**2.11. Biosynthesis of patchouli alcohol (patchoulol)**

The history of patchouli alcohol **39** from its isolation till date has narrated in a recent exhaustive review article [26]. Biosynthetic pathways were proposed based on experimental work for the conversion of FPP to patchouli alcohol **39** (**Scheme 14**).

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**Scheme 13.** Mechanism of pogostol **34** formation from FPP.

**2.10. Biosynthesis of Pogostol**

98 Terpenes and Terpenoids

(**Figure 5**).

tol **34** (**Scheme 13**).

**34** thus represents (+)-pogostol (**Figure 6**).

**2.11. Biosynthesis of patchouli alcohol (patchoulol)**

**Figure 5.** Biosynthesis of Pogostol **34** using isotopomers of mevalonolactone.

for the conversion of FPP to patchouli alcohol **39** (**Scheme 14**).

Biosynthesis of pogostol **34** by the endophytic fungus *Geniculosporium* was investigated by Dickschat and co-workers [22]. In this study, six 13C labeled isotopomers of mevalonolactone were synthesized and used in feeding experiments with the endophytic fungus *Geniarlosperium*. Feeding experiments with **35a** and **35b** gave insights into the stereochemical course of the terpene cyclization. The methyl group of the mevalonolactone that is labeled in these two isotopomers is converted into terminal (z)-methyl group of FPP (C-13). Both feeding experiments showed that the deprotonation step leading to germacrene A **36** proceeds with stereospecific deprotonation of C-13 and not C-12 of FPP

The volatile fraction was extracted by closed loop stripping apparatus followed by direct <sup>13</sup>CNMR analysis (CLSA-NMR) newly developed by the same group. The biosynthesis of pogostol **34** proceeds through initial formation of germacrene-A **36**. Protonation of 4,5 double bond initiates a second cyclization to cation which gets neutralized with water to give pogos-

In view of correlation of (−)-pogostol **37** with (+)-bulnesol **38** with known absolute stereochemistry, (−)-pogostol be represented by the stereostructure **37** [23–25]. The stereostructure

The history of patchouli alcohol **39** from its isolation till date has narrated in a recent exhaustive review article [26]. Biosynthetic pathways were proposed based on experimental work

**Figure 6.** Absolute stereochemistry of (−)-pogostol **37**—correlation of (−)-pogostol **37** and (+)-bulnesol **38**.

**Scheme 14.** Mechanism proposed for cyclization and rearrangement of FPP to patchoulol **39**.

Croteau et al. [27] and Akhila et al. [28] proposed biosynthetic pathways for the conversion of FPP to patchouli alcohol **39** based on experimental work. Croteau et al. reported the 1,3-shift for conversion of **40** to **41** while Akhila et al. proposed two consecutive 1,2-hydride shifts for the same conversion (**Scheme 15**).

Incubation of isotopically pure [2-2H<sup>1</sup>

] (*E,E*)-farnesyldisulfate with recombinant patchoulol

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Recent Developments in Selected Sesquiterpenes: Molecular Rearrangements, Biosynthesis…

] patchouliol is

101

synthase (rPTS) from *Pogostemon cablin* afforded a 65:35 mixture of monodeuterated and didueterated patchouliols and several hydrocarbons of which eight have been identified. This is confirmed by extensive NMR analysis on the labeled patchouliol mixture and comparison with those of unlabeled patchouliol. Deuterium label was located at position C5 (both isotopomers

rationalized through an unknown (so far) hydrocarbon **42** which could incorporate deuterium at C12. This significant observation may have implication on the biosynthesis of nor-patchouliol **43** a congener of patchouliol, the biosynthesis is based on the earlier work [26] (**Figure 7**).

The interesting observation which can be made on the patchouli oil constituents that though α-guaine **44** and α-bulnesene **45** are genuine natural products [26], (+)-guaiol **46** and (+)-bul-

**Figure 7.** Structures of nor-patchouliol **43**, α-guaiene 44, α-bulnesene **45**, (+)-guaiol **46** and (+)-bulnesol **38**.

Pyle et al. [30] reported the first enzymatic synthesis of valerena-4,7(11)-diene **47** (numbering used for valarenic acid) by a unique TPS from *Valeriana officinalis*. They identified two TPS's VoTPS1 and VoTPS2. Transgenic yeast expressing VoTPS1 produced germacrene B **48**, germacrene C **49** and germacrene D **50**. On the other hand, VoTPS 2 produced valerena-4,7(11) diene **47** as a major compound was substantiated by 13CNMR and GC–MS comparison with the synthetic standard. Minor products were identified as bicyclogermacrene **51** and alloaromadendrene **52**. The proposed mechanism involves ring contraction of germacrane ring to a nine-membered intermediate having isobutenyl side chain. Cyclization gives valerena-

Yeo et al. [31] proposed a mechanism wherein the isobutyl side chain is derived by the intermediacy of a caryophyllenyl carbocation **53**. A 1,2-hydride shift followed by opening of the cyclobutyl ring. In this way the two methylene carbons of the isobutenyl side chain are predicted to arise from C1 and C11 of the originating FPP and therefore should become labeled when [1-13C] acetate is incorporated into FPP by mevalonate pathway operating in yeast

Valerina-1-10-diene **47** and related sesquiterpenes retain an isobutyl side chain whose origin has been recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. They identified seven *Valeriana officinalis*, terpene synthase genes (VoTPSs) and two were functionally characterized as sesquiterpene synthase VoTPS1 and

nesol **38** has never been reported to be present in patchouli oil.

**2.12. Biosynthesis of Valerenadiene**

4,7(11)-diene **47** (**Scheme 17**).

(**Scheme 18**).

ca. 100%) and at C12 (minor isotopomer, 30–35%). The formation of [5,12-2H<sup>2</sup>

**Scheme 15.** Biosynthetic pathways for the conversion of [2-2H<sup>1</sup> ]-FPP to patchoulol isotopomer.

The recent isotopic labeling studies of Coates and colleagues [29] unrevealed the biosynthetic pathways for **39** which confirmed the 1,3-hydride shift across the five membered ring ruling out two consecutive 1,2-hydride shifts (**Scheme 16**).

**Scheme 16.** Proposed biosynthesis of patchouliol **39** from deuterated FPP.

Incubation of isotopically pure [2-2H<sup>1</sup> ] (*E,E*)-farnesyldisulfate with recombinant patchoulol synthase (rPTS) from *Pogostemon cablin* afforded a 65:35 mixture of monodeuterated and didueterated patchouliols and several hydrocarbons of which eight have been identified. This is confirmed by extensive NMR analysis on the labeled patchouliol mixture and comparison with those of unlabeled patchouliol. Deuterium label was located at position C5 (both isotopomers ca. 100%) and at C12 (minor isotopomer, 30–35%). The formation of [5,12-2H<sup>2</sup> ] patchouliol is rationalized through an unknown (so far) hydrocarbon **42** which could incorporate deuterium at C12. This significant observation may have implication on the biosynthesis of nor-patchouliol **43** a congener of patchouliol, the biosynthesis is based on the earlier work [26] (**Figure 7**).

**Figure 7.** Structures of nor-patchouliol **43**, α-guaiene 44, α-bulnesene **45**, (+)-guaiol **46** and (+)-bulnesol **38**.

The interesting observation which can be made on the patchouli oil constituents that though α-guaine **44** and α-bulnesene **45** are genuine natural products [26], (+)-guaiol **46** and (+)-bulnesol **38** has never been reported to be present in patchouli oil.
