research communications\(\def\hfill{\hskip 5em}\def\hfil{\hskip 3em}\def\eqno#1{\hfil {#1}}\)

ISSN: 2056-9890

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

aAdvanced Fibres and Chemical Industries, CSIRO Manufacturing, Melbourne, Victoria 3169, Australia, bInfection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton 3800, Australia, and cSchool of Chemistry, Monash University, Clayton 3800, Australia
*Correspondence e-mail:,

Edited by P. C. Healy, Griffith University, Australia (Received 3 July 2017; accepted 6 July 2017; online 21 July 2017)

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)-2a-hy­droxy-1,8-cineole (trivial name) or (1S,4R,6S)-1,3,3-trimethyl-2-oxabi­cyclo­[2.2.2]octan-6-ol (V) (systematic), C10H18O2. In the solid state this compound exhibits an inter­esting O—H⋯O hydrogen-bonding motif.

Keywords: crystal structure.

1. Chemical context

The terpenoid compound commonly known as 1,8-cineole, or less easily identified using systematic nomenclature as 1,3,3-trimethyl-2-oxabi­cyclo­[2.2.2]octane (I)[link] (Fig. 1[link]), 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[link]). 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 transformation of 1,8-cineole (I)[link] 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[Azerad, R. (2014). ChemPlusChem 79, 634-655.]) recently published an extremely useful review article capturing all the oxidation products of 1,8-cineole (I)[link] by providing trivial and systematic names along with characterization data (i.e. melting point, optical rotation and proton and carbon NMR spectroscopic information).

[Figure 1]
Figure 1
Trivial and systematic naming and atom numbering used for compound (I)[link].

In continuing our research activities on the biocatalytic mono-hy­droxy­lation of 1,8-cineole (I)[link] 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[link], 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 1,8-cineole bicyclic ring system, we sought confirmation of the absolute configuration by undertaking X-ray crystallographic studies.

[Figure 2]
Figure 2
Biotransformation of 1,8-cineole (I)[link] by S. yanoikuyae B2 to produce four possible isomeric mono-hy­droxy­lated products (Unterweger et al., 2016[Unterweger, B., Bulach, D. M., Scoble, J., Midgley, D. J., Greenfield, P., Lyras, D., Johanesen, P. & Dumsday, G. J. (2016). Appl. Environ. Microbiol. 82, 6507-6517.]).

2. 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[link]) was solved in the P21 space group and revealed the location of the hydroxyl group to be in the 6 position (IUPAC) (Fig. 1[link]). The absolute configuration was determined by the method of Parsons et al. (2013[Parsons, S., Flack, H. D. & Wagner, T. (2013). Acta Cryst. B69, 249-259.]) and confirmed the proposed stereochemistry (i.e. structure (V) see above, Fig. 2[link]).

[Scheme 1]
[Figure 3]
Figure 3
Mol­ecular structure of (1S,4R,6S)-1,3,3-trimethyl-2-oxabi­cyclo­[2.2.2]octan-6-ol (V) with non-H atoms represented by 50% displacement ellipsoids and H atoms as spheres of arbitrary size.

The presence of the axial hydroxyl substituent in (V) breaks the crystallographic symmetry of the parent 1,8-cineole (I)[link], with P21/m space group (Bond & Davies, 2001[Bond, A. D. & Davies, J. E. (2001). Aust. J. Chem. 54, 683-684.]), resulting in a slight twisting of the mol­ecular framework as shown by the torsion angle C1—O2—C7—C4 of −12.8 (2)° and is presumably steric in origin. For the related 1,8-cineole-5,6-diol, three of the four possible diastereoisomers have been structurally characterized and only the one with the 6α hydroxyl group showed a similar distortion (Farlow et al., 2013[Farlow, A. J., Bernhardt, P. V. & De Voss, J. J. (2013). Tetrahedron Asymmetry, 24, 324-333.]).

3. Supra­molecular features

Individual mol­ecules of (V) are connected by O–H⋯O hydrogen bonds between the hydroxyl and ether moieties (Table 1[link]) and form spiral chains parallel to the b axis (Fig. 4[link]).

Table 1
Hydrogen-bond geometry (Å, °)

O1—H1⋯O2i 0.80 (3) 1.97 (3) 2.7530 (19) 170 (3)
Symmetry code: (i) [-x+1, y-{\script{1\over 2}}, -z+1].
[Figure 4]
Figure 4
Ball-and-stick representation of a hydrogen-bonded chain of mol­ecules of (V). Only selected H atoms are shown and O—H⋯O contacts are indicated as dashed bonds.

4. Database survey

A search of the Cambridge Structural Database (V5.38; Groom et al., 2016[Groom, C. R., Bruno, I. J., Lightfoot, M. P. & Ward, S. C. (2016). Acta Cryst. B72, 171-179.]) for the 1,3,3-trimethyl-2-oxabi­cyclo­[2.2.2]octane (cineole) skeleton gave the parent structure (I)[link] (ref code MOFPAY; Bond & Davies, 2001[Bond, A. D. & Davies, J. E. (2001). Aust. J. Chem. 54, 683-684.]) and the oxidation products, 5,6-di­hydroxy­cineole (three steroisomers: ref codes DIFJAF, DIFJEJ and DIFJIN; Farlow et al., 2013[Farlow, A. J., Bernhardt, P. V. & De Voss, J. J. (2013). Tetrahedron Asymmetry, 24, 324-333.]), 6-(1,3-dioxolan-2-yl)-5-ketocineole and 5-(1,3-dioxolan-2-yl)-6-ketocineole (ref codes DIFHOR and DIFHUX; Farlow et al., 2013[Farlow, A. J., Bernhardt, P. V. & De Voss, J. J. (2013). Tetrahedron Asymmetry, 24, 324-333.]).

5. Synthesis and crystallization

1,8-Cineole (I)[link] was mono-hy­droxy­lated using a recombinant Escherichia coli whole-cell fed-batch process using CYP101J2 in combination with suitable redox partner proteins from S. yanoikuyae B2 to provide a major product (Unterweger, 2016[Unterweger, B. (2016). PhD thesis, Monash University, Victoria, Australia.]). The isolated material was further purified by recrystallization from diethyl ether/petroleum ether to afford white needles. The melting point (this work m.p. 371.2–371.8 K, lit. m.p. 371–372 K (Carman et al., 1986[Carman, R. M., Macrae, I. C. & Perkins, M. V. (1986). Aust. J. Chem. 39, 1739-1746.]), 370, 370, 369, 368, 371–372, 371–372, 371–372 369–372, 372 and 370 K as cited in Azerad (2014[Azerad, R. (2014). ChemPlusChem 79, 634-655.])) and 1H NMR spectrum are in agreement with cited literature values (Azerad, 2014[Azerad, R. (2014). ChemPlusChem 79, 634-655.]) for either compound (IV) and/or (V). Optical rotation {this work [a]D +32.0 (c 1.3, EtOH), lit [a]D +31.9 (c 1.3, EtOH)}. The experimental data for the current material produced from the biotransformation of cineole is well aligned with one set of literature data (Carman et al., 1986[Carman, R. M., Macrae, I. C. & Perkins, M. V. (1986). Aust. J. Chem. 39, 1739-1746.]).

6. Refinement

Crystal data, data collection and structure refinement details are summarized in Table 2[link]. H atoms potentially involved in hydrogen-bonding inter­actions were located by difference methods and were freely refined. Other H atoms were included in the refinement at calculated positions with C—H = 0.95–0.98 Å and treated as riding with Uiso(H) = 1.2Ueq(C) or 1.52Ueq(O or methyl C).

Table 2
Experimental details

Crystal data
Chemical formula C10H18O2
Mr 170.24
Crystal system, space group Monoclinic, P21
Temperature (K) 123
a, b, c (Å) 6.3121 (1), 10.5611 (2), 7.9925 (2)
β (°) 112.126 (3)
V3) 493.57 (2)
Z 2
Radiation type Cu Kα
μ (mm−1) 0.62
Crystal size (mm) 0.25 × 0.10 × 0.02
Data collection
Diffractometer Oxford Gemini Ultra CCD
Absorption correction Multi-scan (CrysAlis PRO; Rigaku OD, 2015[Rigaku OD (2015). CrysAlis PRO. Rigaku Oxford Diffraction, Yarnton, England.])
Tmin, Tmax 0.650, 1.000
No. of measured, independent and observed [I > 2σ(I)] reflections 6839, 1746, 1728
Rint 0.027
(sin θ/λ)max−1) 0.596
R[F2 > 2σ(F2)], wR(F2), S 0.029, 0.076, 1.05
No. of reflections 1746
No. of parameters 113
No. of restraints 1
H-atom treatment H atoms treated by a mixture of independent and constrained refinement
Δρmax, Δρmin (e Å−3) 0.13, −0.11
Absolute structure Flack x determined using 804 quotients [(I+)−(I)]/[(I+)+(I)] (Parsons et al., 2013[Parsons, S., Flack, H. D. & Wagner, T. (2013). Acta Cryst. B69, 249-259.])
Absolute structure parameter 0.07 (9)
Computer programs: CrysAlis PRO (Rigaku OD, 2015[Rigaku OD (2015). CrysAlis PRO. Rigaku Oxford Diffraction, Yarnton, England.]), SHELXS97 (Sheldrick, 2015[Sheldrick, G. M. (2015). Acta Cryst. C71, 3-8.]), SHELXL2016 (Sheldrick, 2015[Sheldrick, G. M. (2015). Acta Cryst. C71, 3-8.]), X-SEED (Barbour, 2001[Barbour, L. J. (2001). J. Supramol. Chem. 1, 189-191.]) and publCIF (Westrip, 2010[Westrip, S. P. (2010). J. Appl. Cryst. 43, 920-925.]).

Supporting information

Computing details top

Data collection: CrysAlis PRO (Rigaku OD, 2015); cell refinement: CrysAlis PRO (Rigaku OD, 2015); data reduction: CrysAlis PRO (Rigaku OD, 2015); program(s) used to solve structure: SHELXS97 (Sheldrick, 2015); program(s) used to refine structure: SHELXL2016 (Sheldrick, 2015); molecular graphics: X-SEED (Barbour, 2001); software used to prepare material for publication: publCIF (Westrip, 2010).

(1S,4R,6S)-1,3,3-Trimethyl-2-oxabicyclo[2.2.2]octan-6-ol top
Crystal data top
C10H18O2F(000) = 188
Mr = 170.24Dx = 1.146 Mg m3
Monoclinic, P21Cu Kα radiation, λ = 1.54184 Å
a = 6.3121 (1) ÅCell parameters from 4695 reflections
b = 10.5611 (2) Åθ = 7.6–66.8°
c = 7.9925 (2) ŵ = 0.62 mm1
β = 112.126 (3)°T = 123 K
V = 493.57 (2) Å3Plate, colourless
Z = 20.25 × 0.10 × 0.02 mm
Data collection top
Oxford Gemini Ultra CCD
1746 independent reflections
Radiation source: fine focus sealed tube1728 reflections with I > 2σ(I)
Mirror monochromatorRint = 0.027
Detector resolution: 10.3389 pixels mm-1θmax = 66.7°, θmin = 7.6°
ω scansh = 77
Absorption correction: multi-scan
(CrysAlis PRO; Rigaku OD, 2015)
k = 1212
Tmin = 0.650, Tmax = 1.000l = 99
6839 measured reflections
Refinement top
Refinement on F2Hydrogen site location: mixed
Least-squares matrix: fullH atoms treated by a mixture of independent and constrained refinement
R[F2 > 2σ(F2)] = 0.029 w = 1/[σ2(Fo2) + (0.0408P)2 + 0.077P]
where P = (Fo2 + 2Fc2)/3
wR(F2) = 0.076(Δ/σ)max < 0.001
S = 1.05Δρmax = 0.13 e Å3
1746 reflectionsΔρmin = 0.11 e Å3
113 parametersAbsolute structure: Flack x determined using 804 quotients [(I+)-(I-)]/[(I+)+(I-)] (Parsons et al., 2013)
1 restraintAbsolute structure parameter: 0.07 (9)
Special details top

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.

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) top
O10.3440 (2)0.06028 (14)0.5824 (2)0.0344 (4)
H10.421 (5)0.000 (3)0.584 (4)0.049 (8)*
C10.3290 (3)0.28323 (16)0.5241 (2)0.0245 (4)
O20.3425 (2)0.37248 (12)0.39090 (15)0.0273 (3)
C20.3671 (3)0.15220 (17)0.4598 (2)0.0268 (4)
C30.1930 (3)0.13171 (17)0.2663 (3)0.0327 (5)
C40.0233 (3)0.24262 (19)0.2122 (3)0.0311 (4)
C50.0898 (3)0.2491 (2)0.3515 (3)0.0357 (5)
C60.0917 (3)0.28989 (18)0.5342 (3)0.0299 (4)
C70.1531 (3)0.36548 (19)0.2146 (2)0.0277 (4)
C80.0064 (4)0.4834 (2)0.1933 (3)0.0394 (5)
C90.2605 (3)0.3681 (2)0.0727 (2)0.0360 (4)
C100.5167 (4)0.3200 (2)0.7013 (3)0.0377 (5)
Atomic displacement parameters (Å2) top
O10.0373 (8)0.0291 (7)0.0455 (8)0.0094 (6)0.0253 (7)0.0102 (6)
C10.0236 (9)0.0271 (10)0.0230 (8)0.0020 (7)0.0089 (7)0.0028 (7)
O20.0264 (6)0.0293 (7)0.0244 (6)0.0054 (5)0.0076 (5)0.0019 (6)
C20.0226 (8)0.0290 (9)0.0315 (9)0.0034 (7)0.0133 (7)0.0045 (8)
C30.0341 (10)0.0280 (11)0.0349 (10)0.0016 (8)0.0116 (8)0.0065 (8)
C40.0226 (8)0.0352 (11)0.0303 (9)0.0013 (7)0.0041 (7)0.0033 (8)
C50.0217 (9)0.0414 (11)0.0444 (11)0.0002 (8)0.0128 (8)0.0045 (9)
C60.0300 (9)0.0296 (10)0.0357 (9)0.0051 (7)0.0187 (8)0.0028 (8)
C70.0253 (8)0.0329 (9)0.0223 (8)0.0028 (8)0.0061 (6)0.0001 (8)
C80.0438 (12)0.0399 (12)0.0381 (11)0.0136 (9)0.0194 (10)0.0101 (9)
C90.0347 (9)0.0462 (11)0.0279 (9)0.0039 (10)0.0127 (7)0.0026 (9)
C100.0380 (11)0.0445 (12)0.0266 (9)0.0091 (9)0.0078 (8)0.0018 (8)
Geometric parameters (Å, º) top
O1—C21.425 (2)C5—H5A0.9900
O1—H10.80 (3)C5—H5B0.9900
C1—O21.448 (2)C6—H6A0.9900
C1—C101.515 (2)C6—H6B0.9900
C1—C21.526 (2)C7—C81.523 (3)
C1—C61.532 (2)C7—C91.525 (3)
O2—C71.4665 (18)C8—H8A0.9800
C2—C31.539 (3)C8—H8B0.9800
C3—C41.535 (3)C9—H9A0.9800
C4—C71.531 (3)C10—H10A0.9800
C4—C51.534 (3)C10—H10B0.9800
C5—C61.540 (3)
C2—O1—H1109 (2)H5A—C5—H5B108.4
O2—C1—C10106.18 (14)C1—C6—C5109.29 (14)
O2—C1—C2106.40 (13)C1—C6—H6A109.8
C10—C1—C2112.35 (16)C5—C6—H6A109.8
O2—C1—C6109.76 (13)C1—C6—H6B109.8
C10—C1—C6112.00 (15)C5—C6—H6B109.8
C2—C1—C6109.91 (14)H6A—C6—H6B108.3
C1—O2—C7114.93 (12)O2—C7—C8107.90 (15)
O1—C2—C1108.43 (14)O2—C7—C9106.51 (13)
O1—C2—C3112.08 (15)C8—C7—C9108.91 (16)
C1—C2—C3108.81 (14)O2—C7—C4107.07 (14)
O1—C2—H2109.2C8—C7—C4113.06 (15)
C1—C2—H2109.2C9—C7—C4113.05 (16)
C4—C3—C2109.53 (15)C7—C8—H8B109.5
C7—C4—C5110.26 (16)C7—C9—H9B109.5
C7—C4—C3109.25 (15)H9A—C9—H9B109.5
C5—C4—C3107.20 (16)C7—C9—H9C109.5
C4—C5—C6108.51 (14)C1—C10—H10B109.5
C10—C1—O2—C7171.24 (15)C3—C4—C5—C668.1 (2)
C2—C1—O2—C768.89 (16)O2—C1—C6—C563.37 (18)
C6—C1—O2—C750.00 (18)C10—C1—C6—C5178.95 (17)
O2—C1—C2—O1177.12 (13)C2—C1—C6—C553.32 (19)
C10—C1—C2—O167.09 (18)C4—C5—C6—C111.6 (2)
C6—C1—C2—O158.34 (18)C1—O2—C7—C8109.18 (16)
O2—C1—C2—C354.97 (17)C1—O2—C7—C9134.02 (16)
C10—C1—C2—C3170.76 (15)C1—O2—C7—C412.81 (18)
C6—C1—C2—C363.81 (17)C5—C4—C7—O265.78 (18)
O1—C2—C3—C4113.35 (17)C3—C4—C7—O251.77 (18)
C1—C2—C3—C46.56 (19)C5—C4—C7—C852.9 (2)
C2—C3—C4—C761.95 (19)C3—C4—C7—C8170.47 (16)
C2—C3—C4—C557.52 (19)C5—C4—C7—C9177.24 (15)
C7—C4—C5—C650.8 (2)C3—C4—C7—C965.2 (2)
Hydrogen-bond geometry (Å, º) top
O1—H1···O2i0.80 (3)1.97 (3)2.7530 (19)170 (3)
Symmetry code: (i) x+1, y1/2, z+1.


The authors are grateful for financial support from Advanced Fibres and Chemical Industries Program at CSIRO Manufacturing and Monash University X-ray facilities.


First citationAzerad, R. (2014). ChemPlusChem 79, 634–655.  CrossRef CAS Google Scholar
First citationBarbour, L. J. (2001). J. Supramol. Chem. 1, 189–191.  CrossRef CAS Google Scholar
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First citationUnterweger, B., Bulach, D. M., Scoble, J., Midgley, D. J., Greenfield, P., Lyras, D., Johanesen, P. & Dumsday, G. J. (2016). Appl. Environ. Microbiol. 82, 6507–6517.  CrossRef CAS PubMed Google Scholar
First citationWestrip, S. P. (2010). J. Appl. Cryst. 43, 920–925.  Web of Science CrossRef CAS IUCr Journals Google Scholar

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ISSN: 2056-9890
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