Doubly deuterium-labeled patchouli alcohol from cyclization of singly labeled [2-2H 1]farnesyl diphosphate catalyzed by recombinant patchoulol synthase

Juan A. Faraldos, Shuiqin Wu, Joe Chappell, Robert M. Coates

Research output: Contribution to journalArticlepeer-review

40 Scopus citations

Abstract

Incubations of isotopically pure [2-2H1](E,E)- farnesyl diphosphate with recombinant patchoulol synthase (PTS) from Pogostemon cablin afforded a 65:35 mixture of monodeuterated and dideuterated patchoulols as well as numerous sesquiterpene hydrocarbons. Extensive NMR analyses ( 1H and 13C NMR, 1H homodecoupling NMR, HMQC, and 2H NMR) of the labeled patchoulol mixture and comparisons of the spectra with those of unlabeled alcohol led to the conclusion that the deuterium label was located at positions (patchoulol numbering system) C5 (both isotopomers, ca. 100%) and C12 (minor isotopomer, 30-35%), that is, an approximately 2:1 mixture of [5-2H1]- and [5,12- 2H2]-patchoulols. Low-resolution FIMS analyses and isotope ratio calculations further corroborated the composition of the mixture as mainly one singly deuterated and one doubly deuterated patchoulol. From a mechanistic point of view, the formation of [5,122H2] patchoulol is rationalized through the intermediacy of an unknown exocyclic [7,10:1,5]patchoul-4(12) ene (15-d1), which could incorporate a deuteron at the C-12 position on the pathway to doubly labeled patchoulol. The corresponding depletion of deuterium content observed in the hydrocarbon coproducts, β-patchoulene and α-guaiene (55% d0), identified the source of the excess label found in patchoulol-d2. Comparison of the PTS amino acid sequence with those of other sesquiterpene synthases, and examination of an active site model, suggested that re-orientation of leucine 410 side chain in PTS might facilitate the creation of a 2-pocket active site where the observed deuteron transfers could occur. The retention of deuterium at C5 in the labeled patchoulol and its absence at C4 rule out an alternative mechanism involving two consecutive 1,2-hydride shifts and appears to confirm the previously proposed occurrence of a 1,3hydride shift across the 5-membered ring. Anew, semisystematic nomenclature is presented for the purpose of distinguishing the three different skeletal structures of the patchoulane sesquiterpenes.

Original languageEnglish
Pages (from-to)2998-3008
Number of pages11
JournalJournal of the American Chemical Society
Volume132
Issue number9
DOIs
StatePublished - Mar 10 2010

ASJC Scopus subject areas

  • Catalysis
  • Chemistry (all)
  • Biochemistry
  • Colloid and Surface Chemistry

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