**2.12. Biosynthesis of Valerenadiene**

**Scheme 15.** Biosynthetic pathways for the conversion of [2-2H<sup>1</sup>

out two consecutive 1,2-hydride shifts (**Scheme 16**).

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

the same conversion (**Scheme 15**).

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]-FPP to patchoulol isotopomer.

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 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

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

> 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 (**Scheme 18**).

> 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

VoTPS7. VoTPS7 encodes for a synthase that biosynthesizes germacrene C **49** (90%) whereas VoTPS 1 catalyzes conversion of *E,E*-FPP to valerena-1-10-diene **47**. Overexpression of VoTPS produced valarena-1-10-diene **47** on the basis of one and two dimensional NMR analysis, further confirmed by comparison with published spectral data, GC retention time and EIMS fragmentation pattern. The most characteristic feature of the [1-13C] acetate is the FPP derived from the incorporation of [1-13C] acetate had labels located at C1, C3, C5, C7, C9 and C11 as expected using a yeast expression system, specific labeled [1-13C] acetate. FPP was catalytically cyclized (using VoTPS1) and produce valeriana-1,10-diene **47** whose 13C labels were found at C3, C5, C7, C9, C1 and C11. Of these C1 and C11 were adjacent carbons of the isobutyl side chain. The proposed mechanism involves an intermediate of a caryophyllenyl carbocation **53**, 1,2-hydride shift followed by cleavage of C10-C11 bond generates a neutral monocyclic triene **54**. The proposed scheme also indicates formation of other sesquiterpenes

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Based on the experimental labeling data of Pyle et al. [30] and Yeo et al. [31], Paknikar et al. [4] proposed a new alternate biosynthetic route (**Scheme 19**) from IPP to valerenadiene **47** which fits the unusual 13C labeling found in valerian and avoids the previously unreported triene **54**.

In **Scheme 19**, the 2-1-10-11 sequence of carbons in the first cyclic intermediate **57** from *E,E*-FPP becomes 2-10-1-11 in valerenadiene **47** which fits the 13C labeling pattern formed from [1-13C] acetate [4]. The biosynthetic pathway involves one neutral intermediate; bicyclogermacrene **36** found in valerian [32]. The key reaction is a cyclopropylcarbinyl cation-cyclopropylcarbinyl cation rearrangement (CCR) analogues to a key reaction in the biosynthesis of squalane from resqualene [3]. Structure interrelationships of the congeners of valerenadiene **47** including bicyclogermacrene **36**, aromadendrene **51**, germacrene C **49**, germacrene D **50**,

through intermediates tamariscenyl cation **55** and valerenyl cation **56**.

α-gurjunene **58** and malliol **59** were considered in this alternate pathway.

**Scheme 19.** A cyclopropropane route to valerenadiene **47** (numbering based on FPP).

**Scheme 17.** Biosynthesis pathway for valerena-4,7(11) diene **47** and other sesquiterpenes from VoTPS1 and VoTPS2.

**Scheme 18.** Three biosynthetic pathways for valerena-4,7(11) diene **47** and other sesquiterpenes from VoTPS1.

VoTPS7. VoTPS7 encodes for a synthase that biosynthesizes germacrene C **49** (90%) whereas VoTPS 1 catalyzes conversion of *E,E*-FPP to valerena-1-10-diene **47**. Overexpression of VoTPS produced valarena-1-10-diene **47** on the basis of one and two dimensional NMR analysis, further confirmed by comparison with published spectral data, GC retention time and EIMS fragmentation pattern. The most characteristic feature of the [1-13C] acetate is the FPP derived from the incorporation of [1-13C] acetate had labels located at C1, C3, C5, C7, C9 and C11 as expected using a yeast expression system, specific labeled [1-13C] acetate. FPP was catalytically cyclized (using VoTPS1) and produce valeriana-1,10-diene **47** whose 13C labels were found at C3, C5, C7, C9, C1 and C11. Of these C1 and C11 were adjacent carbons of the isobutyl side chain. The proposed mechanism involves an intermediate of a caryophyllenyl carbocation **53**, 1,2-hydride shift followed by cleavage of C10-C11 bond generates a neutral monocyclic triene **54**. The proposed scheme also indicates formation of other sesquiterpenes through intermediates tamariscenyl cation **55** and valerenyl cation **56**.

Based on the experimental labeling data of Pyle et al. [30] and Yeo et al. [31], Paknikar et al. [4] proposed a new alternate biosynthetic route (**Scheme 19**) from IPP to valerenadiene **47** which fits the unusual 13C labeling found in valerian and avoids the previously unreported triene **54**.

In **Scheme 19**, the 2-1-10-11 sequence of carbons in the first cyclic intermediate **57** from *E,E*-FPP becomes 2-10-1-11 in valerenadiene **47** which fits the 13C labeling pattern formed from [1-13C] acetate [4]. The biosynthetic pathway involves one neutral intermediate; bicyclogermacrene **36** found in valerian [32]. The key reaction is a cyclopropylcarbinyl cation-cyclopropylcarbinyl cation rearrangement (CCR) analogues to a key reaction in the biosynthesis of squalane from resqualene [3]. Structure interrelationships of the congeners of valerenadiene **47** including bicyclogermacrene **36**, aromadendrene **51**, germacrene C **49**, germacrene D **50**, α-gurjunene **58** and malliol **59** were considered in this alternate pathway.

**Scheme 17.** Biosynthesis pathway for valerena-4,7(11) diene **47** and other sesquiterpenes from VoTPS1 and VoTPS2.

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**Scheme 18.** Three biosynthetic pathways for valerena-4,7(11) diene **47** and other sesquiterpenes from VoTPS1.

**Scheme 19.** A cyclopropropane route to valerenadiene **47** (numbering based on FPP).

Bicyclogermacrene **36** appears also to be an intermediate in the biosynthesis of related set of sesquiterpene with different stereochemistry found in *Valeriana officinalis*, including tamariscene **60**, pacifigorgiol **61** and (+)-pacifigorgia-1,10-diene **62** (**Scheme 20**). In this scheme also the key reaction is again cyclopropylcarbinyl cation-cyclopropylcarbinyl cation rearrangement (CCR) with this time with a different stereoisomer.

**Scheme 21.** Proposed new consolidated mechanism for the biosynthesis of valerenadiene **47**.

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**Figure 8.** Structural interrelations among the congeners of *Xylaria* sp. and the sequence of formation of isolated

metabolites **65**–**73**.

**Scheme 20.** Biosynthetic pathway of tamariscene **60**, pacifigorgiol **61** and (+)-pacifigorgia-1,10-diene **62** from bicyclogermacrene **36**.

Based on the results of three groups [4, 30, 31] a new consolidated mechanism for the biosynthesis of valerenadiene **47** from FPP *via* bicyclogermacrene **36** through alloaromadendryl cation **63** and CCR is presented which also explains formation of alloaromadendrene **64** (**Scheme 21**) replace alloaromadendryl cation with allo-aromadendryl cation.
