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

Collis, G.E.; Unterweger, B.; Dumsday, G.J.; Forsyth, C.M.

doi:10.1107/S2056989017010015

research communications

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ISSN: 2056-9890

Volume 73| Part 8| August 2017| Pages 1242-1245

https://doi.org/10.1107/S2056989017010015

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

Gavin E. Collis,^a ^* Birgit Unterweger,^a,^b Geoff J. Dumsday ^a and Craig M. Forsyth ^c ^*

^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: gavin.collis@csiro.au, craig.forsyth@monash.edu

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.

CCDC reference: 1560548

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) (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 transformation 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).

Figure 1
Trivial and systematic naming and atom numbering used for compound (I)

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 1,8-cineole bicyclic ring system, we sought confirmation of the absolute configuration by undertaking X-ray crystallographic studies.

Figure 2
Biotransformation of 1,8-cineole (I)

by S. yanoikuyae B2 to produce four possible isomeric mono-hydroxylated products (Unterweger et al., 2016

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) was solved in the P2₁ 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).

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), with P2₁/m space group (Bond & Davies, 2001 ), 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 ).

3. Supramolecular features

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

Table 1
Hydrogen-bond geometry (Å, °)

D—H⋯A	D—H	H⋯A	D⋯A	D—H⋯A
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
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 ) for the 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane (cineole) skeleton gave the parent structure (I) (ref code MOFPAY; Bond & Davies, 2001) and the oxidation products, 5,6-dihydroxycineole (three steroisomers: ref codes DIFJAF, DIFJEJ and DIFJIN; Farlow et al., 2013), 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).

5. Synthesis and crystallization

1,8-Cineole (I) 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 ). 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 ), 370, 370, 369, 368, 371–372, 371–372, 371–372 369–372, 372 and 370 K as cited in Azerad (2014)) and ¹H NMR spectrum are in agreement with cited literature values (Azerad, 2014) 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).

6. Refinement

Crystal data, data collection and structure refinement details are summarized in Table 2. 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 U_iso(H) = 1.2U_eq(C) or 1.52U_eq(O or methyl C).

Table 2
Experimental details

Crystal data
Chemical formula	C₁₀H₁₈O₂
M_r	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)
V (Å³)	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 $[Rigaku OD (2015). CrysAlis PRO. Rigaku Oxford Diffraction, Yarnton, England.]$ )
T_min, T_max	0.650, 1.000
No. of measured, independent and observed [I > 2σ(I)] reflections	6839, 1746, 1728
R_int	0.027
(sin θ/λ)_max (Å⁻¹)	0.596

Refinement
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
Δρ_max, Δρ_min (e Å⁻³)	0.13, −0.11
Absolute structure	Flack x determined using 804 quotients [(I⁺)−(I⁻)]/[(I⁺)+(I⁻)] (Parsons et al., 2013)
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 ), SHELXL2016 (Sheldrick, 2015), X-SEED (Barbour, 2001 ) and publCIF (Westrip, 2010 ).

Supporting information

CCDC reference: 1560548

Crystal structure: contains datablocks I, global. DOI: https://doi.org/10.1107/S2056989017010015/hg5490sup1.cif

Structure factors: contains datablock I. DOI: https://doi.org/10.1107/S2056989017010015/hg5490Isup2.hkl

Supporting information file. DOI: https://doi.org/10.1107/S2056989017010015/hg5490Isup3.cml

checkCIF report

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

C₁₀H₁₈O₂	F(000) = 188
M_r = 170.24	D_x = 1.146 Mg m⁻³
Monoclinic, P2₁	Cu Kα radiation, λ = 1.54184 Å
a = 6.3121 (1) Å	Cell parameters from 4695 reflections
b = 10.5611 (2) Å	θ = 7.6–66.8°
c = 7.9925 (2) Å	µ = 0.62 mm⁻¹
β = 112.126 (3)°	T = 123 K
V = 493.57 (2) Å³	Plate, colourless
Z = 2	0.25 × 0.10 × 0.02 mm

Data collection top

Oxford Gemini Ultra CCD diffractometer	1746 independent reflections
Radiation source: fine focus sealed tube	1728 reflections with I > 2σ(I)
Mirror monochromator	R_int = 0.027
Detector resolution: 10.3389 pixels mm^-1	θ_max = 66.7°, θ_min = 7.6°
ω scans	h = −7→7
Absorption correction: multi-scan (CrysAlis PRO; Rigaku OD, 2015)	k = −12→12
T_min = 0.650, T_max = 1.000	l = −9→9
6839 measured reflections

Refinement top

Refinement on F²	Hydrogen site location: mixed
Least-squares matrix: full	H atoms treated by a mixture of independent and constrained refinement
R[F² > 2σ(F²)] = 0.029	w = 1/[σ²(F_o²) + (0.0408P)² + 0.077P] where P = (F_o² + 2F_c²)/3
wR(F²) = 0.076	(Δ/σ)_max < 0.001
S = 1.05	Δρ_max = 0.13 e Å⁻³
1746 reflections	Δρ_min = −0.11 e Å⁻³
113 parameters	Absolute structure: Flack x determined using 804 quotients [(I⁺)-(I^-)]/[(I⁺)+(I^-)] (Parsons et al., 2013)
1 restraint	Absolute 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

	x	y	z	U_iso*/U_eq
O1	0.3440 (2)	0.06028 (14)	0.5824 (2)	0.0344 (4)
H1	0.421 (5)	0.000 (3)	0.584 (4)	0.049 (8)*
C1	0.3290 (3)	0.28323 (16)	0.5241 (2)	0.0245 (4)
O2	0.3425 (2)	0.37248 (12)	0.39090 (15)	0.0273 (3)
C2	0.3671 (3)	0.15220 (17)	0.4598 (2)	0.0268 (4)
H2	0.525368	0.147597	0.459797	0.032*
C3	0.1930 (3)	0.13171 (17)	0.2663 (3)	0.0327 (5)
H3A	0.273802	0.126032	0.181704	0.039*
H3B	0.109504	0.051339	0.259698	0.039*
C4	0.0233 (3)	0.24262 (19)	0.2122 (3)	0.0311 (4)
H4	−0.095250	0.227897	0.088837	0.037*
C5	−0.0898 (3)	0.2491 (2)	0.3515 (3)	0.0357 (5)
H5A	−0.152629	0.165173	0.363326	0.043*
H5B	−0.217014	0.310951	0.312108	0.043*
C6	0.0917 (3)	0.28989 (18)	0.5342 (3)	0.0299 (4)
H6A	0.060505	0.377427	0.562909	0.036*
H6B	0.085833	0.233283	0.631090	0.036*
C7	0.1531 (3)	0.36548 (19)	0.2146 (2)	0.0277 (4)
C8	0.0064 (4)	0.4834 (2)	0.1933 (3)	0.0394 (5)
H8A	−0.121668	0.480975	0.076148	0.059*
H8B	−0.052847	0.486220	0.290291	0.059*
H8C	0.099284	0.558972	0.199665	0.059*
C9	0.2605 (3)	0.3681 (2)	0.0727 (2)	0.0360 (4)
H9A	0.139808	0.363569	−0.048097	0.054*
H9B	0.347117	0.446788	0.084261	0.054*
H9C	0.363568	0.295535	0.090656	0.054*
C10	0.5167 (4)	0.3200 (2)	0.7013 (3)	0.0377 (5)
H10A	0.515033	0.262157	0.796667	0.056*
H10B	0.665254	0.314862	0.689156	0.056*
H10C	0.491166	0.406855	0.732737	0.056*

Atomic displacement parameters (Å²) top

	U¹¹	U²²	U³³	U¹²	U¹³	U²³
O1	0.0373 (8)	0.0291 (7)	0.0455 (8)	0.0094 (6)	0.0253 (7)	0.0102 (6)
C1	0.0236 (9)	0.0271 (10)	0.0230 (8)	−0.0020 (7)	0.0089 (7)	0.0028 (7)
O2	0.0264 (6)	0.0293 (7)	0.0244 (6)	−0.0054 (5)	0.0076 (5)	0.0019 (6)
C2	0.0226 (8)	0.0290 (9)	0.0315 (9)	0.0034 (7)	0.0133 (7)	0.0045 (8)
C3	0.0341 (10)	0.0280 (11)	0.0349 (10)	−0.0016 (8)	0.0116 (8)	−0.0065 (8)
C4	0.0226 (8)	0.0352 (11)	0.0303 (9)	−0.0013 (7)	0.0041 (7)	−0.0033 (8)
C5	0.0217 (9)	0.0414 (11)	0.0444 (11)	0.0002 (8)	0.0128 (8)	0.0045 (9)
C6	0.0300 (9)	0.0296 (10)	0.0357 (9)	0.0051 (7)	0.0187 (8)	0.0028 (8)
C7	0.0253 (8)	0.0329 (9)	0.0223 (8)	0.0028 (8)	0.0061 (6)	−0.0001 (8)
C8	0.0438 (12)	0.0399 (12)	0.0381 (11)	0.0136 (9)	0.0194 (10)	0.0101 (9)
C9	0.0347 (9)	0.0462 (11)	0.0279 (9)	0.0039 (10)	0.0127 (7)	0.0026 (9)
C10	0.0380 (11)	0.0445 (12)	0.0266 (9)	−0.0091 (9)	0.0078 (8)	0.0018 (8)

Geometric parameters (Å, º) top

O1—C2	1.425 (2)	C5—H5A	0.9900
O1—H1	0.80 (3)	C5—H5B	0.9900
C1—O2	1.448 (2)	C6—H6A	0.9900
C1—C10	1.515 (2)	C6—H6B	0.9900
C1—C2	1.526 (2)	C7—C8	1.523 (3)
C1—C6	1.532 (2)	C7—C9	1.525 (3)
O2—C7	1.4665 (18)	C8—H8A	0.9800
C2—C3	1.539 (3)	C8—H8B	0.9800
C2—H2	1.0000	C8—H8C	0.9800
C3—C4	1.535 (3)	C9—H9A	0.9800
C3—H3A	0.9900	C9—H9B	0.9800
C3—H3B	0.9900	C9—H9C	0.9800
C4—C7	1.531 (3)	C10—H10A	0.9800
C4—C5	1.534 (3)	C10—H10B	0.9800
C4—H4	1.0000	C10—H10C	0.9800
C5—C6	1.540 (3)

C2—O1—H1	109 (2)	H5A—C5—H5B	108.4
O2—C1—C10	106.18 (14)	C1—C6—C5	109.29 (14)
O2—C1—C2	106.40 (13)	C1—C6—H6A	109.8
C10—C1—C2	112.35 (16)	C5—C6—H6A	109.8
O2—C1—C6	109.76 (13)	C1—C6—H6B	109.8
C10—C1—C6	112.00 (15)	C5—C6—H6B	109.8
C2—C1—C6	109.91 (14)	H6A—C6—H6B	108.3
C1—O2—C7	114.93 (12)	O2—C7—C8	107.90 (15)
O1—C2—C1	108.43 (14)	O2—C7—C9	106.51 (13)
O1—C2—C3	112.08 (15)	C8—C7—C9	108.91 (16)
C1—C2—C3	108.81 (14)	O2—C7—C4	107.07 (14)
O1—C2—H2	109.2	C8—C7—C4	113.06 (15)
C1—C2—H2	109.2	C9—C7—C4	113.05 (16)
C3—C2—H2	109.2	C7—C8—H8A	109.5
C4—C3—C2	109.53 (15)	C7—C8—H8B	109.5
C4—C3—H3A	109.8	H8A—C8—H8B	109.5
C2—C3—H3A	109.8	C7—C8—H8C	109.5
C4—C3—H3B	109.8	H8A—C8—H8C	109.5
C2—C3—H3B	109.8	H8B—C8—H8C	109.5
H3A—C3—H3B	108.2	C7—C9—H9A	109.5
C7—C4—C5	110.26 (16)	C7—C9—H9B	109.5
C7—C4—C3	109.25 (15)	H9A—C9—H9B	109.5
C5—C4—C3	107.20 (16)	C7—C9—H9C	109.5
C7—C4—H4	110.0	H9A—C9—H9C	109.5
C5—C4—H4	110.0	H9B—C9—H9C	109.5
C3—C4—H4	110.0	C1—C10—H10A	109.5
C4—C5—C6	108.51 (14)	C1—C10—H10B	109.5
C4—C5—H5A	110.0	H10A—C10—H10B	109.5
C6—C5—H5A	110.0	C1—C10—H10C	109.5
C4—C5—H5B	110.0	H10A—C10—H10C	109.5
C6—C5—H5B	110.0	H10B—C10—H10C	109.5

C10—C1—O2—C7	−171.24 (15)	C3—C4—C5—C6	68.1 (2)
C2—C1—O2—C7	68.89 (16)	O2—C1—C6—C5	63.37 (18)
C6—C1—O2—C7	−50.00 (18)	C10—C1—C6—C5	−178.95 (17)
O2—C1—C2—O1	−177.12 (13)	C2—C1—C6—C5	−53.32 (19)
C10—C1—C2—O1	67.09 (18)	C4—C5—C6—C1	−11.6 (2)
C6—C1—C2—O1	−58.34 (18)	C1—O2—C7—C8	109.18 (16)
O2—C1—C2—C3	−54.97 (17)	C1—O2—C7—C9	−134.02 (16)
C10—C1—C2—C3	−170.76 (15)	C1—O2—C7—C4	−12.81 (18)
C6—C1—C2—C3	63.81 (17)	C5—C4—C7—O2	65.78 (18)
O1—C2—C3—C4	113.35 (17)	C3—C4—C7—O2	−51.77 (18)
C1—C2—C3—C4	−6.56 (19)	C5—C4—C7—C8	−52.9 (2)
C2—C3—C4—C7	61.95 (19)	C3—C4—C7—C8	−170.47 (16)
C2—C3—C4—C5	−57.52 (19)	C5—C4—C7—C9	−177.24 (15)
C7—C4—C5—C6	−50.8 (2)	C3—C4—C7—C9	65.2 (2)

Hydrogen-bond geometry (Å, º) top

D—H···A	D—H	H···A	D···A	D—H···A
O1—H1···O2ⁱ	0.80 (3)	1.97 (3)	2.7530 (19)	170 (3)

Symmetry code: (i) −x+1, y−1/2, −z+1.

Acknowledgements

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

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