolated from Japanese SUIFU tobacco.[ 50] The predicted C14 key metabolite 68 may also be generated through the 7,11and/or 7,12-dihydroperoxides, but via a route illustrated in Scheme 17, The resulting C14 aldehyde 68 is the precursor of the diol 69 and the hydroxy-acid 70. The hydroxy-acid 70, in turn, would give the acids 71 and 72 by dehydration. Both the aldehyde 68 and its  daughter compounds 69-72 have been isolated from tobacco.[ 51 ] As shown in Scheme 18, oxidative cleavage of the 6,7 double bond in secoaldehyde 54 [49] or an acid-induced cleavage of the 6,7 double bond in 33 [ 34] leads to the C13 key metabolite, solanone 73, which is an abundant and important flavor constituent in  tobacco. Subsequent metabolism via hydration and epoxidation leads to the hydroxyketone 74 and the epoxide 75. The epoxide 75 undergoes a stereo-controlled rearrangement 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 the formation of C12 degraded cembranoids. Among the C12 metabolites, norsolanadione 78 is a predominant constituent of the tobacco volatiles and an important precursor of many other C12 constituents [ 53, 54 ]. For instance, the formation of 78 can take place by oxidative  breakage 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 can rearrange to the dioxabicyclo-[3.2.1]-octanes 84 and 85 [4, 53 ]. The epoxy-diol 83, a compound isolated from Greek tobacco [ 51 ], may be generated by reduction of the epoxy-  26  1 _^ HO =^*OH HOO  I HOO +  HOO 64  I MI  66 HO OH H + .0H  O- ^  HO —/ 67  Scheme 16, Formation of the C15 compounds  27  1  I +  )c  I  0 OH x  H 0, / N.f 68  >1111‹  4 A 70  1  69  A 72 Scheme 17, Formation of the C14 compounds  \ COOH  28  HO  C  0  54  r I  33 >< ^ O/ 73  I  Scheme 18, Formation of solanone 73 and the other C13 compounds  29 dione 80. Norsolanadione 78 is also the precursor of several C8 to C11 tobacco compounds, such as the recently encountered degraded products 82 and 86 [ 55 1. It is noteworthy that 82 and 86 are the first degraded cembranoids encountered in tobacco in which the isopropyl-bearing carbon atom also carries oxygen.  0 /N 79 OH  44  ^  ZN  O/ 78  80  O HOOCV\  /N. 81  86  Scheme 19, Formation of nor-solanadione 78 and other C12 compounds  OH  30 1.3 Plant tissue culture 1.3.1 Applications of plant tissue cultures in biotransformation studies Plants are not only the most important sources for foods, oils and fibres but also an immense repository of chemicals including flavours, essences, pigments, fine chemicals, pharmaceuticals and novel biologically active substances. Plants and their extracts have been used by mankind for centuries and thousands of organic compounds whose chemical syntheses are really a challenge to organic chemists have now been directly isolated from plants. Vinblastine 87 and taxol 88, for example, are two important anticancer drugs which could be isolated from Catharanthus roseus and Taxus brevifolia, respectively. But most of these compounds occur in the plants only in minute amounts and localize strictly in specific organs of whole plants, such as roots, flowers and leaves. Their production by field plantation is highly dependent on environmental factors such as climate, season as well as destruction by pests and disease, all of which can influence plant growth and the build-up of specific metabolites in the plants. The isolation processes for such natural products from other co-occurring substances are usually very difficult, lengthy and costly as they involve the purification of small amount of substances from a very large mass of plant material. In addition, many plants grow in some inaccessible regions and may take years 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 economically viable. In summary all of these factors make it undesirable to obtain such expensive fine chemicals from field plants and often inappropriate for commercial production. Chemical syntheses can provide an approach for obtaining supplies of these compounds. The modern spectroscopic techniques have aided structural determination and facilitated the elucidation of biochemical pathways. However, even in the case where synthetic routes are well established, the total syntheses of the desired product cannot  31 always be achieved economically. Actually, they are rarely useful in any practical production of such complex natural products, often required in large scale for their use as drugs, etc.  OH  N  H H3 COOC  CH 0  1  CH 3  OCOCH2 COOCH 3  Vinblastine, 87  0 ..•OH ....  NH  0= C  Taxol, 88  Fortunately, many studies indicate that plant natural products can be obtained alternatively from in vitro culture systems, including plantlets, specific plant organs or cells growing in solid or liquid nutrient medium [ 56]. Also there is evidence that such plant cell  32 cultures retain an ability to transform specific exogenous substrates administered to the cell cultures [ 57 ]. Therefore, plant cell cultures, if successively developed for large scale fermentation, can be considered not only as substitutes for some field plants, but also serve as useful "tools" to transform inexpensive and plentiful substances into rare and expensive substances. Cell cultures have now been established for many plants. Scheme 20 gives an outline of the processes involved in establishing a typical plant cell culture.  Whole plant I  it Explant^I  Callus culture  IT Suspension culture Scheme 20, The development of a plant cell suspension culture The initial stage for preparing such a culture involves the removal of a section of plant tissue under aseptic conditions. This material, known as explant, is then placed on nutrient agar which consists of carbohydrates such as sucrose, inorganic salts and small amounts of plant growth auxins. The explant may be a piece of root, leaf or other parts of the plant. Successful growth of the plant tissue on the solid nutrient medium results in a  33 callus culture which is a mass of disorganized cells. The callus culture can be maintained for a long time through sub-culturing, a process in which the sample of callus is removed and placed on a fresh agar medium where they continue to grow. If a sample of callus is placed into a liquid nutrient medium, a suspension culture can be obtained. The liquid nutrient medium is prepared using similar constituents which are initially used to produce the nutrient agar. The cell suspension cultures can be sub-cultured in the same way as the callus cultures. Once a cell culture has been established, growth may be rapid, and environmental factors such as light, temperature and aeration can be controlled to promote cell growth and metabolite accumulation. Compared with field crops, cell cultures offer the following major advantages: 58 [  a)  ]  Compounds could be produced year-round under controlled laboratory conditions, assuring a steady supply without seasonal fluctuation.  b)  Metabolic processes can be regulated and thus, yields of the compounds of interest can be maximized.  c)^Cells could be genetically modified and thus accumulate specific intermediates or other metabolites through biosynthesis or biotransformation The potential of plant cell cultures has been demonstrated by the production of the cardiac drug ubiquinone-10 using suspension culture of Nicotiana tabacum in amounts of 15 mg/L or about 1.9 mg/g dry cell. This yield is ten times greater than that in the intact plant [ 59 ]. As a matter of fact, Shikonin, an antiseptic and dyestuff used in Asia since ancient times, is currently being produced commercially in Japan by using cell culture of Lithospermum erythrorhizon . In this instance, yields exceeding 2 g/L (12.4% of dry  weight) 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 very exciting prospect. In these so called biotransformation studies, plant cell suspension  34 cultures are very often used. In preparation of such a culture, as mentioned above, the undifferentiated callus tissue is dissociated into a fine cell suspension by putting the callus into liquid nutrient medium and allowing it to grow in rotary shakers. The cells will grow to a stationary phase after an appropriate time period and are then ready for use. A typical biotransformation experiment involves addition of substrate to the plant cell culture under sterile conditions, incubation for a certain period of time, harvest and isolation of products. If the products are present in the culture broth, the cells can be filtered and products can then be extracted from the broth. However, if the products are trapped in the cells, the cell material must be homogenized to break up the cells so that the products can be extracted. The advantages of using whole cell suspensions include the homogeneity of the culture environment, the ease of manipulation of culture conditions and the rapid generation of large volumes of relatively uniform tissue. Also, the cofactors necessary for the various enzyme functions are generated in situ and need not to be added externally. Despite the tremendous versatility of the system, some problems still can arise when this whole cell biotransformation technique is used. The lack of specific enzymes is frequently responsible for the inability of the cell culture to biotransform the foreign substrates. Also the substrate must have a certain degree of solubility in the cell culture growth medium, and must be able to diffuse or transport through the cell membrane. In addition, the isolation of products may be difficult if they remain inside the cells and do not diffuse into the medium. Since there are many enzymes present in the cell culture, the production of the desired product by specific enzymes may be complicated if competing enzymes can transform the substrate to undesirable end products. Many other reactions and subsequent degradation can occur to produce undesirable by-products. Despite these obstacles, this methodology holds much promise as shown by the semi-continuous conversion of 13-methyldigitoxin, a highly toxic byproduct obtained in the extraction of cardenolides from Digitalis lanata , to the highly valuable cardiac drug D-methyldigoxin by  35 Digitalis lanata cell culture. In the latter study, an 80% yield was obtained in a 300 litre  airlift-bioreactor.{ 62, 63 ]. Cell free systems provide another approach used in biotransformation studies. This involves the isolation and use of the enzymes, thus maintenance of the biomass is not required and any problems connected with cell membrane impermeability are also eliminated. Typically, the cell culture is filtered and the cells are disrupted by homogenization in the presence of a buffer at a low temperature. This homogenate is then centrifuged to remove the cell debris. The supernatant thus obtained is called cell free extract (CFE) and can be considered as a solution of enzymes originally present in the culture but now in a cell-free form (Scheme 21). The purification is often limited to a removal of buffer-insoluble material, such as cell walls, but it may also include chromatography to isolate the required enzymes.  Cell suspension culture Filtration Cells I Homogenization in buffer Homogenate I Centrifugation  Pellet (Cell debris)  Supernatant (Cell Free Extract) CFE  Scheme 21, The preparation of Cell Free Extract (CFE)  36 The advantage is that the reaction condition can be determined precisely because it is carried out in a homogeneous solution rather than inside a cell. The isolation of products is relatively easier than that in whole cell biotransformation because much less cell material is present in this case. It is evident that the substrate and any necessary cofactors for the reaction must be supplied directly to the CFE mixture as these can no longer be synthesized  in situ. This requirement is a major consideration for a potential commercial process. For example, biotransformation employing costly nicotinamide adenine dinucleotide phosphate (NADPH), a cofactor for the oxidative enzyme cytochrome P-450, would be practical only if particularly expensive final products were involved. Other drawbacks of this approach include the possibility of loss of enzymatic activity during CFE preparation as many enzymes are very labile and can be denatured by osmotic shock, pH change or temperature. The complexity of the overall procedure may not 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 a purified and immobilized form which would permit their recovery and re-use after each biotransformation. To date over 30 classes of compounds have been produced or transformed by using plant cell cultures including steroids, alkaloids, terpenoids and quinones [  56 ].  These data  suggest a bright future for this technology. An illustration of such a biotransformation in our laboratory was the preparation of 3',4'-anhydrovinblastine 91, a precursor of the anticancer drug vinblastine 87, using Catharanthus roseus cell culture (Scheme 22). Incubation of catharanthine 89 and vindoline 90 with C. roseus CFE in the presence of hydrogen peroxide as a cofactor gave 3',4'-anhydrovinblastine 91 in 25% yield [64 ]. This yield increased to 38% when immobilized enzymes from the same culture were employed [65]  .  37 +  CH 3 0  1 NH^  OCOCH 3  cH 3 COOCH 3  Catharanthine 89 C. roseus H2 0 2  CFE  Vindoline 90  V  N H H3 COOC \ N C3 0^N  1H  CH3  OCOCH 3 COOCH 3  3',4'-anhydrovinblastine 91 Scheme 22, Production of 3 ,4 -anhydrovinblastine 91 by CFE from C. roseus 1  1  1.3.2^Oxidative reactions in biotransformation studies using plant cell cultures As mentioned above, plant cell cultures are capable of producing and/or transforming a variety of chemical compounds. This is due to the fact that different kinds of enzymes are present in plant cells and act as catalysts for those reactions leading to specific products. Among those bio-reactions, the oxidative processes are very often encountered. As oxidative reactions are concerned, it is worth to mention cytochrome P-450. The  38 cytochromes P-450, abbreviated from "Pigment with an absorption at 450 nm", are hemoproteins with a characteristic absorbance at 450 nm in the UV spectra of their CO adducts and are well known enzymes common to many plants and animals. They play an important role in such processes as steroid metabolism, drug detoxification and the carcinogenic activation of polycyclic aromatic hydrocarbons. The levels of these hemoproteins can be influenced by many chemicals and the enzymes, in turn, are capable of metabolizing many compounds. Because of the diversity of substrates and the variety of transformations that these enzymes execute, this family of cytochromes has attracted the attention of researchers in many fields, including organic and inorganic chemistry, biochemistry and pharmacology. Extensive research has led to the isolation and characterization of many apparently distinct forms of cytochrome P-450. Now the complete primary structures of many P-450s have been elucidated through protein and DNA sequences. The active site of P-450 has long been known to contain the ferroprotoporphyrin IX prosthetic group. (Figure 4).  Figure 4, Structure of iron protoporphyrin IX and catalytic cycle of cytochrome P-450  39 A general accepted catalytic cycle for P-450 is shown in Figure 4 [ 661. The features include: I.  Binding of the substrate to give a ferric complex  II.  One-electron reduction of the iron to the iron(11) state  III.  Binding of oxygen to generate the oxy form SFe 3 +02 -  IV. A second one-electron reduction to yield the iron peroxo species Fe 3+022V.  Formal heterolysis of the 0-0 bond with generation of the reactive oxidant [FeO] 3 + and a molecule of water  VI. A two electron oxidation of substrate to produce SO and regenerate the ferric state of the enzyme  During metabolism or biotransformation, cytochrome P-450 behaves as a monooxygenase. The mono-oxygenation occurs according to Equation 1 and involves the incorporation of one oxygen atom into the substrate. Obviously, the reaction requires the input 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 reducing cofactor. Due to the fact that the pyridine nucleotides are two electron donors, while P-450 can only accept one electron at a time, other transfer agents must be involved in the process. Such agents usually are flavin mononucleotide (FMN) or flavoadenine dinucleotide (FAD). These flavoproteins can undergo a single electron transfer to the P-450 system which is then able to oxidize the substrate. Cytochrome P-450s are responsible for many oxidative reactions and the reported results can be clas , ified into six categories.[ 67]  40 1. Carbon hydroxylation: formation of an alcohol at a methyl, methylene, or methine position. 2.  Heteroatom release: oxidative cleavage of the heteroatomic portion of a molecule  3. Heteroatom oxygenation: conversion of a heteroatom-containing substrate to its corresponding heteroatom oxide 4.  Epoxidation: formation of oxirane derivatives of olefins and aromatic compounds  5.  Oxidative group transfer: a 1, 2 carbon shift of a group with concomitant incorporation of oxygen as a carbonyl at the C-1 position  6.  Olefinic suicide destruction: inactivation of the heme of cytochrome P-450 by an enzyme product or an enzyme intermediate  For instance, cytochrome P-450pB_B [ 68 ] can catalyze all six types of oxidative reactions 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 hydroxylation are two most important and very often encountered reactions. These reactions have received considerable attention in biotransformation studies. It was reported that the suspension cultures of Nicotiana tabacum have the ability to hydroxylate the trans-methyl group in the isopropylidene moiety of linalool 115 and its acetate 116 to give the corresponding 8hydroxyderivatives 117 and 118.[ 74 ] Such an ability was also investigated with the monoterpenoids having terminal, endocyclic and exocyclic C-C double bonds, such as aterpineol 119 and its acetate 120, f3-terpineol 126 and its acetate 127 and y-terpinyl acetate 133 as showed in Scheme 23. The terpineols were hydroxylated at the carbon atoms allylic to the C-C double bond to yield the corresponding allylic alcohols.  41  R I  117, R=OH 118, R=OAc  115, R=OH 116, R=OAc HO,, +  119, R=OH 120, R=OAc  121, R=OH 122, R=OAc  +  123, R=OH 124, R=OAc  125, R=OAc  +  126, R=OH 127, R=OAc  128, R=OH 129, R=OAc  130, R=OH 131, R=OAc  132, R=OAc  OAc^OAc +  +  OH 133  134  135  136  Scheme 23, Some examples of allylic hydroxylation using plant cell cultures  42  On the other hand, terpinyl acetates were hydroxylated, not only at the allylic positions, but also at the C-C double bond to give glycols as the major productsP 41 The process of glycol formation was investigated in the biotransformation of aterpinyl acetate 120 and y-terpinyl acetate 133 in the cultured cells of N. tabacum. It was found that glycols were formed from epoxidation of the C-C double bond, followed by hydrolysis of the resulting epoxides.[ 75] 1.3.3 Reductive reactions in biotransformation studies using plant cell cultures  The reductions of C-C double bonds and of carbonyls to alcohols are very popular in biogenesis and biotransformations. These reactions are catalyzed by dehydrogenases utilizing pyridine or flavin coenzymes and considered as the reverse processes of the oxidations using dehydrogenases. Therefore, these reactions obey the specificity requirements of the dehydrogenases. S + AH2 > SH2 + A (3) There are many reports on the reductions of ketones and aldehydes to the corresponding alcohols with plant cell cultures. One of these examples is the biotransformation of monoterpenoids by plant cell suspension cultures of Lavandula angustifolia. [ 76 ] It was found that this plant cell culture can reduce monoterpenoid  aldehydes and structurally related compounds to the corresponding primary alcohols. Some of the structures are given in Scheme 24. Citronellal 137, geranial 139 and perillaldehyde 141 were reduced to the corresponding alcohols 138,140 and 142. It was also found that acyclic monoterpenoid alcohols 138 and 140, once formed, disappeared from the cultures over about a 15 hr period and were metabolized into unidentified compounds. In contrast, the cyclic monoterpenoid alcohol, perillyl alcohol 142, remained unmetabolized in the cultures over a 72h period.  43 )  .,CHO  OH  138  137 CHO  139  OH  ^  140  CHO  141  ^  142  Scheme 24, Some examples of reduction using plant cell cultures 1.3.4 Plant cell lines developed and available in our laboratory  Tremendous efforts have been directed in our laboratory towards the production of pharmaceutically important chemicals from plant tissue cultures. As a result, several stable cell lines have been established. For example, the AC3 cell line derived from Catharanthus roseus is capable of producing the indole alkaloid vinblastine 87, a clinically used drug for  cancer chemotherapy, and biotransforming the indole alkaloids catharanthine 89 and vindoline 90 into 3',4'-anhydrovinblastine 91, a precursor of vinblastine 87  [  77  ].  The  TRP4a cell line derived from Tripterygium wilfordii , a well known Chinese herbal plant, can produce the diterpene tripdiolide  [  78  ]  which is a cytotoxic compound and reveals  44 significant male contraceptive activity. A cell line derived from Podophyllum peltatum is used to produce podophyllotoxin and demethylpodophyllotoxin, which could be utilized as intermediates 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 and several promising results have been achieved. For example, an oxidative coupling reaction of 117 catalyzed by enzymes in CFE prepared from C. roseus and in the presence of hydrogen peroxide as cofactor affords an essentially quantitative yield of the ring-closed product 118 (Scheme 25), thereby providing a route for the syntheses of lignans 971 [  HO  HO  MeO  CFE^MeO  0111^  .  C. roseus H20 2  CH 3 O^OCH3^CH3O OH OH 117 118 Scheme 25, Oxidative coupling leading to lignan 118 1.4 Objectives of the project  Since the chemical investigations on tobacco cembranoids reported so far were based exclusively on isolation, structure elucidation and chemical transformation, no biotransformation studies on tobacco cembranoids using plant cell cultures had been reported. Therefore, it was of considerable interest to examine if similar interconversions could be executed, more regiospecifically and hopefully in high yield, by enzymatic processes. With various plant cell lines and biotransformation technology in hands, our intention to perform biotransformation studies on tobacco cembranoids was initiated. One of the aims of this project was to test the hypotheses of the proposed metabolic pathways as discussed previously. Another important objective was to see if it is possible to produce  45 molecules which had potential use in the area of aroma and fragrance chemicals, for example, norsolanadione 78, via metabolism of the major cembranoids, diols 1 or 2, using our plant cell cultures (Scheme 26).  41.  HO  7  107  7R  Scheme 26, Proposed objective for cembranoid biotransformation  46 2 RESULTS AND DISCUSSION 2.1 Biotransformation of cembranoids using the TRP4a cell line  The major reasons for selecting the TRP4a cell line in a first attempt to perform the biotransformation studies on the tobacco cembranoids were not only the stability and availability of this plant cell line in our laboratory, but also the documented capability to carry out oxidative degradations.[78,  80, 81]  This plant cell line was well established in our  laboratory 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 the cembranoids, biotransformation experiments using this cell line were performed. As discussed in the Introduction of this thesis, three olefinic linkages are present in the starting diols 1 and 2. From the chemical point of view, the disubstituted 2,3 double bond is expected to be less reactive for oxidation than the trisubstituted 7,8 and 11,12 double 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. Our  biotransformation studies confirmed such a proposal. The suspension culture of TRP4a showed a high degree of regio-selectivity toward tobacco cembranoids. It reacted only with the 11,12 double bond to give the epoxide as the major product when either diol 1 or diol 2 was used as substrate. The biotransformation results showed that incubation of diol 1 with growing cells of the TRP4a cell suspension culture and with cells resuspended in phosphate or TrisHC1 buffers afforded recovered diol 1 and products 8, 4, 92 ,92 and 93 in order of increasing polarity as determined by thin layer chromatography (Scheme 27). The (11S, 12S)-epoxide 4 was the predominant product in 40-50% yield in our optimized conditions while the other  compounds were obtained as minor products in less than 10% yield. Among the five products, triols 92, 93 and 94 are new compounds not previously isolated from the living  47  1 TRP4a  8^  92  ^  4  93  ^  94  Scheme 27, Biotransformation of the diol 1 using TRP4a cell culture plants of Nicotiana species, the triols 92 and 93 were assigned as a pair of diastereomers with stereochemistry at C-10 undefined at present. The structure elucidations of the products were accomplished largely on the information obtained from 1 H, 13 C NMR and MS spectra. The mass spectra are informative even though the 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 the starting diol 1 and a molecular ion peak at m/z 322 was observed. This implied that one oxygen atom was introduced into the starting material. In its 1 H NMR spectrum, there were three methyl groups (singlets at 5 1.20, 1.37 and 1.78) and an isopropyl group (two doublets at 5 0.80 and  48 0.85), characteristic signals of cembranoids. The 13 C NMR spectrum of 4 revealed that there were 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 of the trisubstituted double bonds, (7,8 or 11,12), had been involved in the bioconversion and strongly suggesting epoxide formation. Since the 1 H NMR spectrum indicated that the 7,8 double bond was still present (a doublet of triplets at 8 4.48 for H-6), it was clear that the 11,12 double bond was involved in epoxide formation. In accord with this assignment, both signals normally arising from the proton and carbon atoms of the 11,12 double bond were now shifted upfield in the corresponding NMR spectra. A signal due to H-11 was observed at 8 2.88 as a doublet of doublets in the 1 H NMR spectrum of 4 and two signals due to C-11 and C-12 were also observed at 8 61.3 and 60.1 in the 13 C NMR spectrum. By direct comparison of the spectral data with the published results [10] , it was found that this biotransformation product was identical to (1S, 2E, 4S, 6R, 7E, 11S, 12S)-11,12-epoxy-2,7-cembradiene-4,6-diol and was thus identified as 4. Table 3, 13 C NMR chemical shifts (ppm) of cembranoids in CDC13 (1) (4) (8) (92) (93) (94) (1) (4) (8) (92) (93) (94)  C-1  C-2  C-3  C-4  C-5  C-6  C-7  C-8  C-9  C10  46.4 47.1 50.9 47.5 46.3 46.2  127.7 127.7 127.4 127.7 127.7 127.5  137.5 138.4 138.3 137.8 137.4 137.4  72.4 72.3 74.2 72.8 72.4 72.4  52.2 52.9 47.2 50.0 52.0 52.7  66.2 66.1 69.6 66.8 65.0 66.1  130.6 132.7 128.3 129.7 132.6 131.3  136.6 135.2 135.2 138.6 137.9 136.2  38.8 35.6 40.8 45.6 48.3 38.7  23.3 24.9 124.7 67.2 65.8 22.9  C-11  C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20  124.4 61.3 138.9 128.4 129.0 128.1  133.3 60.1 73.9 134.5 133.6 135.2  36.8 36.6 40.1 39.2 36.3 77.4  27.9 28.6 26.4 28.8 27.2 35.7  33.0 33.1 30.2 34.0 33.0 33.2  19.3 19.1 17.8 19.2 19.4 19.5  20.7 20.9 21.9 20.4 20.5 20.4  30.1 30.0 31.8 30.9 30.1 30.1  16.1 16.0 18.1 16.8 16.6 15.9  15.0 16.3 30.1 19.0 14.7 9.6  49 Epoxide 4 has been isolated from various tobacco plant sources 36 and postulated [  ]  as the key biogenetic intermediate for the metabolism of cembranoids.[ 3 ] The isolation of the epoxide 4 as a major biotransformation product confirmed that the enzymatic epoxidation was a principal biogenetic pathway in the metabolism of tobacco cembranoids. The mass spectrum of another product, the tertiary alcohol 8, also showed a molecular ion at m/z 322. The 13 C NMR spectrum indicated that there were three double bonds and two tertiary hydroxyl groups (5 73.9 and 74.2 ppm) in the molecule. It also showed 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 newly formed double bond, possibly a disubstituted one, was the result of the biotransformation reaction. Because the signal at 5 4.80 (t, J=7 Hz) due to 11-6 was still observed in the 1 11 NMR spectrum, the 7,8 double bond was unattacked. Therefore the newly introduced tertiary hydroxyl group was assigned to be located on C-12 and the 11,12 trisubstituted double bond was shifted to form a disubstituted 9,10 double bond. In accord with this assignment, a signal at 5 2.70 (due to two diastereomeric protons at C-9) was observed as a doublet of doublets in the 1 H NMR spectrum. It was found that the spectroscopic properties 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 isolated from tobacco [ 83 ]. This compound can be obtained by photooxygenation of the diol 1 with singlet oxygen followed by reduction with triethyl phosphite.[ 10 ] In conclusion, this product was assigned structure 8. The biotransformation products 92, 93 and 94 had not yet been isolated from natural sources so direct comparison with published data was not possible. The mass spectra 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 double bonds (two trisubstituted and one disubstituted) were still present, these three compounds were novel hydroxylated products of diol 1.  50 Product 92 formed a diacetate on treatment with acetic anhydride in pyridine. Since it already contained a tertiary hydroxyl group (5 72.8 for C-4 in 13 C NMR), this product could be a triol with two secondary hydroxyl groups. This suggestion was reflected in its 13 C NMR spectrum since an additional signal at 5 67.2 ppm could be seen. In comparing its MS fragmentation patern (Scheme 29) with that of diol 1 (Scheme 28), especially the fragmentation pathway of m/z 322 --> m/z 304 --> m/z 223 vs. m/z 306 --> m/z 288 --> m/z 207, it was clear that the fragment ions at m/z 304, m/z 221 and m/z 223 carry the newly introduced 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 newly introduced hydroxyl group at the C-10 position was most reasonable. Conformation for such placement came from the NMR data. In agreement with this assignment, the C-10 signal at 5 23.3 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 1 H 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 was identical with that of the synthetic sample 92 obtained in an independent synthetic program at the Swedish Tobacco Company, Stockholm.[ 84 ] Based on the available information, the stereochemistry 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 was also located at C-10 and the signals in the 13 C NMR were very similar to those in product 92 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 a diastereomer of 92 with opposite stereochemistry at C-10. The differences in the 13 C NMR chemical shifts for C-10 in 92 and 93 are obviously due to the stereochemical orientation of 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. Although the above data established the structures of 92 and 93, the specific stereochemical  51 assignment at C-10 remains ambiguous. Various attempts to obtain a satisfactory crystal for X-ray analysis failed.  I m/z=306  0 1  +.  1 -H2O  l  H ,i  m/z=288 -H2O  C6H9 + m/z=81 C14H23 0 m/z=207  m/z=288  -CH3CO  m/z=270  -CH3  m/z=245 m/z=43 +.  --/ •  C51170 miz=83  C9H15  m/z=123  Ci5H25  m/z=288 C101-1 17 m/z=205^m/z=137  -C3H7 m/z=273  m/z=245  McLafferty rearrangement  m/z=227  McLafferty rearrangement  )()c^>^<1 ' ,  C9 11 1 40 m/z=138  >^<  ,'N__  -  C101-1 16 m/z=136  -CH3^-CH3C0  m/z=123  ^  m/z=95  m/z=121  nil-L=93  Scheme 28, The mass spectrometric fragmentation pattern of diol 1  1÷  52  92 m/z=322  1 -H20  O  m/z=261 m/z=43 m/z=304  H  -H20 C6H9+ m/z=81 ^(  OH - CH3  -C3H7  m/z=223 -H20  m/z=289  m/z=261  )  >^<  C91-1140 m/z=138  1  Clair m/z=304 m/z=137  m/z=123  -H201 McLafferty rearrangement  m/z=243 Ci5H23  McLafferty ^0 141421 0^rearrangement m/z=205  ON  C91115  OH C15H250 m/z=221  - CH3CO  1^  -  C51170 m/z=83  m/z=286  m/z=304  CI4H23%-/2  +  m/z=203  ,  .  >^<  )\___  -  1+  C 1011 16  m/z=136  m/z=121^m/z=93  Scheme 29, The mass spectrometric fragmentation pattern of triol 92 Product 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. The hydroxylation of the cembrene skeleton was evident from the molecular ion at m/z 322 and  53 the fragment ions at m/z 304 and m/z 223. Even though the signal at 8 77.4 ppm arising from the carbon bearing the newly introduced hydroxyl group was not easily observed due to the solvent peaks (a triplet at 8 76.64, 77.07 and 77.49 due to CDC13), it was clearly seen in an APT experiment. This downward signal indicated that the newly introduced hydroxyl group was attached to a secondary carbon atom. Because a new one-proton signal at 8 3.98 ppm as a doublet of doublets was also observed, the newly introduced hydroxyl group was assigned to be attached at C-13. In accord with this assignment, the C-13 signal at 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-20 signal in the 13 C NMR spectrum (9.6 ppm vs 15.0 ppm in diol 1) provided further evidence for the placement of the hydroxyl function at C-13. However, the above data do not establish the stereochemistry at C-13 and it remains unknown. In contrast to allylic hydroxylation at C-10 which afforded two isomeric viols 92 and 93, only one C-13 hydroxylated product 94 was obtained. Fortunately, it was found that the spectral data of biotransformation product 94 were identical with those of a recent synthetic sample 94 provided by the Swedish Tobacco Company [K. The newly created carbinol centre was then assigned to have the S-configuration. The mechanisms for the formation of the epoxide 4 and the allylic alcohols 92, 93 and 94 are not clear. They could be cytochrome P-450-catalyzed reactions since these types of 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 double bond was oxidized to give the epoxide 95 in approximately 50% yield (Scheme 30). Its identity was established by comparing the spectral data of 95 with the published data.[ 361 The remaining fractions of the chromatography, constituting approximately 20% of the original mixture, was an inseparable mixture of several compounds with identical retention times on a TLC plate. Attempts to separate the mixture met with failure and eventually the compounds decomposed.[ 851  54  TRP4a  2^ 95 Scheme 30, Biotransformation of (3-diol 2 using TRP4a cell culture 2.1.1 Biotransformation of the diols 1 and 2 using the whole cells Since the abilities of the TRP4a cell suspension culture to execute oxidative reactions vary considerably with the reaction parameters, a series of experiments were carried out where conditions such as cell age, buffer, pH, incubation time and substrate concentration were altered. In the biotransformation experiments performed, the substrate was dissolved in ethanol and added to the cell culture in one of the following four different ways: 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 a peristaltic pump. 2.1.1.1 Addition of substrate to the cells resuspended in buffer Since plant cells are capable of producing various kinds of natural chemical substances, the presence of these metabolites in the culture broth could interfere with the analysis of the biotransformation results. Therefore the cell resuspension method was first considered. In this series of experiments, TRP4a cell suspension cultures grown to stationary phase (monitored by pH and refractive index of the spent medium ) in PRDCo medium were selected. The broth of the cell suspension culture was removed by filtration and the cells were then resuspended in a buffered medium (phosphate or TrisHC1)  55  containing sucrose (8%) as an osmotic balancing agent. The substrate dissolved in ethanol was added in one batch to the above resuspended cell culture. The mixture was then Cell suspension culture 1)Filtration through Miracloth 2) Wash with distilled water 3) Resuspension in buffer (pH 6.3) 4) Substrate addition 5) incubation Work-up Filtration through Miracloth  Filtrate EtOAc extraction  Cell material 1)Homogenization in EtOAc, (24,000rpm) 2) Filtration  1 EtOAc extracts  aqueous layer (discard)  Cell material^EtOAc (discard)^extracts  1) Wash with water, brine 2) Dry over anhydrous Na2SO4 3) Concentration in vacco 4) Chromatographic separation  Products Scheme 31, Procedures for biotransformation using resuspended culture of TRP4a  56 incubated on a rotary shaker at 26°C and 135 rpm without illumination for an appropriate time. The procedures are outlined in Scheme 31. Steps 1-5 (prior to workup) were performed under aseptic conditions in a sterile room. Progress of the reaction was monitored by TLC. After an appropriate incubation time, the biotransformation mixture was harvested by filtration, followed by extraction of broth and cells with ethyl acetate as outlined in Scheme 31. Chromatography of the crude extracts on silica gel afforded recovered substrate and biotransformation products. In order to isolate some polar products which may not be extracted by ethyl acetate and remain in the aqueous layer, the aqueous layer, after extraction, was subjected to freeze-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, the aqueous layer was not processed. 2.1.1.1.1 Differences in reactivities between diol 1 and diol 2  Structurally, diol 1 differs from diol 2 only in the chirality at C-4, however, marked differences in reactivities towards the TRP4a cell suspension culture were observed. The a-diol 1 was found to react with the cell suspension at a much slower rate when 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 after 120 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 biotransformation  Due the fact that both diols 1 and 2 were unstable under acidic conditions as discussed in the Introduction [ 47 ], TrisHC1 buffer at pH 7.5 was chosen for our initial studies. Later on, it was found that the biotransforamtion could be carried out more efficiently at a lower pH and the incubation time was reduced dramatically without any change in product yields (Tables 4 and 5). Therefore, subsequent biotransformation studies  57 were carried out in a phosphate buffered medium at pH 6.3. In all studies, TrisHC1 and phosphate buffers contained 8% of sucrose as an osmotic balancing agent. Table 4, Effect of pH on biotransformation of diol 1 Expt No. Cell age (days) pH  nD25  1^2^3 19^19^19 5.20^5.50^5.15 1.3330^1.3330^1.3331  4 19 5.50 1.3331  5^6 16^19 5.68^5.61 1.3336^1.3332  Substrate 1 /EtOH (mg/ ml ) Volume of culture (L) Resuspension medium :^type pH Incubation time (h) Yield (%)^1 4 92 93 94 Total recovery (%)  186/15^22.5/5^45/5 1.5^0.5^0.5 TrisHC1^Phosph. TrisHC1 7.5^6.3^7.5 120^72^144 20.5^0^5.8 33.9^50.2^41.6 0^0^0 12.5^24.0^14.9 12.3^13.0^12.2 79.2^87.6^74.4  25/5 0.5 TrisHC1 9.0 144 35.6 29.8 0 20.4 8.0 93.8  314/20^400/20 2.5^2.0 TrisHC1 Phosph. 7.5^6.3 168^144 21.7^4.6 28.7^35 9.1^12.3 8.9^13.8 4.1^5.2 72.4^70.9  Table 5, Effect of pH on biotransformation of diol 2 Expt. No. Cell age (days) pH 25  nD  Substrate 2/EtOH(mg/ ml) Volume of culture (L) Resuspension medium :^type pH Incubation time (h) Yield (%)^2 95 mixt Total recovery (%)  7^8 19^19 5.50^5.15 1.3330^1.3331 25/5^50/5 0.5^0.5 Phosphate^TrisHC1 6.3^7.5 72^144 0^0 53.6^52.6 16.8^24.8 70.4^77.4  9 19 5.25 1.3330 25/5 0.5 TrisHC1 9.0 144 30.4 36.8 9.2 76.4  10 19 5.20 1.3330 196/15 1.5 TrisHC1 7.5 120 8.4 48.7 19.7 76.3  58 It was found that biotransformations using phosphate buffer at pH 6.3 proceeded much faster when compared to those using TrisHCl buffer at pH 7.5 and 9. In an experiment with 200 mg of diol 1 per litre of cell suspension with a phosphate buffer at pH 6.3 (Entry 6), only 4.6% of the starting material was recovered after 144 h. However, in another experiment with a substrate level of 126 mg per litre of cell suspension with TrisHC1 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 further retarded when TrisHC1 buffer at pH 9 was used (Entry 4). A similar result was obtained when diol 2 was used as substrate. 2.1.1.1.3 Effect of substrate concentration on biotransformation  In the next series of experiments, the amounts of substrate administrated to the cell suspension cultures were increased progressively in order to assess the optimum substrate concentration for the cells to biotransform. When the amount of substrate was increased to the level of above 600 mg per litre of cell suspension, a significant portion of the starting material was found to be unreacted after 5-6 days. With the hope that prolonged reaction time could lead to a complete substrate consumption, the incubation time was therefore extended. 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 the usual products were found to be lower, but also a mixture of unidentified biotransformation products was obtained (Table 6). In the worst case, when 1000 mg of diol 1 was incubated with 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 only a total recovery of 53.9% (in general, recovery of material was between 75-85%). A complex mixture , not extracted in the usual procedure, was also obtained in addition to the normal product mixture. It was possible that the initial products formed were further  59 biotransformed to a mixture of unidentified products. Therefore, the optimum conditions for the biotransformation of diols 1 and 2 seemed to be a reaction using 16-19 days old cells with a substrate concentration below 400 mg per litre of cell suspension at pH 6.3 and an incubation time not exceeding 120 h. In summary, the technique of using resuspended cells in TrisHC1 and phosphate buffers does provide the opportunity to achieve regioselective enzymatic attack of the diol 1 to afford products 4, 92, 93 and 94. With 16-19 days old cells in which the enzyme systems were properly developed, relatively efficient biotransformations were achieved. At lower 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. Cell age (days) pH 25  nD  11 18 5.25 1.3330  12 19 5.30 1.3332  13 19 5.30 1.3331  14 18 5.35 1.3332  Substrate 1/Et0H(mg/ ml) Volume of culture (L) Substrate concentration (mg/L) Resuspension buffer: type pH Incubation time (h) Yield (%) 1 4 92 93 94  1010/25 2.5 400 Phosphate 6.3 168  600/30 800/20 1 1 600 800 Phosphate Phosphate 6.3 6.3 216 144  1000/10 1 1000 Phosphate 6.3 240  0 38.7 4.3 9.2 9.4  2.9 28.5 10.5 9.0 8.9  22.5 26.9 3.9 7.0 7.2  18.2 23.2 4.5 3.9 4.0  Total recovery (%)  61.6  59.9  67.5  53.9  60 2.1.1.2^Addition of substrate to the growing cell suspension culture in one batch  In the previous experiments, the "spent medium", which was the nutrient growth medium left after filtration of cells, was just discarded. Based on the considerations that such "spent medium" may contain some "new" enzymes which were generated during cell growth and relevant to biotransformation, and also in order to reduce the tedious procedures for cell resuspension, biotransformation experiments with direct addition of substrate to the growing cells were performed. The procedures for this series of experiments were generally the same as the procedures outlined in Scheme 31 except that the initial steps 1 to 3 were omitted. The diol 1 dissolved in ethanol was added to the growing cell suspension culture (younger cells  with 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 days old) in the PRDCo nutrient medium (Entry 18), only 24% of the starting material was recovered unreacted after only 24 h. It was of interest to note that, in addition to the usual products 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 11 NMR and 13 C NMR data as discussed earlier. Compound 8 has been isolated from tobacco plants and its synthetic equivalent has also been prepared chemically by reaction of diol 1 with singlet oxygen [ 83 }. Comparison of the spectral data of the biotransformation product 8 with the published data indicated their identity. Surprisingly, the results indicated that the reaction proceeded at a faster rate when the substrate was applied directly to the growing cell suspension in the PRDCo medium and a better recovery of materials was also observed. Moreover, the metabolites produced by the cell suspension did not cause any problems for our analysis of the results.  61 2.1.1.2.1^Effects of cell age on biotransformation of diol 1  With the encouraging results from the application of substrate directly to the cell suspension, attention was transferred to an investigation of the effects of different cell ages on biotransformation of diol 1. In a series of experiments, the diol 1 was directly incubated with the growing cells of various ages. When the diol 1 was administrated during the inoculation (0 day) of the cell suspension, no biotransformation was observed and the cells were found to stop growing completely. However, when the diol 1 was applied to cell cultures at 5 days, 11 days and 13 days of growth, not only were the incubation times dramatically reduced to less than 48 h, but also the recovery of material was found to be higher (Table 7). It was revealed that more efficient biotransformation was achieved with younger cells (11-13 days), at a low ratio of substrate to cell suspension (53 mg in 500 ml suspension culture) and in a relatively short incubation time (Entry 17). The overall yield of the 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 the rate of reaction or the yields of the products, indicating that enzyme systems were already present in the young cells. However, using the 5 day old cell culture seemed to be the best turn around time for the whole process. Therefore, this technique provided an attractive alternative in performing the biotransformation of tobacco cembranoids. Not only was the tedious resuspension procedure being avoided, but also the reaction was found to proceed at a faster rate. Clearly, an additional enzyme system responsible for C-12 hydroxylation was produced during the short incubation time with younger cells.  62 Table 7, Effect of cell age on the biotransformation of diol 1 with direct substrate application to the cell suspension. Exp No. Cell age (days) pH (initial) 45 (initial) Substrate 1/EtOH (mg/ ml) Volume of culture (L) Substrate concentration (mg/L) Incubation time (h) Yield (%)^1 8 4 92 93 94  Total recovery (%)  15 0 5.12 1.3370 50/5 0.5 100 98 83.0  -  83.0  16 5 4.90 1.3362 50/5 0.5 100 24 32.4 8.6 27.1 3.5 7.0 7.4 86.0  17 11 5.25 1.3347 53/5 0.5 106 42 13.2 14.9 33.4 4.0 9.8 10.0 85.3  18 13 5.20 1.3331 50/5 0.5 100 24 24.0 10.4 33.4 3.7 7.5 7.5 86.4  19 13 5.25 1.3331 50/5 0.5 100 24 38.0 6.2 25.4 3.0 6.1 6.3 85.0  2.1.1.2.2 Effects of substrate concentration on biotransformation of diol 1  The effects of increasing substrate concentrations were also investigated by applying different levels of substrate directly to the 5 day old cell suspensions. The results are summarized in Table 8. It is revealed that very young cells, in which the enzyme systems are not so well developed, are still capable of achieving biotransformation provided that low concentrations of substrate are involved (Entry 20). The low yields seen in Entry 22 may also relate to the short incubation time and high substrate concentration. Under similar conditions, more starting material was recovered as a result of more substrate application to the cell suspensions. At this stage, 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 was recovered unreacted after 24 h.  63 Table 8, Effects of substrate concentration on biotransforamtion of diol 1 Exp No. Cell age (days) pH (initial) 45 (initial) Substrate 1/EtOH (mg/ ml) Volume of culture (L) Substrate concentration (mg/L) Incubation time (h) Yield (%)^1 8 4 92 93 94  Total recovery (%)  20 5 5.12 1.3370 50/5 0.5 100 24 27.2 7.8 29.5 3.3 7.5 7.7 83.0  21 5 5.09 1.3370 100/5 0.5 200 24 52.3 4.7 15.8 1.8 3.1 3.2 80.9  22 5 5.12 1.3371 200/5 0.5 400 24 70.2 2.1 7.2 1.3 1.4 1.4 83.6  2.1.1.3 Batchwise additions of substrate to growing cell suspension culture  Results obtained from the experiment using 0 day old cells (Entry 15) suggested that the substrate concentration at this level (100 mg/L of culture) could be too high and indeed 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 be diminished. The toxicity problems encountered prompted the investigation of sequential additions of substrate in smaller quantities to the cell suspension with the hope that the cells could endure the toxicity and ultimately handle larger amounts of substrate. Therefore, batchwise addition of substrate was undertaken. In this procedure, the substrate was dissolved in ethanol, divided into several batches and added in batches to the cell suspension over a certain period of time.  64 In a series of experiments performed, the diol 1 was dissolved in ethanol, divided into 5 portions and added twice a day to the cell suspension. After the addition of the last batch, the incubation was allowed to continue for a further 24 h. Unfortunately, the result showed 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 culture 24  25  26  27  Cell age (days) 5 pH (initial) 5.12 25^ nD (initial) 1.3370 Volume of culture (L) 0.5 Substrate 1/EtOH (mg/ml) 10/1 x5a Total 1 added (mg) 50 100 Concentration (mg/L) Total incubation time (h) 120  5 4.92 1.3369 0.5 10/1 x5b 50 100 72  12 5.12 1.3351 0.5 10/1 x5b 50 100 72  12 5.42 1.3351 0.5 50/2 x5b 250 500 72  12 5.81 1.3348 0.5 50/2 x 5b 250 500 72  Yield (%) 1 8 4 92 93 94  10.8 9.6 28.0 3.2 5.8 6.0  14.6 11.6 29.5 3.3 6.6 6.8  17.4 10.8 23.2 2.4 6.0 6.2  30.7 7.5 20.5 2.2 4.6 4.8  38.9 14.3 22.2 2.5 3.5 3.5  Total recovery (%)  63.4  72.4  66.2  70.4  84.9  Exp No.  23  .^• •  a^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 (10am and 4pm).  65 2.1.1.4 Semi-continual addition of substrate via a peristaltic pump  By employing a mechanical peristaltic pump, a series of experiments with controlled semi-continual addition of substrate to the growing cell suspension were performed. The substrate was added to the cell suspension in much smaller batches and in a more frequent manner. 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 pump Exp No.  28  29  30  31  32  33  34  6 Cell age (days) 5.02 pH (initial) 25^ n D (initial) 1.3362 Volume of culture (L) 0.5 Substrate 1/EtOH (mg/m1) 100/12 Rate of addition (ml/hr) 0.5/1 24 Time for addition (h)  6 4.75 1.3361 0.5 100/12 0.5/2 46  12 5.33 1.3345 0.5 100/12 0.5/1 24  6 4.95 1.3359 0.5 250/12 0.5/1 24  6 4.95 1.3362 1.0 100/12 0.5/1 24  12 5.36 1.3349 1.0 100/12 0.5/1 24  2 5.60 1.3368 0.5 100/12 0.5/1 24  Incubation time after last addition (h) Total incubation time (h) Yield: (%)^1  24  2  24  24  24  24  24  48 33.5 18.6 27.4 3.0 5.6 5.7 93.8  48 46.5 9.8 19.2 2.1 5.7 5.9 89.2  48 35.2 5.8 23.6 7.0 3.9 8.6 84.1  48 16.1 2.1 34.4 1.3 5.8 2.1 61.8  48 22.8 4.3 33.1 1.1 8.3 1.6 71.2  48 26.7 5.2 18.3 3.1 6.5 3.6 63.4  48 13.8 2.0 20.8 0 2.8 0 39.4  .^• •  8 4 92 93 94  Total recovery (%)  In one particular experiment (Entry 28), diol 1 (100mg) was added to the cell suspension culture (0.5 L) via a peristaltic pump over 24 h and the incubation was allowed to continue for a further 24 h after the last addition. The unreacted starting material was isolated  66 in 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 to be extremely good in this case (93.8%). It was revealed that the young cells appear to generate large amounts of the C-12 "hydroxylase" enzyme since the ratio of C-12 alcohol 8 to epoxide  4 was higher (Entry 28 and 29). Very young cells (2 days old, Entry 34), even though incapable of producing any significant amounts of allylic alcohols, were still capable of producing epoxide 4 and clearly it already possessed the enzyme system for epoxidation. In summary, this technique allowed higher substrate to suspension culture ratios although the overall benefits in terms of biotransformation yields were marginal.  2.1.1.5 Analytical method for evaluating cembranoid biotransformation The above results obtained from the various biotransformation experiments relied on the actual isolated yields of recovered starting material and products as determined by flash chromatography on silica gel. Since the structures of products and their behaviours towards the absorbent are very similar, considerable difficulties were encountered in resolving these mixtures. In order to analyse the biotransformation results more effectively and quickly, an analytical method employing GC was developed in our laboratory. In this analysis, the underivatized products obtained from biotransformation of cembranoids were used directly even though there were reports that underivatized cembranoids were not stable at the high temperature employed with GC column and derivatizations were necessary. [ 14 ] These underivatized compounds did not seem to undergo decomposition under our experimental conditions and the retention times were reproducible. Obviously, this analytical system was superior to the reported method based on derivatization in which bis(trimethylsily1)trifluoroacetamide (BSTFA) 14 By careful selection of methyl docosanoate as internal [  ].  standard and calibration of the GC detector responses, quantifications of the underivatized biotransformation products were carried out. Table 11 listed the retention times and GC conditions.  67  Table 11, GC conditions and retention times Compound^Retention Time (min) 1^6.1 Methyl docosanoate^8.7 11.3 4 12.0 8 92 12.6 93 13.3 94 14.4  GC conditions Column:^DB-1701(15m x 0.262 mm) Carrier Gas:^Helium (0.68 ml/min) 250°C Injection temperature: 220°C Oven temperature: 300°C Detector temperature: FID Detector: Concentration of internal standard: 0.25 mg/ml  With the development of the analytical GC method on cembranoids in hand, the analysis of the biotransformation results was performed more efficiently. Entry 35 in Table 12 shows the results obtained at different incubation times from an experiment where the diol 1 was added to the suspension culture at a much slower rate (0.5 ml/3 h) via a peristaltic pump and incubated for a longer time (168 h of total incubation time). Table 12, GC results of biotransformation of diol 1 Exp No. Cell age (days) pH (initial) nD25 (initi al) Volume of culture (L) Substrate 1/Et0H (mg/ ml) Rate of addition (nil/ hr) Incubation time (h) GC Yield: (%)^1 8 4 92 93 94 Total recovery (%)  72 61.9 0 19.1 1.1 4.3 + 86.4  96 48.1 5.7 24.2 4.7 6.3 + 89.0  35 18 6.30 1.3339 0.5 500/12 0.5/3 120 144 30.7 15.7 9.0 6.1 33.0 39.4 6.8 5.0 9.0 11.3 4.2 1.1 84.9 86.4  168 8.9 9.2 42.7 7.0 13.1 4.5 85.4  36 17 5.65 1.3332 0.1 15/3 168 76.6 1.1 2.7 1.7 5.4 87.5  68 In order to destroy the enzyme system, the plant cell culture was autoclaved at 120 0C for 15 minutes and thus used in biotransformation experiment, almost no biotransformation was observed and the starting material was recovered in good yield (Entry 36). Therefore, it was clear that the described biotransformations were being achived by enzymatic processes.  2.1.1.6 Conclusion The above results indicated that diols 1 and 2, two major tobacco cembranoids, can be biotransformed efficiently into a series of products, mainly epoxide 4 and epoxide 95 along with 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 allylic alcohol. These two distinctive groups of products could be derived from different reaction pathways catalyzed by different enzyme systems. Among the products isolated from the biotransformation experiments, compounds 92 and 93 are assigned as a pair of diastereomers with opposite stereochemistry at C-10, the newly created chiral centre, based on 1 H NMR and 13 C NMR studies. Since attempts to obtain the required crystals of both 92 and 93 failed, X-ray analyses of these compounds are impossible at present. Attempts at obtaining derivatives of 92 and 93 may be considered in the near future in 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, marked differences in reactivities towards the cell derived enzymes were observed. The a-diol 1 reacted with 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 substrate plays an important role in the rate of cembranoid biotransforamtion. Reactions involving resuspension in phosphate buffer at pH 6.3 were found to proceed much faster than those involving TrisHCl buffer at pH 7.5 and 9. When the substrate concentration was increased to and/or above 600 mg per litre of cell suspensions at pH 6.3, a significant portion of starting material remained unreacted after 120 h  69 and extended incubation time resulted in poor overall recovery of material. The optimum conditions for carrying out the biotransformation of cembranoids 1 and 2 seemed to be a reaction with a substrate concentration below 400 mg per litre of cell suspension at pH 6.3 and with an incubation time not exceeding 120 h. Application of substrate directly to the growing cell suspension culture provided an attractive alternative in performing cembranoid biotransformation, not only was the tedious resuspension procedure being eliminated, but also the reaction was found to proceed much faster. No significant differences in terms of rate of substrate consumption and product yields were observed when cultures of different ages (between 5-13 days) were used. The optimum level 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 the cells leading to cell injury or indeed cell death thereby reducing the production of the required enzymes for the biotransformation. The experiments with batchwise addition of substrate in smaller quantities or semi-continual addition of substrate via a peristaltic pump in even much smaller batches and in a more frequent manner over a longer period of time showed that higher substrate concentration was allowed (500 mg/L) and total recovery was good (as high as 93.8%) although the overall benefits in terms of biotransformation yields were marginal. The above studies have provided evidence that the TRP4a cell line, originally propagated for the production of di- and triterpenes as part of a study relating to Chinese herbal medicine [78, 801, can tolerate the cembranoid diols 1 and 2 as "foreign" substrates which are unrelated in structure to those normally produced by this T. wilfordii cell line. It is clear that enzymatic functionalization of the 11,12 double bond is predominant to afford the corresponding epoxides 4 and 95 as major products. Other enzyme-catalyzed hydroxylations at 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, some differences.  70 2.1.2 Chemical studies on the cembranoids: preparation of cembranoid analogues In order to pursue one of our objectives of degrading cembranoid diols 1 or 2 into the low molecular weight compounds responsible for the tobacco aroma and facilitate structure elucidation of the biotransformation products in our studies, several cembranoid analogues were prepared from diol 1 by chemical methods. These analogues were either subjected to further biotransformation studies or used as standard compounds for comparison purposes. 2.1.2.1 Conversion of diol 1 to norsolandione 78 and methyl ester 103 The diol 1 was converted to norsolanadione 78, a target molecule in our biotransformation studies, and the methyl ester 103 prepared according to the published procedures (Scheme 32) [ 34, 35 ] with slight modifications. Treatment of diol 1 with acetic anhydride and triethylamine in dichloromethane at room temperature gave monoacetate 97 in 95% yield. Oxidation of this acetate 97 with osmium tetroxide in pyridine afforded the triol 98 in 86% yield. Cleavage of 98 with lead tetraacetate in benzene gave the secoaldehyde 99 in 83% yield. Oxidation of 99 with Jones' reagent in acetone afforded the corresponding acid which was derivatized as the methyl ester 100 by reaction with ethereal diazomethane in methanol in 80% yield. Subsequent hydrolysis of acetate 100 with potassium carbonate in methanol gave diol 101 in 86% yield. Oxidation of 101 with pyridinium chlorochromate (PCC) in dichloromethane followed by retro-aldol fragmentation 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 by GC and 1 H NMR. This mixture was inseparable by conventional chromatography and appeared as one spot on TLC under several solvent systems. The 1 H NMR spectrum of 103 showed that there were two singlets at 8 6.03 and 6.05 ppm due to the olefmic proton,  71  Ac20^  0s04 ^ Pyridine Et N^ CI Cl 2^86% 95% H.  98  97  1  Pb(OAc) 4 Benzene 83%  1, Jones' 2, CH 2 N 2 80% 100 99 PCC CH 2 C1 2  +  Me0 'O 103  78  Scheme 32, Chemical conversion of diol 1 to norsolanadione 78 two singlets at 8 1.89 and 2.13 ppm attributed to the vinyl methyl (H-8) and two almost superimposed 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 reaction  72  0s04 Pyridine 76% 104  1  Pb(OAc) 4 Benzene 86%  HQ OH :^• PCC^ CH 2 C1 2 57%  >< l  K 2 CO 3 Me0H 52%  Scheme 33, Alternative route to norsolanadione 78 with osmium tetroxide in pyridine for 5 h. Cleavage of this glycol 104 with lead tetraacetate in benzene at room temperature gave the seco-aldehyde 32 in 86% yield. Oxidation of this allylic alcohol 32 with pyridinium chlorochromate in dichloromethane at room temperature afforded the enone 106 in 57% yield. Upon reaction with potassium carbonate in methanol, it underwent a retro-aldol fragmentation to give the norsolanadione 78 in 52% yield. This compound was well characterized and used in our biotransformation studies as a standard compound for GC calibration.  73 2.1.2.2 Conversion of diol 1 to seco-diketone 44 In order to cleave the 4,5 bond, diol 1 was oxidized to enone 43 in 81% yield using PCC in dichloromethane. The retro-aldol reaction of enone 43 with potassium carbonate in methanol afforded seco-diketone 44 in 85% yield (Scheme 34).  1^  43  ^  44  Scheme 34, Conversion of diol 1 to seco-diketone 44 2.1.2.3 Conversion of epoxide 4 to 7 and 107 Epoxide 4, which was the major product obtained in the previous biotransformation experiments using the TRP4a cell line, was subjected to similar chemical conversion as diol 1. It was first converted to enone 7 using PCC in dichloromethane in 69% yield and then to seco-epoxide 107 in 84% yield under basic condition via retro-aldol reaction (Scheme 35). PCC CH 2 C1 2 69%  4  ^  K2CO3 Me0H 84%  7  ^  Scheme 35, Conversion of epoxide 4 to 7 and 107  107  74 2.1.2.4^Conversion of seco-aldehyde 32 to triol 110  The seco-aldehyde 32 was reduced to the corresponding triol 110, in 69.5% yield with sodium borohydride in methanol (Scheme 36).  NaBH4 CH 3 0 H 69.5%  32  ^  110  Scheme 36, Conversion of seco-aldehyde 32 to triol 110  Among the compounds obtained in these chemical studies, 7, 78, 103, 107 and 110 have been used as reference samples in our investigation for the biodegradation of cembranoids and 32, 43, 44 and 104 were subjected to further biotransformation studies later on. 2.1.3 Biotransformation of diol 1 using cell free extract (CFE) obtained from the TRP4a cell line Although the biotransformation results with the TRP4a cell line showed that the whole cells were capable of transforming diols 1 and 2 into corresponding epoxides and allylic alcohols, the locations of the enzyme systems within the cells and responsible for such 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 did not contain enzymes capable of such transformation, or the enzymes within the cells were  75 inaccessible, perhaps membrane bound in a manner that the substrate could not reach the active site. For this reason, CFE techniques were considered since obviously the available enzymes were now released from the cells and into appropriate buffer systems. 2.1.3.1 Preparation of CFE  The general procedures for CFE preparation and biotransformation are outlined in Scheme 37. Steps 1 to 4 were performed at 0-4°C to avoid the protein or enzyme denaturation. The homogenization was performed with an IKA Ultra-Turrax Disperser T25 fitted with an S25N-25F rotor/stator at 24,000 rpm for 30 seconds and the same procedure was repeated 3 times. In order to avoid overheating, a 1 minute break was allowed between each operation. The homogenate was then subjected to centrifugation at 10,000 g (8,000 rpm) for 30 minutes in an automatic refrigerated centrifuge. The clear supernatant was collected as crude CFE. In order to ascertain the quality of the CFE, both soluble protein concentration and peroxidase activity of the crude CFE were determined [86, 87] The soluble protein concentration was assayed by the Bio-Rad procedure and expressed as mg/ml. In this assay, 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 protein concentration of the CFE can be calculated from the standard curve which was obtained by dissolving known amounts of bovine serum albumin (BSA) powder in the same buffer to produce a set of standard solutions and measuring their absorbances at 595 nm, with the assumption that the extinction coefficient of the dye-CFE protein was identical to that of dye-BSA protein complex. The peroxidase activity of CFE was assayed by the pyrogallol-purpurogallin method and expressed as units/ml using the definition that one unit of peroxidase will form  76 Cell suspension culture 1)Filtration 2) Wash with distilled water 3) Homogenization in buffer (pH 6.6) 4) Centrifugation  Cell pellet^Supernatant ( CFE ) 1)Substrate administration with cofactor (H202 ) 2) Incubation Work-up Filtration through Celite  Filtrate  Celite pad 1) Sonication in EtOAc 2) Filtration I Cell material  EtOAc extraction  EtOAc extracts  EtOAc extracts I^  1) wash with water, brine 2) dry over Na2 SO4 3) Concentration in vacuo 4) Chromatographic separation  Products Scheme 37, Preparation of CFE and biotransformation  77  1.0 mg of purpurogallin from pyrogallol in 20 seconds and 20 0 C. The standard curve was obtained by dissolving known amounts of purpurogallin in ether to produce a set of standard solutions and measuring their absorbances at 420 nm.  In the following biotransformation studies, the CFE was used in the amount of 25 units 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 from theTRP4a cell line  In order to evaluate the effects of cell ages on cembranoid biotransformation, CFEs were prepared from the cell culture of different ages grown in PRDCo medium and biotransformation experiments using such CFEs were performed. Unfortunately, all the CFEs prepared from 2-18 day old cultures failed to convert the diol 1 into any expected products after 3 h of incubation even in the presence of hydrogen peroxide (2.16 equiv.) as a cofactor. An overall recovery of 85-95% of diol 1 was observed with a negligible amount of epoxide 4 as determined by GC analysis (Table 13).  The inability of CFE to transform the diol 1 into any products is in sharp contrast with the previous results obtained from the whole cell experiments. Presumably, the procedures for CFE preparation could have destroyed the activities of the enzyme systems required for the expected biotransformation or the CFE does not contain the relevant enzymes and cofactors which are necessary for the biotransformation of cembranoids. The enzymes could have remained in the pellet after centrifugation.  78 Table 13, Biotransformation of diol lwith CFE prepared from TRP4a grown in PRDCo medium Expt. No. Age (days) n D25 pH Volume of culture (L)  37^38^39^40^41^42^43^44 2^4^7^9^11^14^16^18 1.3370 1.3368 1.3361 1.3353 1.3347 1.3332 1.3330 1.3331 5.55^5.10^4.85^5.35^5.60^5.50^5.50^5.60 1.5^1.0^0.5^0.5^0.5^0.5^0.5^0.5  ^ ^ ^ ^ ^ ^ ^ CFE (m1)^92 73 82 86 93 86 96 230 ^ ^ ^ ^ ^ ^ ^ peroxidase activity (unit/ml) 3.43 3.53 4.15 3.83 5.51 5.17 5.90 3.94 ^ ^ ^ ^ ^ ^ ^ protein concentration (mg/ml)0.98 1.58 3.48 1.21 1.26 0.72 0.90 0.58 ^ ^ ^ ^ pH^6.50 6.50 6.80 6.50 6.55 6.55 6.60 6.60 Incubation conditions: Substrate 1/EtOH (mg/ml) Volume of CFE (ml) Buffer added (ml) Distilled water(ml) 0.24% H202(m1) Incubation time (h) 0.5 1.5 3.0  5/1 4.5 15 7 1.0 99.0 80.1 78.1  ^ ^ 10/2 10/2 10/2 ^ ^ 7.0 6.0 6.5 ^ ^ 35 35 35 ^ ^ 15 15 15 ^ ^ 1.0 1.0 1.0 ^recovered substrate 86.0^89.5^84.3 91.2^92.5^91.9 82.7^87.4^92.9  10/2^10/2^50/5 50/5 4.5^4.8^21.0^32.0 35^35^175^175 15^15^75^75 1.0^1.0^5.0^5.0 1 (%,by GC) ^ 95.8^93.0^89.6^90.2 95.3^90.3^93.2^91.7 93.8^92.2^92.1^92.0  2.1.4 Biotransformation of diol 1 using the cell homogenate and the pellet prepared from the TRP4a cell line 2.1.4.1 Preparation of cell homogenate and resuspended pellet  During the CFE preparation, the pellet obtained after centrifugation was discarded previously. In order to examine the possibility that some enzymes still remain in the pellet after centrifugation, a series of experiments were performed with the cell homogenate, which was the total "enzyme mixture" obtained by homogenizing the cells in the phosphate buffer, and with resuspended pellet, that is, with pellet obtained after centrifugation and resuspension in phosphate buffer, as shown in Scheme 38.  79 Cell culture 1) Filtration 2) Wash with distilled water 3) Homogenization in buffer (pH 6.6) (24,000rpm, 30sec.x3) Cell homogenate  1) Substrate administration with cofactors (H2 0 2 , FMN, MnC1 2 ) 2) Incubation  Centrifugation (10,000g, 30min)  Cell pellet Work-up as before  ^  Addition of buffer Resuspended pellet 1) Substrate administration with cofactors (H 20 2 , FMN, MnC1 2) 2) Incubation  Supernatant ( CFE ) 1) Substrate administration with cofactors (H20 2 , FMN, MnC1 2 ) 2) Incubation  Work-up as before  Work-up as before Scheme 38, Preparation of the cell homogenate and resuspended pellet and biotransformation The cells were harvested and homogenised as before in the phosphate buffer (0.1 M, pH 6.6). The homogenate was then divided into two portions. One portion was used directly in the biotransformation experiments. The remaining portion was then centrifuged at 10,000 g (8,000 rpm) for 30 minutes as in the preparation of CFE. The supernatant was  80  collected as CFE for biotransformation experiments. The pellets were resuspended in the same buffer and used directly in biotransformation experiments. The peroxidase activity and protein concentration were measured as before for the homogenate, CFE and the resuspended pellet, respectively. Cofactors were also added and based on earlier studies in our laboratory in which peroxidase enzymes were isolated from Catharanthus roseus cell cultures, manganous chloride and flavin mononucleotide (FMN) were chosen for this purpose. 2.1.4.2 Biotransformation of diol 1 using cell homogenate and resuspended pellet A series of simutaneous biotransformation experiments with diol 1 were then  performed using CFE, cell homogenate and resuspended pellet (Table 14). It was found that the pellet obtained from the 18 day old TRP4a culture with the supplement of hydrogen peroxide, FMN and manganous chloride appeared to be an optimum condition for the formation of the epoxide 4 from diol 1. Thus, when diol 1 (50 mg in 10m1 ethanol) was added 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 chloride and 0.5 equivalents of FMN and incubated at room temperature for 48 h, the epoxide 4 was 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 be comparable with those obtained from the whole cell studies, indicating that the enzymes were indeed present in the pellet portion of the homogenate. Comparing these results with those obtained in the biotransformation of diol 1 with resuspended pellet in which hydrogen peroxide is present as sole cofactor (Table 15), it was clear that without manganous chloride and FMN, the yields of epoxide 4 were low, implying that these cofactors were necessary for the conversion of diol 1 to epoxide 4.  81  Table 14, Biotransformation of diol 1 with TRP4a cell homogenate, pellet and supernatant Expt. No. Cell age (days) n D25 pH Vol. of culture (L) Fresh weight (g) Buffer added (ml)  45 7 1.3335 5.15 2.0 249 200  46 12 1.3332 6.09 2.0 503 480  47 14 1.3332 5.70 3.0 964 732  48 18 1.3342 6.30 2.0 416 480  47H 1670 3.86 0.80 6.50  48H 880 4.50 1.19 6.00  Homogenate  Expt. No. Volume (ml) peroxidase activity (unit/nil) protein concentration (mg/m1) pH  45H^46H 400 950 5.28 4.33 2.12 1.00 6.55 6.55  Incubation conditions  Substrate 1 /Et0H (mg/m1) Homogenate used (m1) Buffer added (pH6.6, ml) Distilled water (ml) Cofactors^H202 (eq) FMN (eq) MnC12 (eq)  50/10 23.7 175 75 2.16 0.5 0.5  50/10 29.0 175 75 2.16 0.5 0.5  50/10 32.4 175 75 2.16 0.5 0.5  50/10 28.0 175 75 2.16 0.5 0.5  Incubation time (h)  24  24  24  24  80.0 6.2  78.5 14.2  80.2 0.9  79.8 11.9  Yield (%)  1 4  82 Table 14, Biotransformation of diol 1 with TRP4a cell homogenate, pellet and supernatant (continued) Supernatant (CFE) 48S 47S 46S 45S Expt. No. 4.05 4.72 4.72 4.95 peroxidase activity (unit/m1) 1.23 0.64 0.67 1.69 protein concentration (mg/ml) 6.55 6.45 6.53 pH 6.45 Incubation conditions 50/10 50/10 50/10 50/10 Substrate 1/EtOH (mg/ml) 31.0 26.5 26.5 25.3 Supernatant used (ml) 175 175 175 175 Buffer added (pH6.6, ml) 75 75 75 75 Distilled water (ml) 2.16 2.16 2.16 2.16 Cofactors^H202 (eq) FMN (eq) MnC12 (eq) Incubation time (h) Yield (%)^1 4 Expt. No. peroxidase activity (unit/In]) protein concentration (mg/ml) pH Incubation conditions Substrate 1/Et0H (mg/ml) Resuspension used (ml) Buffer added (pH6.6, ml) Distilled water (ml) Cofactors^H202 (eq)  0.5 0.5  0.5 0.5  0.5 0.5  0.5 0.5  24 81.6 6.5  24 87.2 4.3  24 68.8 6.9  24 84.8 7.7  Resuspended pellet 45R^46R 2.40 1.80 0.51 1.07 6.00 6.50  47R 2.50 0.80 6.55  48R 2.60 0.83 6.00  50/10 25.0 175 75 2.16  50/10 90 175 75 2.16  50/10 100.0 175 75 2.16  50/10 50.0 175 75 2.16  FMN (eq)  0.5  0.5  0.5  0.5  MnC12 (eq)  0.5  0.5  0.5  0.5  Incubation time (h)  48  48  48  48  Yield (%)^1  66.5  75.4  71.6  38.2  4  19.7  12.2  +  40.1  83 Table 15, The effects of manganous chloride and FMN on biotransformation of diol 1 with resuspended pellet Expt. No. Cell age (day) Fresh weight of pellet (g) Buffer added (ml) Vol. of resuspension (ml) peroxidase activity (unit/ml) protein concentration (mg/ml) pH Incubation conditions: Substrate 1/EtOH (mg/ml) Resuspended pellet used (ml) Buffer added (pH6.6, ml) Distilled water (ml) Cofactors^H202 (eq) Incubation time (h) Yield (%): 1 4  49 7 38 100 134 1.80 1.07 6.50  50 12 301 250 415 2.40 0.51 6.00  51 18 84 200 266 2.60 0.83 6.00  50/10 25.0 175 75 2.16 48  50/10 90 175 75 2.16 48  50/10 50.0 175 75 2.16 48  77.3 11.7  93.9 1.9  81.9 12.4  2.1.4.3 Conclusion  Even though cell homogenate and CFE prepared from the TRP4a cell line gave very low yields of biotransformation products, the pellet resuspended in phosphate buffer (pH 6.6) afforded reasonable conversion of diol 1 to epoxide 4 when such cofactors as hydrogen peroxide, manganous chloride and FMN were added. The results indicated that the 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 the enzymes responsible for such biotransformation processes.  84 2.2 Biotransformation of cembranoids using the T-43-T cell line 2.2.1 Biotransformation of diol 1 using the T-43-T whole cells  Because none of the three enzyme systems, ie, whole cells, CFE and resuspended pellet of TRP4a, can convert diols 1 or 2 into any ring cleavage products, the tobacco cell line T-43-T was then initiated from the seed of Nicotiana sylvestris, a species in which diols 1 and 2, and many other cembranoids are encountered. This cell line was used in our biotransformation studies with the hope that it might be more effective in biotransformation of the tobacco cembranoids than TRP4a . Initial biotransformation studies with the T-43-T cell line were performed with whole cells harvested from culture reaching stationary phase and resuspended in phosphate buffer (pH 6.3) containing sucrose (8%) as those described in the experiments using TRP4a. However, this method was deemed inappropriate because of the rapid deterioration of the cell suspension. It was observed that the color of precursor-treated cells changed from green to dark brown within 20 minutes and the Evan's Blue stain test showed no viable cells [88]. Subsequent biotransformations were then performed by adding the substrate into the growing cell cultures directly as in the case of TRP4a. Due to the toxicity of substrate to plant cells, both batch-wise addition and semi-continual addition methods were employed again in order to reduce such toxic shock to cells. The procedures were the same as those used 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 with varying substrate addition rates and incubation times. Based on recovery of substrate and overall yield of biotransformation products, longer incubation times were preferable. It was interesting to note that epoxide 4, which was the major product in experiments using TRP4a growing cells, was a minor component in this study. It was possible that the enzymes within the cells of T-43-T cell line performing hydroxylation at C-10 and C-12 to  85 Table 16, Biotransformation of diol 1 with batch-wise addition of substrate to T-43-T cell suspension Expt No.  52  53  54  55  56  Cell age (days) n D25 pH Volume of culture (L) Substrate 1 (mg) Incubation time (h) Yield (%)  12 1.3358 5.31 0.5 200a 60  12 1.3355 5.35 0.5 90b 60  12 1.3355 5.35 0.5 90b 72  12 1.3355 5.35 0.5 90c 126  12 1.3356 5.25 0.5 90d 158  62.5 3.8 2.9 0 3.6  37.3 14.9 12.6 5.6 12.3  41.6 6.0 12.8 1.4 7.2  30.8 14.2 19.8 0 21.4  8.9 19.8 12.4 6.9 29.3  1 8 4  92 93  a The substrate in 25ml of EtOH was divided into 5 batches and added to suspension culture 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 to suspension culture in 77 h. d The substrate in 12m1 of Me0H was divided into eight 11.25mg batches and added to suspension 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 the biotransformations observed. Several experiments were performed in which an alcoholic solution of diol 1 was added slowly in a controlled semi-continual manner by means of a peristaltic pump. It was felt that this approach may provide a more desirable biotransformation particularly if substrate toxicity was a problem. However, the results were not sufficiently encouraging to justify further study with this technique.  86 Table 17, Biotransformation of diol 1 with semi-continual addition of substrate to T-43-T cell suspension via peristaltic pump Expt. No.  57  58  59  Cell age (days)  14 1.3352 5.68 350 35/12 0.5 48  11 1.3360 5.50 350 35/6 0.25 48  12 1.3355 5.30 500 90/12 0.5 48  54.7 1.7 0 0 0 2.9  49.0 1.8 2.7 0 0 0.8  59.3 10.0 15.8 3.0 4.8 0  n D25  pH Volume of culture (ml) Substrate 1/EtOH(mg/ml) Addition rate(ml/hr) Incubation time (h) Yield (%) 1 8 4 92 93 94  2.2.2^Biotransformation of diol 1 using CFE prepared from the T-43-T cell line  Because still no expected ring cleavage products were obtained in the whole cell experiments 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 were prepared from the T-43-T cell cultures of various ages (2-11 days old) and subjected to similar biotransformation conditions. Again, no biotransformation was observed when hydrogen peroxide was used as cofactor and only the starting material was recovered in 9096% yields (Table 18).  87 Table 18, Biotransformation of diol 1 with CFE prepared from T-43-T cell line Expt. No. Cell age (days) n D25 pH Volume of culture (L)  60 2 1.3373 5.42 1.0  61 7 1.3366 5.51 1.0  62 11 1.3357 5.39 1.0  Fresh weight (g) Buffer added(ml) CFE^Volume(m1) Protein concentration (mg/m1) Peroxidase activity (units/m1) pH  47 90 117 0.63 3.16 6.55  102 85 160 1.10 4.00 6.53  63 95 148 0.88 4.16 6.55  Substrate 1/EtOH(mg/ml) Phosphate buffer(pH6.6, ml) Distilled water(ml) 0.24% H202 (eq) CFE (m1) Incubation time(h)  10/2 35 15 2.16 8.0 24  10/2 35 15 2.16 6.3 24  20/4 70 30 2.16 6.0 24  Recovered substrate 1 (%)  95.0  95.7  90.7  Incubation^conditions  2.2.3 Biotransformation of diol 1 using cell homogenate and resuspended pellet prepared from the T-43-T cell line  Based on the same consideration as in the case of TRP4a, similar experiments were performed using CFE, the cell homogenate and resuspended pellet prepared from the tobacco T-43-T cell line. The results indicated that the epoxide 4 was formed more efficiently by the reaction of diol 1 with the homogenate and resuspended pellet containing  88 cofactors (hydrogen peroxide, FMN and manganous chloride). Thus, when diol 1 was subjected to incubation with the homogenate and resuspended pellet (prepared from the 12 day old cell culture) containing 0.5 equivalents of FMN, 0.5 equivalents of manganous chloride and 2.16 equivalents of hydrogen peroxide for 48 h, the epoxide 4 was obtained in 57.7% and 61.8%, respectively (Table 19). From the results in Table 19, it is clear that centrifugation of cell homogenate at 10,000g to afford CFE eliminates the enzymes relevant to biotransformation and lowers the biotransformation yields, but the enzymes are indeed present in the pellets left after centrifugation of the cell homogenate and favorable biotransformation yields are obtained in experiments using cell homogenate and resuspended pellet. As indicated in Table 19, the results were dramatically different from those obtained with whole cells. The epoxide 4, which was obtained only in low yields in the whole cell experiments, was the major product with yields of 57.7% and 61.8% for the experiments using cell homogenate and resuspended pellet. In a similar experiment using cell homogenate (Entry 64, Table 20), the incubation time was extended to 120 h and the result indicated that the biotransformation yield was higher. The epoxide 4 was obtained in 70.6% yield, that is 75% based on the starting material 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 cell homogenate, but the enzymes for hydroxylation were probably destroyed during the homogenization procedure. Also, it was clear that, although the toxicity of diol 1, in terms of inhibition of cell growth, was a problem at higher substrate concentration in whole cell experiments (Entry 52, Table 16), the enzymes once liberated from the cells, were able to tolerate reasonably high concentrations of diol 1 in the cell homogenate experiments and very respectable biotransformation yields (72%) could be achieved (Entry 65, Table 20).  89  Table 19, Biotransformation of diol 1 with cell homogenate and resuspended pellet prepared from T-43-T cell line Expt No. Cell age (days) n D25 pH Volume of culture (L) Fresh weight (g) Buffer added (ml)  63 12 1.3354 5.47 1.0 158 150  Homogenate Protein concentration (mg/m1) 1.90 1.77 Peroxidase activity (units/in') pH 6.30 Incubation^conditions: Substrate 1/EtOH (mg/ml) Phosphate buffer (ml) Distilled water (ml) T-43-T Volume (ml) Cofactors 0.24% H202 (eq) FMN (eq) MnC12 (eq) Incubation time (h) Yield (%)^1 4  Supernatant 0.96 4.11 6.35  Resuspended pellet 1.68 2.92 6.55  50/10 175 75 80  50/10 175 75 80  50/10 175 75 60  2.16 0.5 0.5 48 30.1 57.7  2.16 0.5 0.5 48 70.1 18.4  2.16 0.5 0.5 48 28.2 61.8  When the CFE was used with addition of the cofactors (FMN and manganous chloride 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 manganous chloride) did show some effects on the biotransformation of diol 1 to epoxide 4.  90 Table 20, Biotransformation of diol 1 with cell homogenate at a longer incubation time Expt No. Cell age (days) nD 25  pH Volume of culture (L) Homogenate: Protein concentration (mg/m1) Peroxidase activity (units/m1) pH  64 14 1.3345 5.67 0.5  65 14 1.3352 5.62 2.5  1.70 4.39 6.45  1.82 3.10 6.40  50/10 175 75 100 2.16  500/100 1750 750 850 2.16  0.5 0.5  0.5  Incubation^conditions  Substrate 1 /Et0H (mg/ml) Phosphate buffer (0.1 M, pH6.6, ml) Distilled water (ml) Homogenate (ml) Cofactors:^0.24% H202 (eq) FMN (eq) MnC12 (eq)  Incubation time(h) Yield (%) 1  120  0.5 120  5.3  6.0  4 93 94  70.6 0.8 8.5  72.2 + 4.8  In control experiments, it was found that if the cell homogenate was autoclaved at 1200 C for 15 minutes before use in order to deactivate the enzymes, only 9.7% of epoxide 4 was obtained when the above conditions were employed. Therefore the results observed  in 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 (Table 21). Therefore, the cofactors were necessary for such biotransformation.  91 Table 21, Biotransformation of diol 1 with T-43-T cell homogenate without cofactors or with autoclaved cell homogenate. Expt No.^ 66 Cell age (days)^ 15 25 n D^ 1.3347 pH^ 5.95 Volume of culture (L)^ 0.5 Fresh weight (g)^ 129 Buffer added (ml)^ 90 Homogenate: Protein concentration (mg/ml)^1.62 Peroxidase activity (units/m1) ^3.30 pH^ 6.50 Incubation conditions:  Substrate 1/Et0H(mg/m1) Phosphate buffer (0.1 M, pH6.6, ml) Distilled water(ml) Homogenate (m1) Cofactors:^0.24% H202 (eq) FMN (eq) MnCl2 (eq) Incubation time(h) Yield (%, GC)^1 8 4 92 93 94  5/1 17.5 7.5 10 (autoclaved) 2.16 0.5 0.5 48 74.6 0  9.7 1.0  1.0 1.1  5/1 17.5 7.5 10  48 85.5 1.9 5.3 1.0 1.8 1.0  2.2.4^Conclusion  The results obtained from the biotransformation using the T-43-T cell line show that this cell line behaves differently from the TRP4a cell line in some ways. In the case of  92  TRP4a, both whole cells and resuspended pellet give good conversion of diol 1 to epoxide 4 and some allylic alcohols, but cell homogenate and CFE gave almost no  biotransformation. In the case of T-43-T, the biotransformation yields using whole cells are much lower. Epoxide 4 is obtained in relatively low yield compared with its yield in TRP4a experiments and considerable amounts of the C-10 alcohol 93 and the C-12 alcohol 8 are obtained. Cell homogenate and resuspended pellet supplemented with such cofactors as hydrogen peroxide, FMN and manganous chloride give best transformation from diol 1 to epoxide 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 and plant cell culture derived enzymes of the T-43-T cell line. 2.3 Biotransformation of cembranoid analogues using the T 43 T cell line -  -  In the previous biotransformation experiments, no expected ring cleavage products were obtained, indicating that diols 1 and 2 were not suitable substrates for such enzymatic processes. Therefore, several cembranoid analogues obtained by chemical conversion from diol 1 were subjected to biotransformation studies. 2.3.1 Biotransformtion of the epoxide 4 using the T 43 T whole cells -  -  Although the TRP4a cell line was capable of converting the diol 1 into a series of oxidation products (Scheme 27), it was ineffective in transforming the epoxide 4 into other products. When the epoxide 4 was incubated with a TRP4a cell suspension, direct TLC analysis failed to reveal the presence of any new products and starting epoxide 4 was recovered 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 4  93 by an enzymatic process and the epoxide 4 was not a suitable substrate for further biotransformation. On the other hand, the whole cells of T-43-T cell line converted the epoxide 4 into a new product , triol 109, in —55% yield after 16 days of incubation time with recovery of —10% of epoxide 4 (Table 22).  16  OH 17  4  ^  109  Scheme 39, Biotransformation of epoxide 4 to 109 Table 22, Biotransformation of epoxide 4 with T-43-T whole cells Expt No. Cell age (days) n D25 pH Vol. of culture (L) Substrate 4/EtOH (mg/ml) Incubation time (days) Yield (%) 4 109  67 8 1.3360 5.30 0.5 47/5 14  68 10 1.3359 5.30 1.0 100/10 12  69 10 1.3360 5.28 1.5 150/15 16  21 47  12 54  13.3 52.7  Low resolution EI-MS spectrum of the triol 109 showed that the base peak was at m/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-MS spectrum gives its ions at m/z 303(M++H-2H20), 321(M++H-H20),337(M+-H) and 356(M++H+NH3). Therefore, the molecular ion was assigned to m/z 338. In accord with  94 this assignment, a characteristic peak at m/z 279 was found. This ion was due to loss of 59 mass units (C3H70), a process similar to loss of 43 mass units from the isopropyl side chain at C-1 of a typical cembranoid system, thereby indicating that the newly introduced hydroxyl function was attached to the original isopropyl unit at C-15. Placement of the hydroxyl function at the tertiary C-15 position was readily revealed from its 1 H NMR spectrum in which the signals due to H-16 and H-17 became two singlets at 8 1.18 and 1.11 ppm. These methyl protons were seen as doublets at 8 0.80 and 0.85 ppm in the epoxide 4. In its 13 C NMR spectrum, an additional signal for a tertiary carbon possessing a hydroxyl group can be seen at 8 72.2. A comparison of 13 C data with epoxide 4 is provided in Table 23. All of these results supported that a new hydroxyl group had been incorporated into the molecule at the non-activated methine position of the isopropyl side chain, ie, the C-15 position. Therefore, this product was assigned as a 15-hydroxylated epoxide, (1S, 2E, 4S, 6R, 7E, 11S, 12S)-11, 12-epoxy-2,7-cembradiene-4, 6, 15-triol 109. Table 23, 13 C NMR Chemical shifts (ppm) of epoxide 4 and triol 109 determined in CDC13  4 109  4 109  C-1  C-2  47.1 52.0  127.7 138.4 72.3 126.4 132.6 72.3  C-11  C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20  61.3 61.2  60.1 59.5  C-3  36.6 36.6  C-4  28.6 25.6  C-5  C-6  C-7  C-9  C10  52.9 53.0  66.1 66.1  132.7 135.2 35.6 141.2 135.7 36.0  24.9 25.0  33.1 72.2  19.1 26.9  20.9 27.2  C-8  30.0 29.9  16.0 15.8  16.3 16.5  This result provides further evidence that the tobacco T-43-T cell line behaves differently from the TRP4a cell line. Even though T-43-T whole cells do not show very good ability to transform the starting diol 1 into the epoxide 4 as does TRP4a in whole cell  95  experiments, they show capability to hydroxylate the non-activated methine position of the isopropyl side chain in the epoxide 4. This enzymatic introduction of hydroxyl function at C-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, such allylic hydroxylations did occur but introduction of hydroxyl function at C-15 has not been observed before. Unfortunately, still no ring cleavage products were isolated. 2.3.2 Biotransformation of the enone 43 using cell homogenate prepared from the T-43-T cell line  Since the T-43-T cell homogenate failed to convert starting diol 1 into any ringcleavage products, diol 1 was then oxidized using PCC to enone 43 in 81% yield and subjected to biotransformation conditions as before (Entry 65). But unfortunately, no expected retro-aldol type reaction took place, only the epoxidation of the 11,12 double bond was observed again and enone 7 was obtained in 65.3 % yield (Table 24). Since this enone was not a suitable substrate for ring cleavage, no further biotransformation experiments were performed.  1  ^  43  ^  7  Scheme 40, Chemical conversion of diol 1 to enone 43 and biotransformation of 43 to 7  96 Table 24, Biotransformation of enone 43 with T-43-T cell homogenate Expt. No. Age (days) n D25 pH Volume of culture (L) Volume of homogenate (ml) Peroxidase activity (unit/mi.) Protein concentration (mg/ml)  70 16 1.3334 5.74 1.5 825 2.81 0.63  Incubation conditions:  Volume of homogenate (ml) Protein/substrate (mg/mg) Peroxidase/substrate (units/mg) Substrate 43 (mg)/EtOH (ml) Buffer added (ml) Distilled water (ml) 0.24% H202 (eq) FMN (eq) MnC12 (eq) Incubation time (h)  267 3.4 15 50/5 220 95 4.0 0.5 0.5 120 12.8 65.3  Yield (%)^43 7  Table 25, 13 C NMR chemical shifts (ppm) of 43 and 7 determined in CDC13  (1) (43) (7)  (1) (43) (7)  C-1  C-2  C-3  46.4 47.1 47.9  127.7 137.5 72.4 130.2 136.1 72.7 130.6 136.0 72.4  C-11  C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20  124.4 133.3 36.8 122.9 134.8 37.2 58.6 60.1 33.8  C-4  27.9 28.7 26.2  C-5  C-6  C-9  C10  52.2 56.3 54.8  66.2 130.6 136.6 38.8 200.6 127.0 157.9 40.4 200.9 126.4 159.3 38.3  23.3 23.9 24.7  33.0 32.6 31.5  19.3 19.3 19.9  C-7  20.7 20.5 20.3  C-8  30.1 30.6 30.8  16.1 18.7 19.1  15.0 15.7 18.1  97 The spectral data of biotransformation product 7 were identical to those of synthetic product 7 obtained by oxidation of epoxide 4 with PCC and consistent with the published data [42]. The chemical shifts of the starting diol 1, enone 43 and epoxide 7, as obtained in the corresponding 13 C NMR spectra are summarized in Table 25. 2.3.3 Biotransformation of the seco-diketone 44 and seco-epoxide 107 using the cell homogenate O 0  < 43  1 ) 1\13 12  1514 -^17 19^1 ^"C 16  7  ^  ^  N8  9 10  18  20  44  ^  107  Scheme 41, Chemical conversion of 43 into 44 and biotransformation of 44 to 107 Since the enone 43 did not undergo the desired retro-aldol fragmentation, it was further converted to seco-diketone 44 chemically. With the hope that T-43-T cell homogenate could perform appropriate conversions on this ring cleavage substrate, seco-  diketone 44 was subjected to biotransformation conditions (Table 26). Unfortunately, still no further cleavage at 7,8 double bond was observed. The only product obtained was secoepoxide 107 in low yield. Therefore, either in ring closed or ring opened substrates, the most active site was the same, ie, the 11,12 double bond in 1 and the 7,8 double bond in 44. The EI-MS spectrum of 107 showed a molecular ion at m/z 320, suggesting that one oxygen had been incorporated into the substrate molecule. The expected enzymatic attack at the 7,8 double bond, corresponding to 11,12 double bond in the cyclic cembranoid skeleton, was readily evident from 1 H NMR spectrum. The olefinic proton  98 signal at 5.06 ppm (t, J=8 Hz, H-7) and the vinyl methyl proton signal at 1.58 ppm (s, H20) 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 homogenate Expt. No.  71  72  73  Age (days) pH Volume of culture (L) weight of fresh cell (g) Buffer added (ml)  16 1.3340 7.00 1.5 489 440  16 1.3340 5.72 1.0 283 250  16 1.3338 5.74 1.0 353 315  Volume of homogenate (ml) pH Peroxidase activity (unit/ml) Protein concentration (mg/ml)  880 6.58 4.78 0.81  520 6.40 4.45 1.12  640 6.52 3.93 1.02  Incubation conditions: Volume of homogenate (ml) Protein(g)/substrate(mg) Peroxidase units/mg Substrate 44 (mg)/EtOH(ml) Buffer added (ml) Distilled water(ml) 0.24% H202(eq) FMN MnC12 Incubation time (h) Yield (%)^4 4 107  315 5.1 30 50/5 90 40 4.0 0.5 0.5 120 56.2 10.3  169 3.8 15 50/5 175 75 4.0 0.5 0.5 120 21.0 25.2  640 6.5 25 100/10 175 75 4.0 0.5 0.5 120 83.7 4.6  25  nD  99 In order to confirm the structure and the stereochemistry at C-7 and C-8, 107 was synthesized from epoxide 4 by oxidation using PCC followed by retro-aldol reaction with potassium carbonate. Since the 1 H NMR spectrum of biotransformation product 107 was identical 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.  ^  4  ^  7  107  Scheme 42, Chemical conversion of epoxide 4 to 107  2.3.4 Biotransformation of the seco-aldehyde 32 using the cell homogenate Upon incubation of the seco-aldehyde 32 with the buffered solution containing the homogenate and the cofactors (4.0 equivalents of hydrogen peroxide, 0.5 equivalents of FMN 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 to proceed for a further 72 h, TLC indicated that most of the starting material was consumed with the appearance of two major new spots. Subsequent chromatography and spectroscopic studies suggested that the more polar product was the primary alcohol 110, a reduced product, and the less polar one the cyclized ether 111 (Scheme 43). GC analysis indicated that both compounds 110 and 111 exhibited the same retention time and it was conceivable that the tetrahydrofuran derivative 111 could be derived from the primary alcohol 110 through a cyclization involving nucleophilic attack of the 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 column  100 chromatography. The actual isolated yields for 110 and 111 after chromatographic separation were 29% and 32%, respectively.  110 19  OH 9 10  8 17 20^11, 4 ^ ".< 6 18 3 43 12 1[1/4^ 2^14 1 H'rl^ ,, 15  \ 0H 7 i9 5 6r-N0 10 ,s  8 -^17 2 0^1> -<16 0 18 13 12 2 1 0 14 15  32 110 111 Scheme 43, Biotransformation of seco-aldehyde 32 into 110 and 111 Among the two experiments performed, the results were dramatically different in terms of yields of biotransformation products. The only significant difference in the reaction parameters used was the higher level of homogenate volume employed (Entry 75 in Table 27). Apparently, the higher concentration of "reductase" enzymes present in the latter experiment was sufficient to perform the reduction of 32 to 110 and the latter, in turn, 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 at m/z 304, due to loss of two molecules of water from 110. The 1 H NMR spectrum of 110 is very similar to that of starting material 32 except that the aldehyde signal at 8 9.78 ppm (-CH2CHO) in 32 was replaced by a triplet at 8 3.65 ppm (-CH2CLI2OH). In order to aid our structural determination, this primary alcohol 110 was prepared by reduction of the seco-aldehyde 32 with 1.0 equivalent of sodium borohydride in methanol. Spectroscopic data of the synthetic sample 110 were found to be consistent with product 110 isolated from the biotransformation experiment.  101  Table 27, Biotransformation of seco-aldehyde 32 with T-43-T cell homogenate Expt. No. Age (days) pH Volume of culture (L) weight of fresh cell (g) Buffer added (ml)  74 15 1.3351 5.51 0.5 123 100  75 16 1.3345 5.72 1.0 299 250  Volume of homogenate (ml) Peroxidase activity (unit/m1) Protein concentration (mg/ml) pH  210 4.00 1.75 6.40  540 3.55 1.57 6.45  90 3.2 7.2 50/10 175 75 2.4 0.5 0.5 120 73.1 10.4  422 6.6 15.0 100/20 350 150 4.0 0.5 0.5 120 2.3 29.3 31.9  n D25  Incubation conditions:  Volume of homogenate (ml) Protein(mg)/substrate(mg) Peroxidase (units)/substrata(mg) Substrate 32 (mg)/EtOH(ml) Buffer added (ml) Distilled water(ml) 0.24% H202(eq) FMN Mn02 Incubation time (h) Yield (%)^3 2 110 111  The CI-MS spectrum of 111 showed that the molecular ion at m/z 323 ([M+H]+). In comparing the 1 H NMR spectrum of 111 with that of seco-aldehyde 32, the signal at 8 4.70 ppm (dt ,H-6) in 32 was missing and vinyl methyl proton signal at 8 1.71 ppm (s, H20) in 32 was now shifted to high field at 8 1.29 ppm (s, H-20) in the spectrum of 111.  102 The olefinic proton signal at 8 5.25 ppm (doublet, J=8 Hz, H-5) and the proton signal at 8 4.78 ppm (multiplet, H-6) in aldehyde 32 were replaced by two new sets of signals at 8 5.48 ppm (doublet, J=16 Hz, H-5) and 8 5.31 ppm (multiplet, H-6) in the spectrum of 111. Finally, in the 13 C NMR spectrum, the C-4 carbon signal at 8 135.7 ppm was replaced by a new C-4 carbon signal at 8 82 ppm. Table 28 provides a summary of 13 C NMR data for the compounds 32, 110 and 111. Therefore, it was clear that the allylic system was attacked and the structure was assigned as the tetrahydrofuran derivative 111 with the stereochemistry at C-4 unsolved. In accord with this assignment, a peak at m/z 85 can 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 significant amounts and the aldehyde functionality was reduced selectively in the presence of the methyl ketone. This was in sharp contrast with the previous results. Therefore, the cell homogenate of the tobacco cell line T-43-T was capable of performing both chemical oxidation and reduction processes. These contrasting enzymatic properties of the tobacco plant cells could be utilized in other areas of organic synthesis. Table 28, 13 C NMR chemical shifts (ppm) of 32, 110 and 111 determined in CDC13 C-1  C-2  32 201.9 41.8 110 62.4 35.7 111 67.4 37.7 C-11 32 48.6 110 48.6 111 48.7  C-3  C-4  C-5  31.4 29.7 45.8  135.7 128.5 66.1 26.2 137.5 128.6 66.2 27.0 82.0 138.7 129.2 26.2  C-6  C-7  C-8  C-9  C10  73.2 73.1 72.2  139.9 128.7 139.9 127.9 140.3 122.4  C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20 47.5 47.6 48.6  42.1 42.1 42.1  209.0 30.0 210.0 30.3 209.5 30.0  16.7 16.5 25.7  26.9 26.2 32.0  32.0 32.0 32.1  20.6 20.6 19.1  19.1 19.1 20.7  103 2.3.5 Biotransformation of the tetrol 104 using the cell homogenate  Several biotransformation experiments using tetrol 104 as a substrate were performed 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 direct homogenization of the suspension culture with an Ultra-Turrax Disperser T-25 at 24,000 rpm. No cofactors were added in the biotransformation process. When tetrol 104 (50mg/5m1Et0H) was incubated with such a cell homogenate, no biotransformation was observed and precursor was recovered in 75% yield. Because of the solubility problem encountered in the experiment using 104 as a substrate, a large amount of ethanol (50mg/50m1 EtOH) was used in subsequent studies. In such a case, a less polar product was isolated in approximately 20% yield along with recovery of 50-70% of the precursor (Entry 77 and 78). The structure of the new product was assigned as ether 112.  OH^HO 112 ^ 113^ 114  Scheme 45, Biotransformation of tetrol 104  104 The mass spectrum of product 112 shows a molecular ion at m/z 338 which is 2 mass units less than that of substrate 104. The 13 C NMR spectrum indicates that there are one double bond and one carbonyl group in the molecule. The structure for 112 was confirmed by direct comparison of its spectral data with the published result.[ 42] This product could be formed by oxidation at C-6 to an enone followed by Michael addition of the 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 identities were established by comparison the mass spectrometric data with published data.[42,  82]  Table 29, Biotransformation of tetrol 104 with T-43-T cell homogenate Expt. No. Age (days) n D25 pH Volume of culture (L) Peroxidase activity (unit/m1) Protein concentration (mg/ml) Incubation conditions: Substrate 104/EtOH (mg/nil)  Incubation time (h) Yield (%)^104 112 113 11 el  76 20 1.3335 6.85 0.4 3.26 0.67 50/5 120 75 0 0  0  77 16 1.3338 6.35 0.4 4.44 0.63 50/50 120 59 21 0 0  78 19 1.3334 5.80 0.4 4.94 0.64  79 18 1.3334 5.60 1.2 2.33 0.81  50/50 120* 52 23 0 0  150/150 96 63 22 0 0  80 16 1.3339 4.40 0.8 2.44 0.89 100/200 120 58 23 2 2  * Air was bubbled (500m1/L/min) into cell homogenate during the entire period of incubation. 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 optimization conditions were considered and further studies with this substrate were discontinued.  105 2.3.6 Conclusion Five cembranoid analogues obtained either by chemical manipulation or previous biotransformation have been evaluated as substrates in biotransformation experiments with "growing" cells of the T-43-T cell cultures and with the cell homogenates derived from these cultures. Regioselective hydroxylation at C-15, a non-activated methine position in the cembranoid epoxide 4, by whole cells of tobacco T-43-T cell line reveals an interesting novel enzymatic bioconversion and provides a possible method to hydroxylate the nonactivated carbon centres in other substrates. This result could be further explored in other areas of natural products chemistry where oxidation of a non-activated carbon atom is required. In the biotransformation experiments performed using the cell homogenate, enone 43 affords only the corresponding epoxide 7 in low yield and the expected ring cleavage products via retro-aldol reaction are not observed. The seco-diketone 44 undergoes regioselective attack, in a manner reminiscent of the parent cembranoid skeleton, to afford an epoxide 107. When seco-aldehyde 32 is exposed to enzymatic conditions, only reduction of the aldehyde function is noted and the resulting primary alcohol 110 undergoes a precedented cyclization 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 attack when various cembranoid analogues are exposed to the enzymes of N. sylvestris cell cultures. It is clear that this tobacco cell line is capable of performing both oxidation and reduction, namely selective epoxidation of double bond, hydroxylation at allylic position, hydroxylation at non-activated methine position and selective reduction of an aldehyde function to the corresponding alcohol. These contrasting enzymatic properties of the tobacco plant cells could be utilized in other areas of organic synthesis.  106 2.4 Overall conclusions Although the results presented in this thesis do not realize the goal of establishing an efficient route from tobacco cembranoids, mainly two isomeric diols 1 and 2, to norsolanadione 78 which is a compound of potential use in the area of aroma and fragrance chemicals, with plant cell culture technology, biotransformation experiments using plant cell cultures derived from Tripterygium wilfordii and Nicotiana sylvestris do show their capabilities to biotransform the tobacco cembranoids and their analogues into oxidation or reduction products. These studies involving biotransformation of tobacco cembranoids with plant cell cultures afford novel products and provide information relevant to possible biosynthetic 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 favored process 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-10 and C-13 positions were obtained in relatively low yields. The stereochemistry at C-10 position 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 was possible. In addition, selective reduction of an aldehyde function in the presence of ketone function was observed. 2.5 Further Research Directions Since no expected ring cleavage products were obtained in studies using plant cell cultures, efforts have been directed to the biotransformation studies using microorganisms. Biotransformations with both bacteria and fungi are currently under investigation in our laboratory.  107 3 EXPERIMENTAL Melting points were determined using a Reichert melting point apparatus and were not corrected. Infrared spectra were recorded on Perkin Elmer 710 spectrometers using chloroform solutions in sodium chloride cells (0.1 mm path length). Optical rotations were recorded on a Perkin-Elmer 141 polarimeter at ambient temperature using a quartz cell of 10 cm pathlength with the solvent and concentration (in g/100m1) given in parentheses. 1 H NMR spectra were recorded on Bruker WH-400, Bruker AC 200 or Varian XL-300 spectrometers. Chemical shift values were reported in ppm relative to tetramethylsilane as an internal standard. 13 C NMR spectra were recorded on a Varian XL-300 spectrometer at 75.3 MHz or a Bruker AC-200 spectrometer at 50.2 MHz. Mass spectra were recorded on AEI-MS-902 (low resolution ) and Kratos-MS-50 (high resolution) spectrometers. Elemental analyses were performed by Microanalysis Laboratory, University of British Columbia. GC analyses were performed on Hewlett-Packard 5890A Gas Chromatography 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, Injection temperature: 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 without illumination. The medium pH was measured on Electrometer Model 265 while the refractive index was measured using an Officine Galileo refractometer at 25°C. The cell homogenization was accomplished 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 Sorvall superspeed RC2-B automatic refrigerated centrifuge. UV absorbances in protein and peroxidase activity assay were recorded on Bausch and Lomb Spectronic 20 spectrophotometer. Semicontinual addition of precursor was accomplished via a peristaltic pump (Cole-Parmer Model 7401-20 with Master Flex pump drive). Column chromatography was performed using silica gel (230-400 mesh) with air or nitrogen gas pressure to obtain a suitable flow rate, while thin layer chromatography was  108 performed using commercial aluminum-backed silica gel plates (Merck, art. 5554). Visualization was 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 Biological Services of the Department of Chemistry , University of British Columbia. 3.1 Chemical conversions of diol 1 to cembranoid analogues 3.1.1 Conversion of (1S, 2E, 4S, 6R, 7E, 11E)-2,7,11-cembratriene-4,6-diol 1 to (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  ^  98  To a stirred solution of diol 1 (300 mg, 0.98 mmol) and triethyl amine (2 ml) in dichloromethane (50 ml), acetic anhydride (0.5 ml) was added. After being stirred at room temperature for 18 h, water (10 ml) was added and the reaction mixture was allowed to stir for another 2 h. The reaction mixture was then extracted with ethyl acetate (3x100 ml). The extracts were 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 the acetate 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 of  109  sodium bisulfate (500 mg in 50 ml of water) was added and the reaction mixture was allowed to stir for an additional 2 h. The reaction mixture was then diluted with ethyl acetate (50 ml) and water (30 ml). The aqueous layer was extracted with ethyl acetate (2x300 ml). The combined organic 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 (1:1) as eluents gave triol 98 (313 mg, 86%). The physical properties are as follows: mp: 59-60 0 C; [a] D : +17 0 (c =0.45, EtOH); IR (CHC13) V max : 3590, 3450, 1720 and 1250cm ; 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,-1 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 2011 34 0 3 (W-60): 322.2509, found: 322.2514 3.1.2^Conversion of 98 to (4E, 6R, 8S, 9E, 11S)-6-acetoxy-4, 8-dimethy1-8hydroxy-11-isopropy1-14-oxo-4, 9-pentadecadienal 99 [ 35 ]  98  Powdered lead tetra-acetate (45 mg, 0.21 mmol) was added to a solution of triol 98 (70 mg, 0.18 mmol) in benzene (10 ml). After being stirred at room temperature for 10 mins, the reaction mixture was diluted with ethyl acetate (20 ml), saturated sodium thiosulfate solution (5 ml) was added and the resulting solution was allowed to stir for an additional 10 min. The reaction mixture was then separated into two layers and the aqueous layer was extracted with ethyl acetate (3x50 ml). The combined extracts were washed with water and brine and then dried over  110 anhydrous sodium sulphate. Concentration in vacuo and chromatography on silica gel using ethyl acetate and hexanes (1:1) as eluents gave seco-aldehyde 99 (56 mg, 83%). The physical properties are as follows: oil; [a]p: -4.8 0 (c =0.27, EtOH); IR (CHC13) Vmax: 3460, 2740, 1720 and 1250 cm -1 ; 1 11 NMR (CDC13) 8: 0.84(d, J=6.7Hz, 3H)/0.88(d, J=6.7Hz, 3H)(H17/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, H7a), 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, H9), 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. for C22H3404 (M -F-18): 362.4657, found: 362.4650.  3.1.3^Conversion of 99 to methyl (4E, 6R, 8S, 9E, 11S)-6-acetoxy-4, 8dimethy1-8-hydroxy-11-isopropy1-14-oxo-4, 9-pentadecadienoate 100 [ 35 1 AcO  AcO =  ,OH  \ \^>i< ^0 0, z^ H N^ Me0 0  99^  , / ^>"‹ 100  Jones' reagent (chromium trioxide in sulphuric acid, 2.7 M) was added dropwise to a solution of 99 (300 mg, 0.79 mmol) in acetone (50 ml) at -10 0 C until the orange color persisted. The excess Jones' reagent was destroyed by dropwise addition of isopropanol and the reaction mixture was then diluted with ethyl acetate (250 ml). The resulting solution was separated into two layers and the aqueous layer was extracted with ethyl acetate (2x200 ml). The combined organic extracts were washed with water and brine and then dried over anhydrous sodium sulphate. Concentration in vacuo gave the crude product. The crude product was dissolved in  111 methanol (50 ml) and ethereal solution of diazomethane was then added dropwise until the reaction mixture attained a permanent yellow color. Excess diazomethane was removed by passing a stream of nitrogen through the solution. The resulting solution was concentrated in vacuo to give the crude mixture of the methyl ester. Chromatography on silica gel using hexanes  and ethyl acetate (3:1) as eluents gave ester 100 (260 mg, 80%). The physical properties are as follows: oil; [alp: -4.2 0 (c =0.81, EtOH); IR(CHC13) V max : 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, 8dimethyl-ll-isopropyl-14-oxo-4, 9-pentadecadienoate 101 [ 35 1  Ac0.  OH  %r ^ 0  Me0  0  100  ><  HO  PH  0, z \N7  >w<  Me0 0 101  Powdered potassium carbonate (50 mg) was added to the solution of 100 (240 mg, 0.585 mmol) in methanol (50 ml). After being stirred at room temperature for 2 h, the reaction mixture was diluted with ethyl acetate (250 ml) and washed with water until neutral pH. The organic layer was then washed with brine (100 ml) and dried over anhydrous sodium sulphate. Concentration in vacuo and chromatography on silica gel using hexanes and ethyl acetate (3:1) as eluents gave  101 (185 mg, 86%). The physical properties are as follows: oil; [ta] D : +260 (c =0.57, CHC13);  112 IR (CHC13): Vmax 3680, 3600, 3480, 1730, 1710 cm -1 ; 1 11 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 ] OH 0 Mee()^Me0 0 101^  102  >< 78  Pyridinium chlorochromate (250 mg, 0.55 mmol) was added to the solution of 101 (180 mg, 0.49 mmol) in dichloromethane (30 ml). After being stirred at room temperature for 2 h, the solution 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) was dissolved 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 (100 ml), washed with water, then brine and dried over anhydrous sodium sulphate. Concentration in vacuo and chromatography on silica gel using hexanes and diethyl ether (2:1) as eluents gave  initially 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,  113  H-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) V max : 1675, 1715 cm -1 ; 1 H 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= 9 and 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); HRMS calcd. for C12H2002: 196.1464, found: 196.1458. 3.1.6^Conversion of diol 1 to (1S, 2E, 4S, 6R, 7E, 11S, 12S)-2,7cembradiene-4,6,11,12-tetrol 104 [ 35 ]  1  Osmium tetroxide (420 mg, 1.64 mmol) was added to a solution of diol 1 (500 mg, 1.63 mmol) in pyridine (25 ml). After being stirred at room temperature for 3 h, sodium bisulfite (800 mg) in water (20 ml) was added to the reaction mixture and the resulting solution was allowed to stir for another 3 h. The reaction mixture was then diluted with ethyl acetate (50 ml) and washed with water (100 ml). The aqueous layer was further extracted with ethyl acetate (2x200 ml) and the organic extracts were combined and washed with brine, then dried over anhydrous sodium sulphate. Concentration in vacuo and chromatography on silica gel using ethyl acetate as eluent afforded tetrol 104 (420 mg, 76%) as a white foam. The physical properties of 104 are as follows: mp: 133-4 0 C; [a]p: +6.5 0 (c =1.3, EtOH); IR (CHC13) V max : 3682, 3404 cm -1 ; 1 H NMR (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-  114  20), 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, H11), 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 0 2011 3403 (M+-18): 322.2508, found: 322.2513; Elemental analysis calcd. for C20113604: C 70.55, H 10.66, found: C 70.40, H 10.46. 3.1.7^Conversion of tetrol 104 to (4E, 6R, 8S, 9E, 11S)-4,8-dimethy1-6,8dihydroxy-11-isopropy1-14-oxo-4,9-pentadecadienal 32 [ 33 ] HO  HO/ ' co ,  104^  H/ 0  ;OH  ><  32  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, the reaction mixture was diluted with ethyl acetate (100 ml), then saturated sodium thiosulfate solution (50 ml) was added and the resulting solution was allowed to stir for an additional 10 mins. The reaction mixture was then separated into two layers and the aqueous layer was extracted with ethyl acetate (200 ml). The combined organic extract was washed with water and brine and then dried over anhydrous sodium sulphate. Concentration in vacuo and chromatography on silica gel using hexanes and ethyl acetate (2:1) as eluents afforded 32 (300 mg, 86%). The physical properties of 32 are as follows: mp: 51-2 0 C; [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,  115  J=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, H15), 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); LRMS m/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. for C20H3404: 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-11isopropy1-6,14-dioxo-4,9-pentadecadienal 106 and nor-solanadione 78 0^,OH  HO OH  >11.< H 0^  0  >-  )f)N < ^ ()/  H 0  32^  106  ^  78  Pyridinium chlorochromate (350 mg, 0.77 mmol) was added to the solution of secoaldehyde 32 (250 mg, 0.74 mmol) in dichloromethane (50 ml). After being stirred at room temperature for 1 hr, the solution was diluted with ether (200 ml) and the resulting mixture was then filtered through a layer of silica gel. Concentration in vacuo and chromatography on silica gel using 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) was added. After being stirred at room temperature for 1 hr, the reaction mixture was diluted with ethyl 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) as eluents afforded nor-solanadione 78 (43 mg, 52%), which is identical to that obtained previously.  116  3.1.9^Conversion of diol 1 to (1S, 2E, 4S, 7E, 11E)-2,7,11-cembratriene-4-ol6-one 43 [ 5, 39 ]  43  To a solution of pyridine (0.24 ml, 3 0 mmol) in dichloromethane (10 ml), chromium trioxide (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 h at 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) as eluents gave enone 43 (80 mg, 80.5%). The physical properties of 43 are as follows: mp 778°C; [a]p: +93 0 (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=12 Hz, 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. for C20113202: 304.2402, found: 304.2399; Elemental analysis calcd. for C20113202: C 78.89, H 10.59, found: C 79.00, H 10.73. 3.1.10^Conversion of 43 to (3E, 7E, 11S, 12E)-11-isopropy1-4,8-dimethyl3,7,12- pentadecatriene-2,14-dione 44 [41]  117  43  ^  44  Enone 43 (55 mg, 0.181 mmol) was dissolved in a mixture of aqueous sodium carbonate solution (1.0%, 10 ml) and ethanol (5 ml) and stirred at room temperature for 24 h. The reaction mixture was worked up by extraction with ethyl acetate (3x20 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) as eluents gave  seco-diketone 44 (46.8 mg, 85.1%). The physical properties are as follows: oil; [a]p: 9.5 0 (c 0.4, CHC13); IR(CHC13) Vmax: 1690, 1675, cm -1 ; 1 H 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-isopropyl14-oxo-4,9-pentadecadiene-1,6,8-triol 110 HOHO OH ,-^  .• OH  HO / 110 Sodium borohydride (5.0 mg, 0.13mmol) was added to a solution of 32 (50 mg, 0.148 mmol) in methanol (5 ml) at 0 0 C. After being stirred for 5 min, the reaction mixture was diluted  118  with ethyl acetate (50 ml) and the resulting solution was washed with water, then with brine and dried over anhydrous sodium sulphate. Concentration in vacuo and chromatography on silica gel using 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 :33.6 0 (c =0.3, CHC13); IR Vmax (CHC13): 3600, 1710 cm -1 ; 1 H 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, H20), 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. for C20113202 (M+-36): 304.2402; found: 304.2403. 3.1.12^Conversion of epoxide 4 to (1S, 2E, 4S, 7E, 11S, 12S)-11, 12-epoxy4-hydroxy-2, 7-cembradien-6-one 7 42] [  I I  <  4  7  To a stirred cool solution of pyridinium chlorochromate (35.6mg, 0.165 mmol) in dichloromethane (15 ml) was added a solution of epoxide 4 (50 mg, 0.155 mmol) in dichloromethane (1 ml). The mixture was stirred at 0°C for 2.5 h. Water (40 ml) was then added and the mixture was stirred for a further 5 min. Extraction with ethyl acetate (3x50m1) followed by 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%). The physical properties of 7 are as follows: mp: 66-67°C; [a] D : +85° (c =0.60, CHC13); IR (CHC13) V m a x : 3610, 3470, 1665 cm -1 ; 1 H NMR (CDC13) 8: 0.81 (d, J=6.5 Hz, 3H)/0.84 (d, J=6.5  119  Hz, 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, H19), 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=12.6 Hz, 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-epoxy11-isopropyl-3, 7, 12-pentadecadiene-2, 14-dione 107  I ,  7  K 107  Enone 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 at room temperature for 2 h. Water (40 ml) was then added and the mixture was extracted with ethyl acetate (3x50 ml). The extract was washed with water until neutral pH, then with brine and dried over anhydrous sodium sulphate. Concentration in vacuo and chromatography on silica gel using ethyl acetate and hexanes (1:1) as eluents gave seco-diketone 107 (25.2 mg, 84%). The physical properties of 107 are as follows: oil; [a]D: +18.9 0 (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),  120 194(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 cultures 3.2.1 Propagation of TRP4a cell culture  TRP4a cell line was derived from a leaf explant of Tripterygium wilfordii.[ 801 The suspensions were maintained in PRDCo medium which was the PRL-4 medium of Gamborg and Eveleigh [ 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 at 26°C and 135 rpm without illumination. The growth profiles were monitored by pH and refractive index of the spent medium. 3.2.2 Propagation of T-43-T cell culture  Tobacco cell line T-43-T was initially derived from seeds of Nicotiana sylvestris which were obtained from the Swedish Tobacco Company. The leaf derived tobacco calli were cultured on 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 were subcultured every 3-4 weeks. The cell suspension cultures were inoculated at a rate of 1.8 g dry cell mass/L of liquid MS medium. All suspension cultures were grown at 26°C and 135 rpm without illumination in 1 L Erlenmeyer flasks with 500 ml liquid Murashige-Skoog medium containing 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 and 135 rpm without illumination. The growth profiles were monitored by pH and refractive index of the spent medium.  121 3.3 Biotransformations using TRP4a cell line 3.3.1^Biotransformations using TRP4a whole cells 3.3.1.1^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 the pH 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 L with distilled water. 3.3.1.2^General procedure for Experiments 1-14: addition of substrate to the cells resuspended in buffer  The buffer solutions and all the apparatus used were autoclaved at 120 0 C for 15 minutes before use. All culture manipulations including cell resuspension and biotransformation in this section were performed under aseptic conditions. The suspension culture was filtered through Miracloth and resuspended in the same volume of buffer. The substrate (diol 1 or 2) dissolved in ethanol was then added to the resuspension culture and incubated at 26 0 C and 135 rpm on a rotary shaker for an appropriate time. The biotransformation mixture was worked up by ethyl acetate 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 1 to 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 days old) were filtered through a Buchner funnel equipped with Miracloth, washed with phosphate buffer (400 ml) and subjected to suction to remove excess water. The resulting cells (800 g) were  122  then transferred to a 4 L. beaker containing 2000 ml of phosphate buffer (pH 6.3). After agitating with 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°C and 135 rpm on a rotary shaker. The control experiment was set up in the same way except that no diol 1 was added . After 144 h of incubation time, the biotransformation mixture in each flask was filtered through Miracloth and the filtrate was extracted with ethyl acetate (3x200 ml). The combined extracts were washed with water (200 ml) and brine (200 ml) and then dried over anhydrous sodium sulphate. Concentration of combined extracts from each flask in vacuo afforded a crude mixture of products (318 mg) from the broth. Ethyl acetate (300 ml) was added to the combined cell material and the resulting suspension 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 vacuo afforded a crude mixture of products (105 mg) from the cell mass. The combined extract (423 mg) was chromatographed on silica gel (200 g) using ethyl acetate as eluent and separated into three portions (A: 35 mg, B: 239 mg, C: 89 mg). The portion A was purified again on silica gel (25 g) using ethyl acetate and hexanes (2:1) as eluents to give recovered diol 1 (20 mg, 5%), while portion B was purified again on silica gel (100 g) using acetone and hexanes (1:2) as eluents to give epoxide 4 (140 mg, 35%) and allylic alcohol 92 (64 mg, 14%). Finally, portion C was purified again on silica gel using acetone and hexanes (1:1) as eluents to give C-10 alcohol 93 (20 mg, 5%) and C-13 alcohol 94 (48 mg, 12%). The physical properties of the isolated products are as follows:  123 HO  OH  HO^ pH HO  ^pH  HO^ HO  4  92  93  ^  94  (1S, 2E, 4S, 6R, 7E, 11S, 12S)-11,12-epoxy-2,7-cembradiene-4,6-diol 4 [ 361: mp 99 1000 C. -  [a] D : +113.9 0 (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, H 10.46 (1S, 2E, 4S, 6R, 7E, 1041, 11E)-2,7,11-cembratriene-4,6,10-triol 92: mp: 41-42 0 C; [a]p: +51.3 0 (c =0.60, CHC13); IR Vmax (CHC13): 3625, 3420 cm -1 ; 1 H 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 10.58  124 (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 ; 1 H 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, H18 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); LRMS m/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); HRMS calcd. for C201-13202 (M+-18): 304.2403, found: 304.2401; Anal. calcd. for C20113403: C 74.49, H 10.63; found: C 74.38, H 10.36 3.3.1.2.2^Typical procedure for biotransformation of diol 2: addition of diol 2 to the cells resuspended in buffer (Entry 8, Table-5)  Diol 2 (50 mg) was dissolved in ethanol (5 ml) and added to an Erlenmeyer flask containing 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 after 144 h as before afforded crude product mixture from broth (25 mg) and from cells (45 mg).  125 Chromatography of combined extracts (70 mg) on silica gel (25 g) using ethyl acetate as eluent gave epoxide 95 (26.3 mg, 53%) followed by a mixture (12.4 mg) which could not be purified by conventional silica gel chromatography and was not studied further. The physical properties for 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 : +66.4 0 (c =1.1, CHC13); IR V max (CHC13): 3400, 1390 and 1375 cm -1 ; 1 H 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]±, found304.2389 3.3.1.3 General procedures for Experiments 15-35: addition of substrate to the growing cell suspension culture  In all instances, the diol 1 was dissolved in ethanol and added to the growing cells in one of the following ways: In experiments 15 to 22, the diol 1 was added to the cell suspension culture in one batch while in experiments 23 to 27, the alcoholic solution of diol 1 was divided into several portions and added to the cell suspension culture batchwise over a certain period of time. The mixture thus formed was incubated on a rotary shaker at 135rpm and 26 0 C. In experiments 28 to 35, the substrate was added to the cell suspension culture in a bottom stirred benchtop bioreactor semi-continually via a peristaltic pump and incubated at room temperature. All  126 other parameters (pH, age of culture, etc.) are indicated in the Tables. The work-up procedures were the same as those used in Experiments 1-14. 3.3.1.3.1 Typical procedure for biotransformation with one batch addition of diol 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 flask containing cell suspension culture (500 ml, 13 days old). The resulting mixture was incubated on a rotary shaker for 24 h. The biotransformation mixture was worked up as before to afford crude extracts (67 mg). Chromatography on silica gel (25 g) using ethyl acetate as eluent afforded the recovered diol 1 (12 mg, 24%), tertiary alcohol 8 (5.2 mg, 10%), epoxide 4 (16.5 mg, 33%), C10 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  ><  HO 8  (1S, 2E, 4S, 6R, 7E, 10E)-2,7,10-cembratriene-4,6,12-triol 8[451: mp: 134-135 0 C; [a] D : +420 (c =0.18, CHC13); IR Vmax (CHC13): 3602, 3460 cm -1 ; 1 H 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. for  127 C2043202: 304.2402 (M+-18), found: 304.2398; Anal. calcd. for C20113403: C 74.49, H 10.63; found: C 74.35, H 10.48 ^3.3.1.3.2^Typical procedure for biotransformation with batch-wise addition of diol 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 h intervals 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-up as before afforded a crude extract (60 mg). Chromatography on silica gel (25 g) using ethyl acetate 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 addition of 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 benchtop bioreactor which was bottom stirred with a magnetic stir bar, aerated through a sintered glass disk at 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 a period of 24 h at a rate of 0.5 ml/h. After the last addition, the incubation was allowed to continue for a further 24 h. Work-up as before afforded crude extract (132 mg). Chromatography on silica gel (50 g) using ethyl acetate as eluent afforded recovered diol 1 (22.8 mg, 22.8%), tertiary alcohol 8 (4.3 mg, 4.3%), epoxide 4 (33.1 mg, 33.1%), C-10 alcohol 92 (1.1 mg, 1.1%), C-10 alcohol 93 (8.3 mg, 8.3%) and C-13 alcohol 94 (1.6 mg, 1.6%), respectively.  128 3.3.2 Biotransformations using CFE 3.3.2.1 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 and Na2HPO4 (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 addition of solution B.  3.3.2.2 Preparation of CFE  All procedures were performed at 0-4°C. TRP4a cell suspension culture was harvested by filtering through a Buchner funnel equipped 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 and the resulting suspension was then homogenised with an IKA Ultra-Turrax Disperser T-25 at 24,000 rpm for 30 sec. The same procedure was repeated three times. In order to avoid overheating the suspension, a 1 minute break was allowed between each operation. The homogenate thus obtained was then subjected to centrifugation at 10,000g (8,000 rpm) for 30 min. The clear supernatant was collected as crude CFE and used in biotransformation experiments. The peroxidase activity and soluble protein concentration were assayed according to the 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 diluted with four parts of distilled water. The diluted dye reagent solution (5 ml) was then added to a test-  129 tube containing CFE (0.1 ml) and the solution was mixed thoroughly. After using a reference sample (prepared by mixing phosphate buffer (0.1 ml, 0.1M, pH 6.6) and the diluted dye solution (5 ml)) to adjust the reading of the UV spectrometer at 595 nm to zero, the absorbance of the CFE was then measured at the same wavelength. The protein concentration can be calculated from the standard curve which was produced by dissolving known amounts of bovine serum albumin (BSA) powder in the same buffer to produce a set of standard solutions (0.1 mg/ml to 1.0mg/m1), adding aliquots (0.1 ml) of these solutions to the diluted dye (5 ml) and and measuring 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 pyrogallol solution (2 ml), 0.1 M phosphate buffer (2 ml, pH 6.6), freshly-prepared 0.5% hydrogen peroxide solution (1 ml) and distilled water (14 ml) at 20°C. This mixture was allowed to stand for 20 seconds at 20°C, then 2M sulphuric acid (1 ml) was added to quench the reaction and the solution was then extracted with ether (2x25 ml). After the reading of the UV spectrometer was adjusted to zero at 420 nm by a reference sample which was an ether extract (2x25 ml) from a mixture of 5% pyrogallol solution (2 ml), 0.1M phosphate buffer (3 ml, pH 6.6), freshlyprepared 0.5% hydrogen peroxide solution (1 ml) and distilled water (14 ml), the absorbance of the organic extract from the CFE reaction was then recorded at the same wavelength. The standard curve can be obtained by measuring absorbance at 420 nm of a set of standard solutions prepared by 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 an Erlenmeyer 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.).  130 After being stirred at room temperature for an appropriate interval, 25 ml of ethyl acetate was added and the mixture was allowed to stir for another 5 min. The resulting mixture was then filtered through Celite and the filtrate was extracted with ethyl acetate (3x50 ml). The Celite pad was sonicated with ethyl acetate (50 ml) for 30 min and then filtered. The combined organic extracts were washed with water, then brine and dried over anhydrous sodium sulphate. Concentration in vacuo afforded the crude mixture of products which were then chromatographed on silica gel column. 3.3.2.6^Typical procedure for biotransformation using CFE (Entry 44, Table13) 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 containing precursor (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 room temperature for an appropriate interval, small amounts of reaction mixture (5% by volume) was withdrawn and extracted with ethyl acetate to provide a sample for TLC and GC analysis. After 3 h of incubation, ethyl acetate (125 ml) was added and the mixture was allowed to stir for another 5 min. The resulting mixture was then filtered through Celite and the filtrate was extracted with ethyl acetate (3x250 ml). The Celite was sonicated with ethyl acetate (150 ml) for 30 minutes and then filtered. The combined organic extracts were washed with water and brine and then dried over 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 (37.8 mg, 88.9%). 3.3.3^Biotransformations using cell homogenate, resuspended pellet and supernatant (CFE) with cofactors added 3.3.3.1 Preparation of cell homogenate, resuspended pellet and supernatant  131 The procedures were similar to those mentioned in the preparation of CFE and were performed at 0-4°C. The homogenate obtained as before was divided into two portions. One portion of the homogenate was used directly in the biotransformation experiment. The remaining portion of the homogenate was then centrifuged at 10,000 g (8,000 rpm) for 30 min. The supernatant was collected 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 peroxidase activity and protein concentration were measured as before for the homogenate, CFE and the resuspended 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 Erlenmeyer flask 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 (0.5 equiv.) and manganous chloride (0.5 equiv.). After being stirred at room temperature for an appropriate interval, ethyl acetate (125 ml) was added and the mixture was allowed to stir for another 5 min. The resulting mixture was then filtered through Celite and the filtrate was extracted with ethyl acetate (3x250 ml). The Celite pad was sonicated with ethyl acetate (150 ml) for 30 minutes and then filtered. The combined organic extracts were washed with water, then brine and dried over anhydrous sodium sulphate. Concentration in vacuo afforded the crude mixture of products which were then chromatographed on silica gel column. ^3.3.3.3^Typical procedure for biotransformation of diol 1 using cell homogenate (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 containing  132  diol 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 manganous chloride (0.5 equiv.). After being stirred at room temperature for 24 h, the reaction mixture was worked up as before through ethyl acetate extraction etc. to afford combined crude extracts (89 mg). 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 pellet resuspended in buffer (Entry 48R, Table-14)  The pellet resuspended in phosphate buffer (50 ml, containing 125 units of peroxidase and 42 mg of protein) prepared from 18 day old cell suspension culture was added to an Erlenmeyer flask containing diol 1 (50 mg) dissolved in EtOH (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 manganous chloride (0.5 equiv.). After being stirred at room temperature for 48 h, the reaction mixture was worked up as before through ethyl acetate extraction etc. to afford combined crude extracts (85 mg). Chromatography on silica gel (25 g) using ethyl acetate as eluent gave recovered diol 1 (19.1 mg, 38%) followed by epoxide 4 (20.1 mg, 40%). 3.4 Biotransformations using the T-43-T cell line 3.4.1 Typical procedure for biotransformation with batchwise addition of diol 1 to 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 added to the cell suspension culture at time intervals of 0, 12, 7, 5 and 12 h. After the last addition, the incubation 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%).  133 3.4.2 Typical procedure for biotransformation with addition of diol 1 to T-43-T growing cells via peristaltic pump (Entry 58, Table-17)  The suspension culture (350 ml, 11 days old) was transferred into a 500 ml benchtop bioreactor which was bottom stirred, aerated through a sintered glass disk at 200 ml/L/min and kept at room temperature. Diol 1 (35 mg) was dissolved in ethanol (6 ml) and added into the cell suspension at a rate of 0.25 ml/h over a 24 h period. After the last addition, the incubation was allowed to continue for a further 24 h. Workup with ethyl acetate extraction etc.as before afforded a combined crude extract (79.6 mg). Chromatography on silica gel (25 g) using ethyl acetate as eluent 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 containing diol 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 (0.5 equiv.). After being stirred at room temperature for 120 h, ethyl acetate (125 ml) was added and the mixture was allowed to stir for another 5 min. The resulting mixture was then filtered through Celite and the filtrate was extracted with ethyl acetate (3x250 ml). The Celite pad was sonicated with ethyl acetate (150 ml) for 30 min and then filtered. The combined organic extracts were washed 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 on silica gel (25 g) using ethyl acetate as eluent gave recovered diol 1 (2.7 mg, 5%), epoxide 4 (35.5 mg, 71%), C-10 alcohol 92 (0.4 mg, 1%) and C-10 alcohol 93 (4.3 mg, 9%).  134 3.4.4 Typical procedure for biotransformation of diol 1 using CFE prepared from T-43-T  CFE (80 ml, containing 329 units of peroxidase and 77 mg of protein) prepared from cell suspension culture (12 days old) was added to an Erlenmeyer flask containing diol 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 (0.5 equiv.). After being stirred at room temperature for 48 h, the reaction mixture was worked up as before and a crude mixture of products (59 mg) was obtained. Chromatography on silica gel (25 g) using ethyl acetate 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 pellet resuspended 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 containing diol 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 (0.5 equiv.). After being stirred at room temperature for 48 h, the reaction mixture was worked up as before and a crude mixture of products (88 mg) was obtained. Chromatography on silica gel (25 g) 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 line 3.5.1^Biotransformation of epoxide 4 using T-43-T whole cells (Entry 69, Table-22) 16  OH 17  4  135  Epoxide 4 (150 mg) was dissolved in absolute ethanol (15 ml) and divided into three equal portions. Each portion was added to an Erlenmeyer flask containing the growing cell suspension culture of T-43-T (500 ml, 10 days old). The resulting suspensions were incubated at 135 rpm and 26°C on a rotary shaker. After shaking for 16 days, the cell suspensions were filtered through Miracloth and the filtrate was extracted with ethyl acetate (3x500 ml). The combined organic extracts were washed with water, brine and dried over anhydrous sodium sulphate. Concentration in vacuo afforded the crude broth extract (145 mg). Ethyl acetate (500 ml) was added to the cell material and the resulting suspension was homogenised at 24,000 rpm for 5 min. The homogenate was filtered through Miracloth and the filtrate 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 on silica 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-triol 109 (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 ; 1 11 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=1 and 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: C 70.77, H 9.96. 3.5.2^Biotransformation of (1S, 2E, 4S, 7E, 11E)-4-hydroxy-2,7,11cembratriene-6-one 43 using T-43-T cell homogenate (Entry 70, Table-24)  136  43  ^  7  Enone 43 (50 mg) was dissolved in ethanol (5 ml) and added to a 1 L Erlenmeyer flask containing T-43-T cell homogenate (267 ml, containing 750 units of peroxidase and 168 mg of protein), 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 was incubated at room temperature with stirring for 120 h. Work up as before afforded a crude product 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 physical properties (MS, 1 H and 13 C NMR) of 7 are identical to those of chemical product 7 obtained by oxidation of epoxide 4 with PCC. 3.5.3^Biotransformation of (3E, 7E, 11S, 12E)-11-isopropy1-4,8-dimethy13,7,12-pentadecatriene-2,14-dione 44 using T-43-T cell homogenate (Entry 72, Table-26)  44  107  Seco-diketone 44 (50 mg) was dissolved in ethanol (10 ml) and added to a 1 L Erlenmeyer flask containing T-43-T cell homogenate (169 ml, containing 752 units of peroxidase and 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.),  137 FMN (0.5 equiv.) and manganous chloride (0.5 equiv.) were added and the mixture was incubated at room temperature with stirring for 120 h. The reaction mixture was worked up as before to afford crude product mixture (129 mg). Chromatography on silica gel (30g) using ethyl acetate 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-dihydroxy11-isopropyl-14-oxo-4,9-pentadecadienal 32 using T-43-T cell homogenate (Entry 75, Table-27) HO 1 9  HO ^OH  OH  ''' 9 10 8 20^1  17 <6 18  / HO  /  19 ‘ 0, OH 5 6/-8N91 0 7  >< +3  20 4 0 2  1  _ 11 413 12 1 "  17 16 18  15  32 Seco-aldehyde 32 (100 mg) was dissolved in ethanol (10 ml) and added to a 1 L Erlenmeyer flask containing T-43-T cell homogenate (422 ml, containing 1498 units of peroxidase and 662 mg of protein) prepared from cell suspension culture (16 days old). To this mixture, phosphate buffer (350m1, pH 6.6), distilled water (150 ml), hydrogen peroxide (4.0 equiv.), FMN (0.5 equiv.) and manganous chloride (0.5 equiv.) were added and the mixture was incubated at room temperature with stirring for 120 h. The reaction mixture was worked up as before to afford crude product mixture (285 mg). Chromatography on silica gel (70 g) using ethyl acetate and hexanes (1:1) as eluents gave (44, 5E, 8S, 9E, 11S)-4, 8-dimethyl-1, 4-epoxy-8hydroxy-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-1,6,8triol 110 (29.3 mg, 29%). The spectral data of 110 were identical with those of the chemical product 110 obtained by reduction of 32 with sodium borohydride. The physical properties of  138 another product 111 are as follows: oil; [a] D : 13.0 0 (c =0.96, CHC13); IR Vmax (CHC13): -  1710 cm-1 ; 1 H 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 16 Hz, 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: 304.2403 3.5.5^Biotransformation of (1S, 2E, 4S, 6R, 7E, 11S, 12S)-2,7-cembradiene4,6,11,12-tetrol 104 with T-43-T cell homogenate (Entry 80, Table-29)  112  „OH^HO ^ 114 113  Tetrol 104 (100 mg) was dissolved in ethanol (200 ml) and divided into two equal portions. 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 cell suspension culture (16 days old) by direct homogenization. The mixture was incubated at room  139 temperature with stirring using a magnetic stir bar for 120 h. The reaction mixture was worked up as before to afford crude product mixture (197 mg). Chromatography on silica gel (50 g) using ethyl acetate as eluent gave (1S, 2E, 4S, 8R, 11S, 12S)-8,11-epoxy-6-oxo-2-cembrene-4,12-diol 112 (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-2,6cembradiene-4,12-diol 114 (1.5 mg, 2%) and recovered 104 (58.4 mg, 54%). The physical properties of (1S, 2E, 4S, 8R, 11S, 12S)-8, 11-epoxy-6-oxo-2-cembren-4,12-diol 112 are as follows[ 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 mass spectrometric data with those described in the literature. [82]  5  F  F  F^  3^  2^  Figure-5, 1 H NMR spectrum of dial 1 in CDC1 3 (400 MHz )  F  1^  0 PPm  78  7  3^'  I^  2  1  Figure-6, 1 1-1 NMR spectrum of nor-solanadione 78 in CDCI 3 (400 MHz )  0 P Pin  5  3  ^  ^ 2^ 1 Figure 7, 1 H NMR spectrum of triol 92 in CDC1 3 (400 MHz ) -  0 ppm  93  4  5  3  2  1  Figure- 8 , 1 H NMR spectrum of triol 93 in CDC1 3 (400 MHz )  94  5  3  4  2^  1  Figure- 9, 1 H NMR spectrum of triol 94 in CDC1 3 (400 MHz )  110  I  I^r•-•^  TV^WI'^  f^I  5^4^3^2^1^0 ppm Figure-10, I H NMR spectrum of seco-alcohol 110 in CDC1 3 (400 MHz )  109  CC  to  O ID  z 1.4 11.0^10.0^9.0^8.0  -t  7.0  I  T T^  '  6.0^5.0 PPM  D1 tO f 1 TIlire  4.0^3.0^2.0^1.0  Figure-11, 1 H NMR spectrum of epoxide 109 in CDC1 3 (200 MHz )  0.0  147 REFRENCES 1^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. 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