Transannular cyclisation reactions and the germacrane system mediated by enzymes from Cichorium intybus

Chicory ( <em>Cichorium intybus L.),</em> one of the many species of the Compositae family, has been cultivated for the production of the leaves since 300 BC as a food supplement and since the 16th century as a substitute for coffee. The sprouts of the chicory are appreciated for their bitter taste. This bitter taste is associated with the presence of sesquiterpene lactones. The majority of these sesquiterpene lactones possess a guaiane framework, a small number possesses a eudesmane- or a germacrane framework. The abundance of these sesquiterpene lactones is not limited to the leaves of the plant. Considerable amounts of are also present in the root, currently an agricultural waste product. Not only is the root a rich source of sesquiterpene lactones, it also contains a large amount of inulin ( <strong>1</strong> ), a storage carbohydrate which is based on fructose instead of glucose. Fructose, an interesting sweetener, is a versatile building block in the synthesis of several polymers and natural products. The bitter principles in the chicory may find their application as a bitter tasting additive in consumer goods.The biosynthesis of the sesquiterpene lactones in the chicory is believed to start from a head to tail cyclisation of farnesyl pyrophosphate ( <strong>23</strong> ) into a germacrane, followed by cyclisation into eudesmanes and guaianes. This thesis deals with the cyclisation of germacrane synthons and natural germacranes, induced by a root homogenate of fresh chicory. The goal is to determine the substrate specificity of the germacrane cyclase of chicory and to obtain more insight in the biosynthesis of the sesquiterpene bitter principles in <em>C.intybus.</em>A general introduction on the history, use and contents of the chicory is given in chapter 1. In chapter 2, an overview of the literature on the (bio)synthesis of germacrane sesquiterpenes and their possible biotransformation into a variety of cyclised products, is presented.In chapter 3, the synthesis of two (E,E)-cyclodeca-1,5-dienols possessing the germacrane framework ( <strong>100</strong> and <strong>101</strong> ), is described. The cyclisation behaviour of these compounds and the natural germacrane (+)-hedycaryol ( <strong>39</strong> ) towards a chicory root homogenate is discussed. The cyclising enzymes in this homogenate transform the 10-membered ring compounds into products with a eudesmane skeleton by protonation of the C _{<font size="-2">l</font>} -C _{<font size="-2">10</font>} double bond followed by transannular cyclisation and subsequent stereoselective incorporation of a water molecule at C _{<font size="-2">4</font>} . The flexibility of the 10-membered ring system was demonstrated by the formation of the epimeric diols <strong>117-120</strong> from <strong>100</strong> and <strong>101</strong> . The relatively small hydroxyl function at C _{<font size="-2">7</font>} permitted inversion of the germacrane framework, enabling cyclisation through two different syn- conformations. The large isopropanol group of <strong>39</strong> prohibits this inversion to such an extent that cyclisation takes place only through one conformation to give cryptomeridiol ( <strong>125</strong> ). <img src="/wda/abstracts/i2143_1.gif" height="228" width="600"/>In chapter 4, the synthesis of the three (E,E)-cyclodeca-1,6-dienols <strong>131-133</strong> and their cyclisation by a chicory root homogenate is described. Two kinds of hydroazulene alcohols were obtained in these reactions arising from 1,5- and 1,7-cyclisation. The 1,5-cyclisation products ( <strong>134-136</strong> ) are formed through an internal nucleophilic displacement of the allylic alcohol moiety by the Cj-CjO double bond, while in the formation of the 1,7-cyclisation products ( <strong>137-139</strong> ), an allylic isomerisation reaction of the (E,E)-cyclodeca-1,6-dienol skeleton into an allylic (E,E)-cyclodeca-1,5- dienol skeleton preceded the internal nucleophilic displacement reaction. Hydroazulenes possessing a C _{<font size="-2">6</font>} -C _{<font size="-2">7</font>} double bond like <strong>134</strong> resemble natural products like alismol ( <strong>43)</strong> . <img src="/wda/abstracts/i2143_2.gif" height="419" width="800"/>Recently, the structure of a trinor-guaiane, dictamnol ( <strong>140</strong> ), similar to alismol, was published. The ring fusion of dictamnol ( <strong>140</strong> ) was postulated as cis. Since <strong>140</strong> was already synthesised at our laboratory and major discrepancies were found between our NMR spectral data of <strong>140</strong> and those reported in the literature, serious doubt about the stereochemistry and the ring junction arose. Therefore, natural dictamnol was isolated, its stereochemistry was reinvestigated and a structural revision into a transfused hydroazulene ( <strong>152</strong> ) is proposed.In chapter 5, the biotransformation of derivatives of germacrone, a readily available sesquiterpene germacrane, is described. In a number of cases, enzyme mediated cyclisation of the chemically epoxidised germacrone derivatives, had to compete with spontaneous cyclisation reactions. However, some selectivity was observed, especially in the biotransformation of germacrone-4,5-epoxide ( <strong>48</strong> ) into neoprocurcumenol ( <strong>161</strong> ). Compound <strong>161</strong> is the only product obtained through an enzyme-mediated cyclisation of <strong>48</strong> . The C _{<font size="-2">l</font>} -C _{<font size="-2">10</font>} double bond in <strong>161</strong> is characteristic for guaiane bitter principles in the chicory. In boiled root samples, the only conversion that was observed was a homofragmentation reaction of <strong>48</strong> into curcumenone ( <strong>160</strong> ). <img src="/wda/abstracts/i2143_3.gif" height="430" width="600"/>The synthesis of isogermacrone ( <strong>166</strong> ) paved the way for studying the influence of the position and the stereochemistry of the double bond on the ring fusion of the cyclisation products. The 4,5-epoxides of isogermacrone ( <strong>167</strong> ) and isogermacrene B ( <strong>176</strong> ) were transformed by a chicory root homogenate into a cis-fused eudesmane and two tricyclo[4.4.0.0]sesquiterpenes.Chapter 6 deals with the synthesis of (E,Z)-cyclodeca-1,5-dienone <strong>184</strong> and the biotransformation of <strong>184</strong> and structurally related compounds. Transannular cyclisation reactions of (E,Z)-cyclodeca- 1,5-dienes appear to proceed in a different way as compared to the (E,E)-cyclodeca-1,5-dienes in chapter 3-5. Instead of a carbon-carbon bond formation between both double bonds of the germacrane skeleton, ring substituents are involved in the cyclisation process to relieve ring strain. If no additional ring substituents are present, e.g. in E-epoxide <strong>201</strong> , a cis-fused hydroazulene diol ( <strong>202</strong> ) is obtained. The Z-epoxides <strong>211</strong> and <strong>214</strong> were not or not unambiguously transformed by a chicory root homogenate. <img src="/wda/abstracts/i2143_4.gif" height="159" width="600"/>In chapter 7, the biotransformation of farnesyl pyrophosphate ( <strong>23</strong> ) by a partially purified chicory root homogenate is described. Radio-GC and GC-MS analysis of the incubation products obtained from [1- <sup><font size="-2">3</font></SUP>H]-farnesyl pyrophosphate revealed that <strong>23</strong> was initially transformed into germacrene A ( <strong>36</strong> ). However, the Cope rearrangement product, (β-elemene, 222) and two cyclisation products of <strong>36</strong> , α- and β-selinene ( <strong>223</strong> and <strong>224</strong> ) were the only products that were detected in the assay, since <strong>36</strong> is sensitive towards acid and elevated temperatures. <img src="/wda/abstracts/i2143_5.gif" height="132" width="600"/>In conclusion, the substrate specificity of the germacrane cyclase is discussed and an active site model for the germacrane cyclase in proposed together with two tentative biosyntheses of the sesquiterpene lactones in chicory. The most likely biosynthesis starts with cyclisation of farnesyl pyrophosphate ( <strong>23</strong> ) in germacrene A ( <strong>36</strong> ) followed by several oxidation steps to give intermediate <strong>227</strong> . Enzyme mediated cyclisation of <strong>227</strong> would start with the protonation and subsequent dehydration of the C _{<font size="-2">3</font>} -hydroxyl group giving allylic cation <strong>228</strong> . This cation then would give <strong>229</strong> after a 1,5-cyclisation, followed by a selective deprotonation towards the bridgehead carbon atom. Further oxidation of <strong>229</strong> would give the guaianolides <strong>12-17</strong> . <img src="/wda/abstracts/i2143_6.gif" height="570" width="600"/>Glucosidation of the C _{<font size="-2">3</font>} -hydroxyl function of <strong>227</strong> gives sonchuside A ( <strong>20</strong> ) and cichorioside C ( <strong>21</strong> ) which may be cyclised by germacrane cyclasing enzymes into the corresponding eudesmanolides, e.g. <strong>18</strong> . Presumably, glucosidation of the C _{<font size="-2">3</font>} -hydroxyl group prevents the 1,5-cyclisation process towards the guaianolides.