青蒿异源基因的遗传转化及青蒿生物合成相关基因的克隆

本论文由三部分内容组成，一、药用青蒿的遗传转化，即根癌农杆菌和发 根农杆菌介导的转化系统的建立及其影响参数的研究。二、青蒿素生物合成的 分子调控。三、倍半萜生物合成相关基因的克隆。 一、药用青蒿的遗传转化。建立了Ri质粒介导和Ti质粒介导的两种转基因系统， 其中Ri质粒介导青蒿转基因系统的建立是国际上首次报道;以GFP基因为报 告基因，首次获得高效表达的青蒿转绿色荧光蛋白基因的丛生芽，并对GFP基 因的表达进行了组织和细胞水平的定位。此外，对影响两种转基因系统的主要 参数进行了较为详细的研究。上述研究为青蒿素生物合成的分子调控奠定了坚 实的基础。 二、青蒿素生物合成的分子调控。为探索提高青蒿植株或组织和器官中的青蒿 素含量，首次以棉花中克隆的杜松烯合成酶和法呢基焦磷酸合成酶的 cDNA 为 目的基因导入青蒿，对青蒿中青蒿素的生物合成进行了分子调控研究的尝试。 通过已建立的两种转基因系统，将从棉花中克隆的杜松烯合成酶和法呢基焦磷 酸合成酶的 cDNA 导入青蒿，获得转基因发根和转基因植株。结果表明，外源 基因的表达能够影响青蒿素的生物合成，其中法呢基焦磷酸合成酶基因的过量 表达能够促进青蒿素的生物合成，提高转基因发根和植株中的青蒿素的含量。 转基因发根F-26系中青蒿素含量最高达3.01 mg/g.DW，与对照相比青蒿素含量 提高3～4倍;转基因植株的青蒿素含量最高达10.08 mg/g.DW，与对照相比， 转基因植株的青蒿素产物提高2～3倍。此外，研究还表明，在转基因的发根C -37株系中，外源杜松烯合成酶基因的导入和表达可能相应地促进青蒿转基因 发根自身的法昵基焦磷酸基因的表达。 三、倍半萜生物合成相关基因的克隆。采用 RT-PCR 技术，从马铃薯 (Solanum tuberosum L.) 幼叶中克隆了 HMGRII 亚基因家族的一个新的成员 HMGR-c2（GenBank accession No．AF 096838Southem）;杂交分析表明，该基因至少以 两个拷贝以上形式存在于马铃薯基因组中;RT-PCR分析表明，HMGR -c2的 表达在幼苗期无组织特异性，广泛地存在于根、茎、叶等组织中。以青蒿001 株系的苗期叶片为材料，构建了青蒿苗期的λgtll cDNA文库，以PCR筛库方 法从青蒿中克隆一个法呢基焦磷酸合成酶cDNA (Artfps2 GenBank accession No． AF136602)和一个HMGR cDNA(GenBank accession No．AF142473);以青蒿 025株系的苗期叶片为材料，构建了青蒿苗期部分质粒文库以 PCR 筛库方法从 青蒿中克隆一个法昵基焦磷酸合成酶 cDNA (Artfpsl GenBank accession No.AF112881);此外，还从青蒿中克隆了倍半萜合成酶的 cDNA 片段(GenBank accession No.AF156854)。其中青蒿倍半萜合成酶基因的克隆是目前国际上本研 究领域最受关注的焦点和难点之一。至此，本研究已将与青蒿素生物合成相关 的三个重要的关键酶基因基本克隆，这无疑将加速青蒿素生物合成的基础和应 用研究的进程。

水菖蒲的化学成分研究和忍冬属植物的色谱分析

本论文由三章组成。第一章阐述了藏药水菖蒲的化学成分研究，共分离鉴定了39个化学成分，其中6个为新化合物。第二章报道了几种忍冬属植物的HPLC、HPLC-MS、GC分析以及抑菌活性、重金属含量测定结果。第三章概述了菖蒲属植物的研究进展。 第一章报道了水菖蒲(Acorus calamus L.)化学成分的分离纯化与结构鉴定。采用正、反相硅胶柱层析等分离方法，从水菖蒲的根中共分离出41个化合物，通过红外、质谱、核磁共振及X-ray单晶衍射等波谱方法和模拟计算方法鉴定了其中39个化合物的结构，主要为倍半萜、苯丙素、甾体类化合物。其中含有5个新的倍半萜类化合物和1系列新的甾体皂苷衍生物。经波谱分析将它们的结构鉴定为 1b, 7a(H)-cadinane-4a, 6a, 10a-triol (1), (2R,6R,7S,9S)-1(10), 4-cadinadiene-2, 9-diol (2), 1a, 5b-guaiane-10a-O-ethyl-4b, 6b-diol (7), 6b, 7b(H)-cadinane-1a, 4a, 10a-triol (13)，(1R,4R,6S,10R)-1-hydroxy-7(11)-cadinen-5, 8-dione (14)， 4′-O-正n碳酰基-3-O- β-D-葡萄糖基谷甾醇(n=14, 16, 18, 22) (15)。 第二章包括四个部分。第一部分报道了忍冬属三种植物40个样品的HPLC测定和对主要活性成分绿原酸的定量分析结果，以及运用HPLC-MS技术对色谱图中8个峰进行指认。在此基础上，考察了种植和采收多个因素对绿原酸含量的影响。第二部分报道了忍冬属三种植物27个样品的GC分析，根据样品的挥发性成分的保留时间对不同样品进行了定性比较，并考察了花期及海拔高度对植物挥发性成分的影响。第三、四部分分别阐述了灰毡毛忍冬和红腺忍冬的体外抑菌活性研究和重金属含量测定结果。 第三章全面系统地概述了菖蒲属植物的化学成分和药理活性研究进展。 This dissertation is composed by three chapters. The first chapter elaborates the phytochemical investigation of Acorus calamus L. Thirty-nine compounds including six new compounds were isolated and identified. The second chapter reports the research on genus Lonicera by HPLC, HPLC-MS and GC. Antifungal activity and heavy metals measurement of genus Lonicera were reported. The third chapter is a review about the research progress on the plant family of Acorus. The first chapter focuses on the isolation and identification of chemical constituents from Acorus calamus L.. Forty-one compounds were isolated from the root of Acorus calamus L. by repeat column chromatography over normal and reversed phase silica gel, the structure of thirty-nine compounds was identified by spectroscopic methods and computational methods, including IR, MS, NMR and X-ray. Those compounds mainly belonged to sesquiterpene, phenylpropanoid and steroid. Among them, five are new sesquiterpenes and one series are new steroid glycoside derivatives. Their structure were suggested as 1b, 7a(H)-cadinane-4a, 6a, 10a-triol (1), (2R,6R,7S,9S)-1(10), 4-cadinadiene-2, 9-diol (2), 1a, 5b-guaiane-10a-O-ethyl-4b, 6b- diol (7), 6b, 7b(H)-cadinane-1a, 4a, 10a-triol (13), (1R,4R,6S,10R)-1-hydroxy-7(11)- cadinen-5, 8-dione (14), 4′-O-carbonyl-3-O-β-D-glucosyl-sitosterol (carbonyl = tetradecanoyl, hexadecanoyl, octadecyl, docosanoyl) (15). The second chapter consists of four parts. The first part reports the HPLC analysis of forty samples of the genus Lonicera, and the quantitative investigation of chlorogenic acid in these samples by HPLC analysis. Relationship between the content of chlorogenic acid in different samples and their planting conditions and harvesting time were discussed. Furthermore, eight compounds were identified or tentatively characterized based on their mass spectra and UV spectra profiles. The second part is about qualitative analysis of the volatile constituent in twenty-seven samples of genus Lonicera by GC. The effect of planting altitude and harvesting time on the volatile constituent was also investigated. The third and fourth parts describe the antifungal activity and content of some kinds of heavy metals of L. macranthoides Hand.-Mazz. and L. hypoglauca Miq.. The third chaspter is a review about the research progress of the plant family of Acorus.

Brominated Selinane Sesquiterpenes from the Marine Brown Alga Dictyopteris divaricata

Two new brominated selinane sesquiterpenes, 1-bromoselin-4(14), 11-diene (1) and 9-bromoselin-4(14), 11-diene (2), one known cadinane sesquiterpene, cadalene (3), and four known selinane sesquiterpenes, alpha-selinene (4), beta-selinene (5), beta-dictyopterol (6), and cyperusol C (7), were isolated from a sample of marine brown alga Dictyopteris divaricata collected off the coast of Yantai (China). Their structures were established by detailed MS and NMR spectroscopic analysis, as well as comparison with literature data.

Cadinane Sesquiterpenes from the brown alga Dictyopteris divaricata

Seven new cadinane sesquiterpenes, (-)-(1R,6S,7S,10R)-1-hydroxycadinan-3-en-5-one (1), (+)-(1R,5S,6R,7S, 10R)-cadinan-3-ene-1,5-diol (2), (+)-(1R,5R,6R,7S,10R)-cadinan-3-ene-1,5-diol (3), (+)-(1R,5S,6R,7S,10R)-cadinan-4(11)-ene-1,5-diol (4), (+)-(1R,5R,6R,7R,10R)-cadinan-4(11)-ene-1,5,12-triol (5), (-)-(1R,4R,5S,6R,7S, 10R)-cadinan-1,4,5-triol (6), and (-)-(1R,6R,7S,10R)-11-oxocadinan-4-en-1-ol (7), together with nine known compounds were isolated from the brown alga Dictyopteris divaricata. The structures of the new natural products, as well as their absolute configuration, were established by means of spectroscopic data including IR, HRMS, 1D and 2D NMR, single-crystal X-ray diffraction, and CD. All compounds were inactive against several human cancer cell lines including lung adenocarcinoma (A549), stomach cancer (BGC-823), breast cancer (MCF-7), hepatoma (Bel7402), and colon cancer (HCT-8) cell lines.

Minor sesquiterpenes with new carbon skeletons from the brown alga Dictyopteris divaricata

Five minor sesquiterpenes (1-5) with two novel carbon skeletons, together with a minor new oplopane sesquiterpene ( 6), have been isolated from the brown alga Dictyopteris divaricata. By means of spectroscopic data including IR, HRMS, 1D and 2D NMR, and CD, their structures including absolute configurations were assigned as (+)-(1R, 5S, 6S, 9R)3- acetyl-1-hydroxy-6-isopropyl-9-methylbicyclo[4.3.0] non-3-ene ( 1), (+)-(1R, 3S, 4S, 5R, 6S, 9R)-3-acetyl-1,4-dihydroxy-6- isopropyl-9-methylbicyclo[4.3.0] nonane (2), (+)-(1R, 3R, 4R, 5R, 6S, 9R)-3-acetyl-1,4-dihydroxy-6-isopropyl-9-methylbicyclo[ ;4.3.0] nonane ( 3), (+)-(1S, 2R, 6S, 9R)-1-hydroxy-2-(1-hydroxyethyl)-6-isopropyl-9-methylbicyclo[4.3.0] non-4-en-3-one (4), (-)-( 5S, 6R, 9S)-2-acetyl-5-hydroxy-6-isopropyl-9-methylbicyclo[4.3.0] non-1-en-3-one ( 5), and (-)-( 1S, 6S, 9R)- 4-acetyl- 1-hydroxy-6-isopropyl-9-methylbicyclo[ 4.3.0] non-4-en-3-one ( 6). Biogenetically, the carbon skeletons of 1-6 may be derived from the co-occurring cadinane skeleton by different ring contraction rearrangements. Compounds 1-6 were inactive (IC50 > 10 mu g/mL) against several human cancer cell lines.

Norsesquiterpenes from the brown alga Dictyopteris divaricata

Three bisnorsesquiterpenes (1-3) with novel carbon skeletons and a norsesquiterpene (4) have been isolated from the brown alga Dictyopteris divaricata. By means of spectroscopic data including IR, HRMS, 1D and 2D NMR techniques, single-crystal X-ray diffraction, and CD, their structures including absolute configurations were proposed as (+)-1R,6S,9R)-1-hydroxyl-6-isopropyl-9-methylbicyclo[4.3.0]non-4-en3-one (1), (-)-(1S,6S,9R)-1-hydroxyl-6-isopropyl-9-methylbicyclo[4.3.0] non-4-en-3-one (2), (+)-(5S,6R,9S)5-hydroxyl-6-isopropyl-9-methylbicyclo [4.3.01 non-1-en-3-one (3), and (-)-(1R,7S,10R)-1-hydroxy-1lnorcadinan-5-en-4-one (4). Biogenetically, the carbon skeleton of 1-3 may be derived from the co-occurring cadinane skeleton by ring contraction and loss of two carbon units, and compound 4 from the oxidation of cadinane derivatives. Compounds 1-4 were inactive (IC50 > 10 mu g/mL) against several human cancer cell lines including lung adenocarcinoma (A549), stomach cancer (BGC-823), breast cancer (MCF-7), hepatoma (Bel7402), and colon cancer (HCT-8) cell lines.

Sesquiterpenes from Laurencia similis

One new sesquiterpene, (4E)-1-bromo-5-[(1'S*, 3'R*)-3'-bromo-2',2'-dimethyl-6'-methylenecyclohexyl]-3-methylpent-4-ene-2,3-diol (1), and fifteen known sesquiterpenes, isopalisol (2), luzonensol (3), palisadin B (4), aplysistatin (5), palisadin A (6), 4-hydroxyl-palisudin C (7), 5-acetoxypalisadin B (8), 10-hydroxyaristolan-9-one (9), aristol-8-en-1-one (10), aristolan-9-en-1-one (11), aristolan-1(10)-en-9-one (12), aristolan-1( 10)-en-9-ol (13), aristolan-1(10), 8-diene (14), aristolan-1,9-diene (15) and aristofone (16), were isolated from a sample of marine red alga Laurencia similis. Their structures were established by detailed NMR spectroscopic analysis and comparison with literature data. Compounds 2-9, and 16 were isolated for the first time from this species. All these metabolites were submitted for a cytotoxicity assay against the tumor cell line BEL7402 (human liver adenocarcinoma), but all of them were found inactive (IC50 > 10 mu g/mL).

Permeation of bioactive constituents from Arnica montana preparations through human skin in-vitro

This study investigated and characterised transdermal permeation of bioactive agents from a topically applied Arnica montana tincture. Permeation experiments conducted over 48 h used polydimethylsiloxane (silastic) and human epidermal membranes mounted in Franz-type diffusion cells with a methanol-water (50:50 v/v) receptor fluid. A commercially available tincture of A. montana L. derived from dried Spanish flower heads was a donor solution. Further donor solutions prepared from this stock tincture concentrated the tincture constituents 1, 2 and 10 fold and its sesquiterpene lactones 10 fold. Permeants were assayed using a high-performance liquid chromatography method. Five components permeated through silastic membranes providing peaks with relative retention factors to an internal standard (santonin) of 0.28, 1.18, 1.45, 1.98 and 2.76, respectively. No permeant was detected within 12 h of applying the Arnica tincture onto human epidermal membranes. However, after 12 h, the first two of these components were detected. These were shown by Zimmermann reagent reaction to be sesquiterpene lactones and liquid chromatography/diode array detection/mass spectrometry indicated that these two permeants were 11,13-dihydrohelenalin (DH) analogues (methacrylate and tiglate esters). The same two components were also detected within 3 h of topical application of the 10-fold concentrated tincture and the concentrated sesquiterpene lactone extract.

Volatile terpenoids and C13 norisoprenoids in wines: development of rapid methods of analysis and evaluation of the sesquiterpenoids biological potential

Vitis vinifera L., the most widely cultivated fruit crop in the world, was the starting point for the development of this PhD thesis. This subject was exploited following on two actual trends: i) the development of rapid, simple, and high sensitive methodologies with minimal sample handling; and ii) the valuation of natural products as a source of compounds with potential health benefits. The target group of compounds under study were the volatile terpenoids (mono and sesquiterpenoids) and C13 norisoprenoids, since they may present biological impact, either from the sensorial point of view, as regards to the wine aroma, or by the beneficial properties for the human health. Two novel methodologies for quantification of C13 norisoprenoids in wines were developed. The first methodology, a rapid method, was based on the headspace solid-phase microextraction combined with gas chromatography-quadrupole mass spectrometry operating at selected ion monitoring mode (HS-SPME/GC-qMS-SIM), using GC conditions that allowed obtaining a C13 norisoprenoid volatile signature. It does not require any pre-treatment of the sample, and the C13 norisoprenoid composition of the wine was evaluated based on the chromatographic profile and specific m/z fragments, without complete chromatographic separation of its components. The second methodology, used as reference method, was based on the HS-SPME/GC-qMS-SIM, allowing the GC conditions for an adequate chromatographic resolution of wine components. For quantification purposes, external calibration curves were constructed with β-ionone, with regression coefficient (r2) of 0.9968 (RSD 12.51 %) and 0.9940 (RSD of 1.08 %) for the rapid method and for the reference method, respectively. Low detection limits (1.57 and 1.10 μg L-1) were observed. These methodologies were applied to seventeen white and red table wines. Two vitispirane isomers (158-1529 L-1) and 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN) (6.42-39.45 μg L-1) were quantified. The data obtained for vitispirane isomers and TDN using the two methods were highly correlated (r2 of 0.9756 and 0.9630, respectively). A rapid methodology for the establishment of the varietal volatile profile of Vitis vinifera L. cv. 'Fernão-Pires' (FP) white wines by headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (HS-SPME/GCxGC-TOFMS) was developed. Monovarietal wines from different harvests, Appellations, and producers were analysed. The study was focused on the volatiles that seem to be significant to the varietal character, such as mono and sesquiterpenic compounds, and C13 norisoprenoids. Two-dimensional chromatographic spaces containing the varietal compounds using the m/z fragments 93, 121, 161, 175 and 204 were established as follows: 1tR = 255-575 s, 2tR = 0,424-1,840 s, for monoterpenoids, 1tR = 555-685 s, 2tR = 0,528-0,856 s, for C13 norisoprenoids, and 1tR = 695-950 s, 2tR = 0,520-0,960 s, for sesquiterpenic compounds. For the three chemical groups under study, from a total of 170 compounds, 45 were determined in all wines, allowing defining the "varietal volatile profile" of FP wine. Among these compounds, 15 were detected for the first time in FP wines. This study proposes a HS-SPME/GCxGC-TOFMS based methodology combined with classification-reference sample to be used for rapid assessment of varietal volatile profile of wines. This approach is very useful to eliminate the majority of the non-terpenic and non-C13 norisoprenic compounds, allowing the definition of a two-dimensional chromatographic space containing these compounds, simplifying the data compared to the original data, and reducing the time of analysis. The presence of sesquiterpenic compounds in Vitis vinifera L. related products, to which are assigned several biological properties, prompted us to investigate the antioxidant, antiproliferative and hepatoprotective activities of some sesquiterpenic compounds. Firstly, the antiradical capacity of trans,trans-farnesol, cis-nerolidol, α-humulene and guaiazulene was evaluated using chemical (DPPH• and hydroxyl radicals) and biological (Caco-2 cells) models. Guaiazulene (IC50= 0.73 mM) was the sesquiterpene with higher scavenger capacity against DPPH•, while trans,trans-farnesol (IC50= 1.81 mM) and cis-nerolidol (IC50= 1.48 mM) were more active towards hydroxyl radicals. All compounds, with the exception of α-humulene, at non-cytotoxic levels (≤ 1 mM), were able to protect Caco-2 cells from oxidative stress induced by tert-butyl hydroperoxide. The activity of the compounds under study was also evaluated as antiproliferative agents. Guaiazulene and cis-nerolidol were able to more effectively arrest the cell cycle in the S-phase than trans,trans-farnesol and α-humulene, being the last almost inactive. The relative hepatoprotection effect of fifteen sesquiterpenic compounds, presenting different chemical structures and commonly found in plants and plant-derived foods and beverages, was assessed. Endogenous lipid peroxidation and induced lipid peroxidation with tert-butyl hydroperoxide were evaluated in liver homogenates from Wistar rats. With the exception of α-humulene, all the sesquiterpenic compounds under study (1 mM) were effective in reducing the malonaldehyde levels in both endogenous and induced lipid peroxidation up to 35% and 70%, respectively. The developed 3D-QSAR models, relating the hepatoprotection activity with molecular properties, showed good fit (R2LOO > 0.819) with good prediction power (Q2 > 0.950 and SDEP < 2%) for both models. A network of effects associated with structural and chemical features of sesquiterpenic compounds such as shape, branching, symmetry, and presence of electronegative fragments, can modulate the hepatoprotective activity observed for these compounds. In conclusion, this study allowed the development of rapid and in-depth methods for the assessment of varietal volatile compounds that might have a positive impact on sensorial and health attributes related to Vitis vinifera L. These approaches can be extended to the analysis of other related food matrices, including grapes and musts, among others. In addition, the results of in vitro assays open a perspective for the promising use of the sesquiterpenic compounds, with similar chemical structures such as those studied in the present work, as antioxidants, hepatoprotective and antiproliferative agents, which meets the current challenges related to diseases of modern civilization.

Développement d'un tandem multicatalytique méthylénation - couplage de Heck et Approche synthétique de l'Hodgsonox

Cette thèse comprend deux parties distinctes, dans lesquelles seront décrits tout d’abord, le développement d’un procédé multicatalytique en un seul pot d’une réaction de méthylénation suivie d’un couplage de Heck, puis dans un second temps, une étude vers la synthèse de l’Hodgsonox. Le premier thème de la thèse correspond à la mise en place d’un procédé en un seul pot, basé sur la méthodologie de méthylénation catalysée par un métal de transition, développée au sein du groupe du Pr. Lebel, et sur des couplages de Heck. Différentes études de compatibilité des réactifs mis en présence sont abordées, ainsi que le choix des conditions optimales (Pd(OAc)2 et P(o-tol)3) pour la réalisation d’un tel système qui ne requiert aucun isolement du produit intermédiaire. Il a été démontré que la présence de triphénylphosphine en excès inhibe la réaction de couplage de Heck, ce qui a finalement orienté notre choix vers les sels de cuivre pour la catalyse de la réaction de méthylénation. Le tandem séquentiel a ensuite été appliqué à la synthèse de divers stilbènes, notamment des composés dérivés du Resvératrol, molécule d’intérêt thérapeutique pour les maladies cardiovasculaires, et à la synthèse d’indanes substitués, avec un couplage intramoléculaire, avec de bons rendements. La deuxième partie de cette thèse traite de l’étude menée vers la synthèse de l’Hodgsonox. Cette molécule correspond à une nouvelle classe de sesquiterpènes tricycliques, comportant un dihydropyrane doté d’une fonction éther diallylique. Cette molécule représente un défi synthétique pour le groupe du Pr. Lebel, qui envisage de synthétiser les deux doubles liaisons terminales au moyen de la méthodologie de méthylénation développée au sein du groupe. L’Hodgsonox, dont la biosynthèse utilise la voie MEP, a un potentiel insecticide pour la croissance de la larve de la mouche verte d’Australie, Lucilia cuprina. La synthèse envisagée au cours de ces travaux est basée sur la formation préalable d’un cycle à 5 chaînons, comportant 3 centres stéréogéniques, puis sur la cyclisation du cycle pyranique au moyen d’une réaction d’insertion dans un lien O H. Un dédoublement cinétique dynamique sur une δ butyrolactone substituée permet de fixer la stéréochimie relative de deux centres chiraux dès la première étape. Le cycle à 5 chaînons est ensuite formé par métathèse après 6 étapes avec un rendement de 37%. Une addition conjuguée suivie d’une réaction de Saegusa et d’une réaction d’hydrosilylation introduit le groupement isopropyle de manière syn. Après mise en place d’un groupement céto-ester, un transfert de groupement diazonium permet de préparer le précurseur pour la réaction d’insertion dans un lien O-H. Le bicycle correspondant à la structure de base de l’Hodgsonox a été préparé au moyen de 16 étapes linéaires avec un rendement global de 12%.

Progrès vers la synthèse totale de l'hodgsonox

Ce mémoire présente une poursuite de l’étude vers la synthèse de l’hodgsonox, un sesquiterpénoïde naturel possédant des propriétés insecticides contre la mouche verte d’Australie, Lucilia cuprina. L’hodgsonox comporte six centres stéréogènes et trois cycles : un époxyde fusionné à un cycle à cinq chaînons et une fonction éther cyclique à six chaînons doublement allylique. La stratégie de synthèse de l’hodgsonox proposée comporte dix-neuf étapes linéaires. Elle s’appuie sur les travaux préliminaires de Lise Bréthous, étudiante au doctorat, de Nicolas Lévaray, étudiant à la maîtrise, ainsi que du Dr. Ying Dong Lu et de la Dr. Sonia Diab, qui ont tous travaillé précédemment dans le groupe de la Pr. Lebel. La première étape de cette synthèse consiste en une hydrogénation cinétique dynamique de Noyori permettant d’obtenir un seul diastéréoisomère à partir de l’α-acétylbutyrolactone. Une séquence de six étapes linéaires supplémentaires, comprenant l’ouverture de la lactone ainsi qu’une métathèse d’oléfine, permet d’obtenir le cycle à cinq chaînons avec un rendement global de 37%. L’unité isopropyle est par la suite installée par une addition conjuguée pour former un éther d’énol silylé, qui est directement oxydé en la cétone correspondante avec l’acétate de palladium(II). Une réaction d’hydrosilylation subséquente permet d’obtenir la stéréochimie syn attendue de l’unité isopropyle. Par la suite, la carbonylation d’un intermédiaire triflate permet d’obtenir le squelette de base pour la formation de l’éther cyclique. Enfin, le cycle à six chaînons est formé par insertion O−H intramoléculaire d’un diazo avec un rendement global de 2% sur 17 étapes. Les travaux spécifiques de l’auteure comprennent l’évaluation de conditions catalytiques pour l’oxydation de Saegusa de l’éther d’énol silylé. Les trois dernières étapes ont également été explorées par l’auteure. Il s’agit de l’époxydation de la double liaison endocyclique, de l’insertion dans un lien O−H catalysée par un dimère de rhodium, et de la méthylénation. Enfin, l’exploration d’une voie alternative a été entamée. Cette nouvelle voie consiste à former l’éther cyclique par une substitution nucléophile sur un époxyde. La double liaison exo-cyclique serait installée par une simple réaction de déshydratation.

Flavour characterization of fresh and processed pennywort (Centella asiatica L.) juices

The flavour characteristics of fresh and processed pennywort juices treated by pasteurization, sterilization and high pressure processing (HPP) were investigated by using solid-phase micro-extraction combined with gas chromatography-mass spectrometry. Sesquiterpene hydrocarbons comprised the major class of volatile components present and the juices had a characteristic smell due to the presence of volatile compounds including β-caryophyllene, humulene, E-β-farnesene, α-copaene, alloaromadendrene and β-elemene. All processing operations caused a reduction in the total volatile concentration, but HPP caused more volatile acyclic alcohols, aldehydes and oxygenated monoterpenoids to be retained than pasteurization and sterilization. Ketones were not present in fresh pennywort juice, but 2-butanone and 3-nonen-2-one were generated in all processed juices, and 2-nonanone and 2-hexanone were present in pasteurized and sterilized juices. Other chemical changes including isomerization were also reduced by HPP compared to pasteurization, and sterilization.

Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function

Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases.

Synthesis of labelled dihydroartemisinic acid

[15-(CH3)-C-13-H-2]-Dihydroartemisinic acid (2a), [15-(CH3)-H-2]-dihydroartemisinic acid (2b) and [15-(CH3)-C-13]-dihydroartemisinic acid (2c) have been obtained in good yield and high isotopic enrichment by a reconstructive synthesis from artemisinin. These labelled compounds were designed to be used in biosynthetic experiments to determine the origins of artemisinin and other sesquiterpene natural products from Artemisia annua. (C) 2003 Elsevier Ltd. All rights reserved.