Hydrolase-catalyzed preparation of (R)- and (S)-4-hydroxy-2,6,6- trimethyl-2-cyclohexen-1-ones (phorenols), the key synthetic intermediates for abscisic acid

Article abstract of DOI:10.1016/S0957-4166(99)00391-2

Preparation of both the enantiomers of 4-hydroxy-2,6,6-trimethyl-2- cyclohexen-1-one (phorenol), which are versatile synthetic intermediates for abscisic acid and carotenoids, was achieved by hydrolase-catalyzed hydrolysis of the corresponding chloroaceta

Full text of DOI:10.1016/S0957-4166(99)00391-2

Pergamon
Tetrahedron: Asymmetry 10 (1999) 3811–3817
Hydrolase-catalyzed preparation of (R)- and (S)-4-hydroxy-2,6,6-
trimethyl-2-cyclohexen-1-ones (phorenols), the key synthetic
intermediates for abscisic acid
Hiromasa Kiyota,∗ Miho Nakabayashi and Takayuki Oritani
Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural Science, Tohoku
University, 1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan
Received 30 July 1999; accepted 30 August 1999
Abstract
Preparation of both the enantiomers of 4-hydroxy-2,6,6-trimethyl-2-cyclohexen-1-one (phorenol), which are
versatile synthetic intermediates for abscisic acid and carotenoids, was achieved by hydrolase-catalyzed hydrolysis
of the corresponding chloroacetate. The hydrolysis with esterase SNSM-87 (Nagase) enriched the (S)-ester, while
lipase P (Amano) afforded the (R)-ester. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
(+)-Abscisic acid [(+)-ABA] is a plant hormone with particular activity for environmental stress
response as well as senescence promotion and growth inhibition.1 In particular, ABA acts antagonistically
against other plant hormones. Among plant hormones, only ABA is not supplied practically because of
its high cost, though several syntheses of optically active ABA were reported.2 Thus, development of a
practical synthesis of (+)-ABA is desired for agrochemical use.
4-Hydroxy-2,6,6-trimethyl-2-cyclohexen-1-ones (S)- and (R)-1 (phorenols) are versatile synthetic
intermediates for the synthesis of optically active natural carotenoids3 and degraded carotenoids,3
especially for the synthesis of (+)-ABA.2b Kienzle et al. prepared (S)- and (R)-1 by chemical modification
of the baker’s yeast reduction product 2 (Scheme 1).4 Their procedure was somewhat lengthy and used
manufacturally impractical baker’s yeast. Recently, our group reported the preparation of (S)- and (R)-
1 through enzymatic resolution of diol (ꢀ)-3;5 however, multi-steps were necessary and the yields
and selectivity were low. The Rh metal-catalyzed asymmetric hydrogenation of enol ester 4 has been
reported.6 For the practical preparation of (+)-ABA as well as carotenoids and degraded carotenoids, we
∗
Corresponding author. Tel/fax: +81-22-717-8783; e-mail: kiyota@biochem.tohoku.ac.jp
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00391-2

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have investigated direct enzymatic resolutions7 of (ꢀ)-1 and its ester (ꢀ)-5, which were easily prepared
from commercially available 4-oxoisophorone.
Scheme 1.
2. Results and discussion
2.1. Hydrolysis
(ꢀ)-4-Hydroxy-2,6,6-trimethyl-2-cyclohexen-1-one (ꢀ)-1 was prepared by regioselective reduction
of 4-oxoisophorone with NaBH4–CeCl3 according to Ishihara’s procedure.8 As a trial of the enzyme-
catalyzed acetylation of (ꢀ)-1 or hydrolysis of its acetate ester gave poor results (each E value9 ≈2), we
tried to use the corresponding chloroacetate ester 5.10 The alcohol (ꢀ)-1 was esterified with chloroacetyl
chloride in the presence of pyridine in 95% yield (Scheme 2). The result of hydrolysis of chloroacetate
(ꢀ)-5 is shown in Table 1. Screening of enzymes was done with the guidance of an E value of each
reaction. Among several hydrolases examined, esterase SNSM-8711 (Klebsiella oxytoca, Nagase) and
lipase P (Pseudomonas cepacia, Amano) were adopted for our purpose. Esterase SNSM-87 (Nagase)
preferentially hydrolyzed (R)-5 and (S)-5 remained unchanged, while lipase P (Amano) hydrolyzed
the (S)-enantiomer faster. Further examinations under various conditions of these two enzymes were
investigated. As shown in Table 2, the desired pH for esterase SNSM-87 was around 5.0–6.0 and
diisopropyl ether was the best solvent (Scheme 3). The fact that the reaction temperature did not effect
the selectivity much was supported by the high heat-resistance of this enzyme.
Scheme 2.
The results for lipase P (Amano) are summarized in Table 3. In this case, the initial condition (pH
7.0, 20°C) was the best choice. Finally, (S)- and (R)-5 were hydrolyzed to give desired (S)- and (R)-1,
respectively (Scheme 3).

H. Kiyota et al. / Tetrahedron: Asymmetry 10 (1999) 3811–3817
3813
Table 1
Enzyme-catalyzed hydrolysis of chloroacetate (ꢀ)-5a
2.2. Transesterification
On the other hand, in the case of enzymatic transesterification, esterase SNSM-87 (Nagase) and
lipase P (Amano) did not catalyze the reaction. Among enzymes examined, CHIRAZYME® L-1, c.-
f. (Burkholderia cepacia, Roche), L-2, c.-f., C2 (Candida antarctica) and L-9, c.-f., C2 (Mucor miehei)
worked to give the (S)-chloroacetate (Scheme 4), while crude lipase from Trichoderma viride, lipase
MY (Candida cylindracea, Meito), lipase 2G (Pseudomonas sp., Nagase) and bioprase AL-45 (Bacillus
subtilis, Nagase) afforded (R)-ester (Table 4). However, the E values of each reaction were low.
3. Conclusion
Both enantiomers of 4-hydroxy-2,6,6-trimethyl-2-cyclohexen-1-one (phorenol, 1), the key intermedia-
tes for the synthesis of (+)-abscisic acid and other degraded carotenoids and carotenoids, were prepared.
These were afforded by hydrolysis of the corresponding chloroacetate with different enzymes (esterase
SNSM-87 for S and lipase P for R); however, the transesterification gave poor results. Since this method
could supply (R)- and (S)-1 in four steps from commercially available 4-oxoisophorone, it is applicable
to practical production of (+)-abscisic acid and other carotenoids.

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H. Kiyota et al. / Tetrahedron: Asymmetry 10 (1999) 3811–3817
Table 2
Survey of reaction conditions for esterase SNSM-87a
Scheme 3.
Table 3
Survey of reaction conditions for lipase P (Amano)a

H. Kiyota et al. / Tetrahedron: Asymmetry 10 (1999) 3811–3817
3815
Scheme 4.
Table 4
Enzyme-catalyzed transesterificationa,b
4. Experimental
4.1. General
Optical rotations were measured on a Horiba Sepa-300 polarimeter. UV spectrum was measured
on a Shimadzu UV-1600 spectrometer. IR spectrum was measured on a Jasco Report-100 infrared
spectrometer. 1H NMR spectrum was measured on a Varian Gemini 2000 spectrophotometer (300 MHz)
using tetramethylsilane as an internal standard. Mass spectra were recorded on a Jeol JMS-700 mass
spectrometer. HPLC was performed on a Hitachi L-6000 pump and a Hitachi L-4200 UV–vis detector.
Merck silica gel 60 (70–230 mesh) was used for column chromatography.
4.2. (ꢀ)-4-Oxo-3,5,5-trimethyl-2-cyclohexenyl chloroacetate (ꢀ)-5
To an ice-cooled mixture of 4-hydroxy-2,6,6-trimethyl-2-cyclohexen-1-one (ꢀ)-1 (1.50 g, 9.73 mmol)
and pyridine (1.24 g, 14.8 mmol) in dry ether (30 ml) was added chloroacetyl chloride (1.32 g, 11.7
mmol) and the resulting mixture was stirred for 5 h at 20°C. The reaction mixture was diluted with ether,
washed with water, 2 M HCl aq. solution, satd aq. sodium hydrogen carbonate solution and brine, dried
with anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified with silica gel
column chromatography (hexane/ethyl acetate) to give (ꢀ)-5 (2.13 g, 9.23 mmol, 94.9%) as a colorless
oil.
UV (c=8.7×10−5 mol/l in EtOH) ε=9900 (λmax 228 nm). IR νmax (film): 2960 cm−1 (s), 2930 (s), 2860
(s), 1755 (s, C_O), 1740 (s, C_O), 1680 (s, C_C), 1470 (m), 1450 (m), 1410 (m), 1380 (m), 1350 (m),

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H. Kiyota et al. / Tetrahedron: Asymmetry 10 (1999) 3811–3817
1315 (s), 1280 (s), 1260 (s), 1160 (br. s, C–O), 1105 (m), 1045 (m), 1000 (s), 980 (s), 960 (s), 860 (m),
1
790 (m), 700 (w). H NMR (300 MHz, CDCl3): δ=1.182 (s, 3H, 5-Me), 1.187 (s, 3H, 5-Me), 1.82 (t,
3H, J=1.7 Hz, 3-Me), 2.00 (dd, 1H, J=12.9, 9.3 Hz, 6-H), 2.21 (ddd, 1H, J=12.9, 5.5, 1.9 Hz, 6-H), 4.11
(s, 2H, Cl-CH2), 5.71 (1H, m, 1-H), 6.53 (dq, 1H, J=4.1, 1.4 Hz, 2-H). EIMS m/z: 43, 61, 98, 174, 230
(M+). HREIMS m/z (M+): calcd for C11H15O3Cl: 230.0710; found: 230.0711.
4.3. (S)-4-Oxo-3,5,5-trimethyl-2-cyclohexenyl chloroacetate (S)-5: hydrolysis catalyzed by esterase
SNSM-87
A mixture of (ꢀ)-5 (60.0 mg, 260 µmol) and esterase SNSM-87 (60 mg) in diisopropyl ether (1.5 ml)
and 0.1 M acetate buffer (pH 5.0, 1.5 ml) was stirred for 2 d at 40°C. The reaction mixture was filtered
through a Celite pad and the organic layer was separated. The aq. layer was extracted with ether and
the combined organic layer was washed with satd aq. sodium hydrogen carbonate solution and brine,
dried with anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified with silica
gel column chromatography (hexane/ethyl acetate) to give (R)-1 (27.5 mg, 158 µmol, 60.7%) and (S)-
5 (12.2 mg, 52.9 µmol, 20.3%, 98.8% ee) as colorless oils. (S)-5: [α]24 −73.7 (c=0.455, EtOH). The
D
enantiomeric purity of (S)-5 was determined by HPLC detected at 230 nm at 20°C; column, Daicel
Chiralcel® OB-H, 4.6×250 mm; solvent, hexane:2-propanol (7:1), 1.0 ml/min; tR=11.4 min [99.4%, (S)-
5] and 18.0 min [0.6%, (R)-5].
4.4. (R)-4-Oxo-3,5,5-trimethyl-2-cyclohexenyl chloroacetate (R)-5: hydrolysis catalyzed by lipase P
(Amano)
A mixture of (ꢀ)-5 (60.0 mg, 260 µmol) and lipase P (Amano) (60 mg) in diisopropyl ether (1.5 ml)
and 0.1 M phosphate buffer (pH 7.0, 1.5 ml) was stirred for 2 d at 40°C. The reaction mixture was filtered
through a Celite pad and the organic layer was separated. The aqueous layer was extracted with ether and
the combined organic layer was washed with satd aq. sodium hydrogen carbonate solution and brine,
dried with anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified with silica
gel column chromatography (hexane/ethyl acetate) to give (S)-1 (28.9 mg, 187 µmol, 71.9%) and (R)-5
(16.0 mg, 69.0 µmol, 26.7%, 98.5% ee) as colorless oils. (R)-5: [α]22 +70.0 (c=0.800, EtOH).
D
4.5. (S)-4-Oxo-3,5,5-trimethyl-2-cyclohexenyl chloroacetate (S)-5: acylation catalyzed by
CHIRAZYME® L-1
A mixture of (ꢀ)-1 (20.0 mg, 130 µmol), vinyl chloroacetate (23.5 mg, 195 µmol) and CHIRAZYME®
L-1, c.-f., lyo. (20 mg) in diisopropyl ether (1.0 ml) was stirred for 4 h at 20°C. The reaction mixture was
filtered through a Celite pad and concentrated in vacuo. The residue was purified with silica gel column
chromatography (hexane/ethyl acetate) to give (S)-5 (19.8 mg, 85.8 µmol, 66%, 37% ee) and (R)-1 (6.82
mg, 44.2 µmol, 34%) as colorless oils. (S)-5: [α]23 −22.0 (c=1.57, EtOH).
D
4.6. 4-Hydroxy-2,6,6-trimethyl-2-cyclohexen-1-one 1: alkaline hydrolysis
A mixture of (ꢀ)-5 (300 mg, 1.30 mmol) and potassium carbonate (100 mg, 0.72 mmol) in methanol
(50 ml) was stirred for 4 h at 20°C. The mixture was concentrated in vacuo to remove most of the
methanol. The residue was diluted with water and extracted with ether. The combined organic layer was
washed with satd aq. sodium hydrogen carbonate solution and brine, dried with anhydrous magnesium

H. Kiyota et al. / Tetrahedron: Asymmetry 10 (1999) 3811–3817
3817
sulfate and concentrated in vacuo. The residue was purified with silica gel column chromatography
(hexane/ethyl acetate) to give (ꢀ)-1 (209 mg, 1.19 mmol, 92.3%) as a colorless oil.
(S)-1 was obtained in the same manner; [α]24 −50.0 (c=0.100, EtOH) {lit.4b [α]20 −49.0 (c=1,
D
D
EtOH)}.
(R)-1 was obtained in the same manner; [α]24 +52.7 (c=0.445, EtOH) {lit.4d [α]20 +47.7 (c=1,
D
D
EtOH)}.
Acknowledgements
We thank Nagase and Co. Ltd., Roche Diagnostics K. K., Meito Sangyo Co. Ltd., Toyobo Co. Ltd. and
Amano Pharmaceutical Co. Ltd. for the gift of various lipases. We also thank Dr. Akira Tanaka (Toyama
Prefectural University) for helpful discussions.
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11. Esterase SNSM-87 is available for laboratory use on request. Please contact the Research and Development Center, Nagase
and Co. Ltd.; fax +81-78-992-1050.