Isotopic Labeling Experiments Solve the Hedycaryol Problem

Abstract

Hedycaryol is a widespread sesquiterpene alcohol and important biosynthetic intermediate toward eudesmols and guaiols. A full NMR assignment for this compound has been hampered because of the unique molecular mechanics of its conformers in complex mixtures. This problem was solved through the enzymatic synthesis of isotopically labeled materials using a mutated plant and a bacterial enzyme for access to both enantiomers of hedycaryol, which also allowed us to follow the stereochemical course of its Cope rearrangement.

Mechanism of the Bifunctional Multiple Product Sesterterpene Synthase AcAS from Aspergillus calidoustus

“…The 1,2-hydride shift from D to E (pink H) is relevant for 2-5 and 10, and was investigated through conversion of (2-2 H)FPP [21] and (2-13 C)IPP [23a] with AcAS, yielding triplets for C6 in all cases (Figure S107). Only for 5 peak broadening was observed, like reported for other terpenes before, [1,20,26,27] that is a result of special molecular mechanics associated with slowly interconverting conformers. For 5 this phenomenon prevented the detection of the expected triplet signal.…”

Hedycaryol – Central Intermediates in Sesquiterpene Biosynthesis, Part II

“…For the optical rotation of 2 low negative values between [α] D =−2 and −9.7 are given in the literature, [24,26,27,30,32,43,46,48] while for 1 positive values between [α] D =+24.5 and +32.7 were reported [23,50–52] . The enantiomer (−)‐ 1 is only known from the bacterial hedycaryol synthase (HcS) from Kitasatospora setae ([α] D 25 =−21.3) whose Cope rearrangement gives (+)‐ 2 ([α] D 25 =+10.0) [56] . This finding reflects the observation that also in other cases bacteria and fungi produce the enantiomers of plant terpenes [57–59] …”

“…Because of its strained 10‐membered ring 1 exists as a mixture of three conformers 1 a with both Me groups attached to the ring up (UU) and crossed double bonds, and 1 b and 1 c with parallel double bonds and each one Me group up and one down (DU, UD) (Scheme 3). [60,61] Their fairly slow interconversion causes line broadening in the NMR spectra, and therefore the NMR data assignment was a long standing problem that was only recently solved through a 13 C‐ and stereoselective 2 H‐labelling approach [56] . Complete NMR data for 2 have also been published [47] .…”

“…The biosynthesis of 1 by type I terpene synthases proceeds through the abstraction of diphosphate from FPP to initiate a 1,10‐cyclisation and attack of water to C11 (Scheme 4A). Selective hedycaryol synthases for 1 are known from the plants Populus trichocarpa (PtTPS7), [63] Camellia brevistyla (CbTPS1), [64] and Liquidambar formosana (LfTPS01), [65] in all cases with undetermined absolute configuration, and for (−)‐ 1 from Kitasatospora setae , [56] whose product was initially erroneously assigned as (2 Z ,6 E )‐hedycaryol; for this bacterial enzyme also a crystal structure is available [66] . In addition, the diterpene synthase VenA from Streptomyces venezuelae that converts GGPP into venezuelaene A has a reported side activity with FPP as hedycaryol synthase [67] .…”

Mechanismus der bifunktionellen Multiprodukt‐Sesterterpensynthase AcAS aus Aspergillus calidoustus

“…Die 1,2‐Hydridverschiebung von D nach E (rosa H) ist für 2 – 5 und 10 relevant und wurde durch Umwandlung von (2‐ 2 H)FPP [21] und (2‐ 13 C)IPP [23a] mit AcAS verfolgt, das in allen Fällen Tripletts für C6 ergibt (Abbildung S107). Nur für 5 wurde eine Verbreiterung der Peaks beobachtet, wie zuvor für andere Terpene beschrieben, [1, 20, 26, 27] die auf eine spezielle Molekülmechanik zurückzuführen ist, die mit sich langsam ineinander umwandelnden Konformeren verbunden ist. Für 5 verhinderte dieses Phänomen die Detektion des erwarteten Triplettsignals.…”