Structural elucidation of a hydroxy–cineole product obtained from cytochrome P450 monooxygenase CYP101J2 catalysed transformation of 1,8-cineole

X-ray structure analysis of hydroxy–cineole, derived from the biotransformation of cineole, was undertaken to unambiguously determine the location and stereochemistry of the hydroxyl functionality. In the solid state, weak intramolecular O—H⋯O hydrogen bonding is present, causing the molecules to arrange in spiral chains.

1,8-Cineole is an abundant natural product that has the potential to be transformed into other building blocks that could be suitable alternatives to petroleum-based chemicals. Monohydroxylation of 1,8-cineole can potentially occur at eight different carbon sites around the bicyclic ring system. Using cytochrome P450 monooxygenase CYP101J2 from Sphingobium yanoikuyae B2, the hydroxylation can be regioselectively directed at the C atom adjacent to the methyl-substituted quaternary bridgehead atom of 1,8-cineole. The unambiguous location of the hydroxyl functionality and the stereochemistry at this position was determined by X-ray crystal analysis. The monohydroxylated compound derived from this microorganism was determined to be (1S)-2ahydroxy-1,8-cineole (trivial name) or (1S,4R,6S)-1,3,3-trimethyl-2-oxabicyclo[2.2.2]octan-6-ol (V) (systematic), C 10 H 18 O 2 . In the solid state this compound exhibits an interesting O-HÁ Á ÁO hydrogen-bonding motif.

Chemical context
The terpenoid compound commonly known as 1,8-cineole, or less easily identified using systematic nomenclature as 1,3,3trimethyl-2-oxabicyclo[2.2.2]octane (I) (Fig. 1), is a key component of the leaf oil from eucalypts and is also found in a variety of plant types, such as sage, thyme and fruit extracts, albeit in lower quantities (Fig. 1). Its natural abundance makes it a suitable bio-derived feedstock from which other useful chemical building blocks could be accessed and used as an alternative to petrochemical based-materials. Although continued research into the chemical and biochemical trans- Trivial and systematic naming and atom numbering used for compound (I).
formation of 1,8-cineole (I) is being directed towards accessing high quality and commercial quantities of these derivatives, the naming of these products by using non-systematic nomenclature, coupled with the chiral nature of these products has created inconsistencies and made it challenging to compare data of these derivatives in the literature. To address this Azerad (2014) recently published an extremely useful review article capturing all the oxidation products of 1,8-cineole (I) by providing trivial and systematic names along with characterization data (i.e. melting point, optical rotation and proton and carbon NMR spectroscopic information).
In continuing our research activities on the biocatalytic mono-hydroxylation of 1,8-cineole (I) at the C atom adjacent to the quaternary C1 bridgehead atom (i.e. labelled 6 or 7 following IUPAC rules) four possible stereoisomers [ Fig. 2, compounds (II), (III), (IV) and (V)] could be formed. However, there is no current crystallographic information of these pure materials to support these assignments. Knowing the inconsistencies with the nomenclature of these compounds and to gain a better understanding of how to control the regio-and stereo-chemistry at the different sites around the 1,8cineole bicyclic ring system, we sought confirmation of the absolute configuration by undertaking X-ray crystallographic studies.

Structural commentary
Suitable crystals for X-ray diffraction were prepared by the slow diffusion of petroleum ether into a solution of the compound dissolved in ethyl acetate. The X-ray crystal structure of the purified mono-hydroxylated 1,8-cineole (V) Fig. 3) was solved in the P2 1 space group and revealed the location of the hydroxyl group to be in the 6 position (IUPAC) (Fig. 1). The absolute configuration was determined by the method of Parsons et al. (2013) and confirmed the proposed stereochemistry (i.e. structure (V) see above, Fig. 2).

Supramolecular features
Individual molecules of (V) are connected by O-HÁ Á ÁO hydrogen bonds between the hydroxyl and ether moieties (Table 1) and form spiral chains parallel to the b axis (Fig. 4).

(1S,4R,6S)-1,3,3-Trimethyl-2-oxabicyclo[2.2.2]octan-6-ol
Crystal data Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.