Chemoenzymatic Formation of Oxa-Terpenoids by Sesqui- and Diterpene Synthase-Mediated Biotransformations with 9-Oxy-FPP Ether Derivatives

Abstract

Farnesyl pyrophosphate derivatives bearing an additional oxygen atom at position 5 proved to be very suitable for expanding the substrate promiscuity of sesquiterpene synthases (STSs) and the formation of new oxygenated terpenoids. Insertion of an oxygen atom in position 9, however, caused larger restraints that led to restricted acceptance by STSs. In order to reduce some of the proposed restrictions, two FPP-ether derivatives with altered substitution pattern around the terminal olefinic double bond were designed. These showed improved promiscuity toward different STSs. Four new cyclized terpenoids with an embedded ether group were isolated and characterized. In the case of two cyclic enol ethers, also the corresponding "hydrolysis" products, linear hydroxyaldehydes, were isolated. Interestingly, all cyclization products originate from an initial 1 → 12 cyclization unprecedented when native farnesyl pyrophosphate serves as a substrate. We found that the most suitable FPP derivative with an additional oxygen at position 9 does not carry any methyl group on the terminal alkene, which likely reduces steric congestion when the preferred conformation for cyclization is adopted in the active site.

Scheme 1. Structures of FPP 1, and FPP Ether 2 and 3; Previous Studies on the Promiscuity of Different STSs towards 2 and 3 and Sesquiterpene T-muurolol 8 (OPP is the Abbreviation for Diphosphate Which Is Commonly Employed as Trisammonium Salt Here)

Scheme 2. Syntheses of New FPP Derivatives 9 and 10 Starting from Geraniol (11) via Alcohol 12

Conditions: (a) imidazole, TBDPSCl, N,N-DMF, 0 to rt (97%); (b) SeO₂, salicylic acid, water, t-BuOOH, CH₂Cl₂, 0 °C to rt, then NaBH₄, MeOH, 0 °C (31%); (c), (d) 12 → 13: NaH, allyl bromide, THF, 0 °C to rt (82%) then TBAF, THF, 0 °C to rt (93%); (c), (d) 12→ 14: NaH, tiglyl bromide, THF, 0 °C to rt, then TBAF, THF, 0 °C to rt (32% o2s); (e) for 13 NCS, DMS, CH₂Cl₂, 0 °C then ((n-Bu)₄N)₃P₂O₇H, MeCN, rt (82%) and for 14 NCS, DMS, CH₂Cl₂, −50 °C to −40 °C to 0 °C to rt, then ((n-Bu)₄N)₃P₂O₇H, MeCN, rt (quant.).

Scheme 3. On the Formation of New Oxygenated Terpenoids by Different STSs: (A) Transformation with 3 Using PvHVS1; (B) Transformation with 9 Using Omp7 and PenA; (C) Transformation with 10 Using BcBOT2

Figure 1

¹H–¹H COSY (blue) and HMBC (orange) correlations for bicyclic ether 18. Noteworthy, a NOESY correlation between the two protons at stereogenic centers was not found; we therefore defined the relative orientation of the two protons to be anti.

Scheme 4. (A) Mechanistic Considerations on the Formation of Enol Ethers 15, 17 and 19 and Aldehydes 16 and 20, Respectively; (B) Proposed Mechanism on the Formation of Bicyclic Terpenoid 18 and Differences in Positioning of the Cyclopentane Moieties in 18 and Presilphiperfolan-8-β-ol (28), the Main Product Formed from 1 with BcBOT2

Scheme 5. Biotransformation of FPP Ether Derivative 10 with the F138V Mutant of BcBOT2

Figure 2

Zoom-in of an overlay for the biotransformation of FPP derivative 10 with BcBOT2 WT (black) or the Y211S mutant (blue).

Figure 3

Zoom-in of an overlay for the biotransformation of FPP derivative 10 with BcBOT2 WT (black) or the W118Q mutant (green).

Figure 4

Zoom-in of an overlay for the biotransformation of FPP derivative 10 with BcBOT2 WT (black) or the F138V mutant (orange).