Kinetic resolution of 3-hydroxycyclohexanone using different lipases

Article abstract of DOI:10.1016/j.tetasy.2011.09.011

A new approach to enantiomerically enriched cyclic β-hydroxy ketones was developed. 3-Hydroxycyclohexanone was successfully resolved using lipase catalyzed transesterification. Among the lipases screened PFL, PCL, and PPL-II gave 57%, 39%, and 25% yield o

Full text of DOI:10.1016/j.tetasy.2011.09.011

Tetrahedron: Asymmetry 22 (2011) 1736–1739
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Tetrahedron: Asymmetry
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Kinetic resolution of 3-hydroxycyclohexanone using different lipases
⇑
Sanjib Kumar Karmee , Remco van Oosten, Ulf Hanefeld
Gebouw voor Scheikunde, Afdeling Biotechnologie, Technische Universiteit Delft, Julianalaan 136, 2628 BL Delft, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
A new approach to enantiomerically enriched cyclic b-hydroxy ketones was developed. 3-Hydroxycyclo-
hexanone was successfully resolved using lipase catalyzed transesterification. Among the lipases
screened PFL, PCL, and PPL-II gave 57%, 39%, and 25% yield of the (R)-3-oxocyclohexyl acetate with
52%, 75%, and 91% ee. The PPL-II gave the highest E-value (32). The absolute configuration of the product
was determined by comparison with the literature data.
Received 26 August 2011
Accepted 16 September 2011
Available online 17 October 2011
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Cyclic and acyclic b-hydroxy ketones are important structural
motifs in various biologically active organic compounds.1–5 There-
fore, the synthesis of b-hydroxy ketones in racemic or enantiome-
rically pure form is of current research interest.1–7 In particular, the
chiral b-hydroxy ketones are used as potential building blocks for
the syntheses of amino alcohols,7 diols,8–11 lactones,12 and natural
products.13,14
However, enantiomerically enriched cyclic b-hydroxy ketones
are not easily accessible with the methods reported for the acyclic
b-hydroxy ketones.15,16 Therefore, the development of new routes
to enantiomerically enriched cyclic b-hydroxy ketones remains a
challenge. Along this line, enantiomerically enriched 3-hydroxycy
clohexanone is used as a precursor for the synthesis of 25-hydroxy-
19-norvitamin D3 analogues.17 Notably, the 25-hydroxy-19-nor
vitamin D3 analogs are known for their antiproliferative activities
toward prostate cells.17
2.1. Substrate preparation
The rac-3-hydroxycyclohexanone 2 was obtained through a
minor modification of a known procedure involving the oxidation
of cyclohexane-1,3-diol 1 with Na2Cr2O7ꢁ2H2O/H2SO4 (Scheme 1).19
The rac-3-hydroxycyclohexanone 2 was obtained in 40% isolated
yield after column chromatography. In the next step, rac
-3-hydroxycyclohexanone 2 was acylated to obtain rac-3-oxocyclo-
hexyl acetate 3 in quantitative yield (Scheme 1). The rac-3-oxocy
clohexyl acetate 3 was used as a standard compound for chiral GC
analysis.
OH
O
O
a
O
b
In this regard, the preparation of enantiomerically enriched 3-
hydroxycyclohexanone reported so far is laborious and requires
multistep chemical synthesis. Starting from (ꢀ)-quinic acid, Arai
et al. reported a seven steps synthesis and 46% overall yield of
(S)-3-hydroxycyclohexanone.17 The other reported synthetic path-
way to (S)-3-hydroxycyclohexanone involves a b-boration–oxida-
tion reaction sequence using cyclohexenone as a substrate and
bis(pinacolato)diboron as a reagent.18
Herein we report a straightforward methodology for the syn-
thesis of enantiomerically enriched cyclic b-hydroxy ketones via
lipase catalyzed kinetic resolution. The potential of this method
was demonstrated by using rac-3-hydroxycyclohexanone 2, which
is a key intermediate for the synthesis of 25-hydroxy-19-norvitamin
D3 analogs as described above.
ref. 19
OH
OH
O
1
2
3
Scheme 1. Reagents and conditions: (a) Na2Cr2O7ꢁ2H2O/H2SO4, room temperature,
40%; (b) Ac2O, pyridine, room temperature, quantitative yield.
2.2. Enzymatic resolution of 3-hydroxycyclohexanone
The rac-3-hydroxycyclohexanone 2 obtained was subjected to a
lipase catalyzed kinetic resolution using vinyl acetate 4 as a solvent
and acylating agent, whose properties as a green acylating agent was
recently evaluated.20 Kinetic resolution via hydrolysis of the ester is
unsuitable since the resulting alcohol cannot be extracted from
water. Different lipases, namely, CAL-B, CAL-A, CRL, PFL, Amano
PSD-I, Pseudomonas stutzeri, Alcaligenes sp., PCL, and PPL-II were
⇑
Corresponding author. Fax: +31 15 2781415.
E-mail addresses: S.K.karmee@tudelft.nl, U.Hanefeld@tudelft.nl (S.K. Karmee).
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.09.011

S. K. Karmee et al. / Tetrahedron: Asymmetry 22 (2011) 1736–1739
1737
screened for the kinetic resolution. Screening reactions were carried
O
O
O
out using the same activity (450 U) at 25 °C (all lipases were derived
from different organisms and therefore have different optimal tem-
peratures) of all of the lipases according to the reported guidelines.21
After 15 h the lipases from PFL, PCL, and PPL-II gave 57%, 39%,
and 25% yield of (R)-3-oxocyclohexyl acetate 5 with 52%, 75%,
and 91% ee (entries 1–3). In contrast, for the CRL, Amano PSD-I,
Pseudomonas stutzeri, Alcaligenes sp., and CAL-B high yield (>90%)
of (R)-3-oxocyclohexyl acetate 5 was achieved but with low ee (en-
tries 4–8). The CAL-A gave 47% yield with 4% ee (entry 9).
Shortening of the reaction time to 4 h showed an increase in the
ee of the (R)-3-oxocyclohexyl acetate 5 with Amano PSD-I (41% ee,
entry 10), Pseudomonas stutzeri (10% ee, entry 11), Alcaligenes sp.,
(23% ee, entry 12) and CAL-B (6%, entry 13) lipase. The difference
in E values for Amano PSD-I catalyzed reactions could be due to
the fact that at high conversion a slight error affected the E value
significantly (entries 5 and 10). Among the lipases screened, PPL-
II gave the enantiomerically enriched (R)-3-oxocyclohexyl acetate
5 in 30% yield and 91% ee (Table 1, entry 3). The E values of the li-
pase catalyzed kinetic resolution were between 1 and 32 (Table 1).
The PPL-II gave the highest E-value of 32 as the best result. Never-
theless, given the almost symmetrical nature of the compound this
E value is remarkably good.
b
O
a
ref. 17
OTBS
O
OH
8
7
5
Scheme 2. Reagents and conditions: (a) CAL-B, EtOH/MTBE, 25 °C, 66%; (b) TBSOTf,
2,6-lutidine, CH2Cl2, 55%.
enantiomerically enriched 3-(tert-butyldimethylsiloxy) cyclohexa-
none was found to be (R) ½a D25
¼ þ4:2 (c 0.96, CHCl3). The isolated
ꢂ
(R)-3-hydroxycyclohexanone 7 was also used for the stereoselec-
tivity determination of the Michael hydratase alcohol dehydroge-
nase (MhyADH).22,23
3. Conclusion
In conclusion, the enantiomerically enriched (R)-3-oxocyclo-
hexyl acetate 5 was synthesized in 30% yield and 91% ee via the
PPL-II catalyzed kinetic resolution. The absolute configuration of
enantiomerically enriched 3-oxocyclohexyl acetate 5 was deter-
mined by derivatization into a known compound. In contrast to
the chemo-catalyzed multi-step synthesis of cyclic b-hydroxy ke-
tones, the present methodology is straightforward, using commer-
cially available lipases.
2.3. Determination of the absolute configuration
Kazlauskas’ rule is difficult to apply to this molecule; so, it is
important to determine the absolute stereochemistry indepen-
dently. The enantiomerically enriched (R)-3-oxocyclohexyl acetate
5 was isolated in 25% yield after the PPL-II catalyzed acylation of
rac-3-hydroxycyclohexanone 2. The acetate group was deprotected
using CAL-B and ethanol/MTBE at 25 °C (Scheme 2). After the eth-
anolysis reaction, the enzyme was filtered off and the enantiome-
rically enriched (R)-3-hydroxycyclohexanone 7 was obtained in
66% yield. In the following step, the TBS-protected 3-hydroxycyclo-
hexanone 8 was synthesized in 55% yield (Scheme 2).17 The abso-
lute configuration was assigned by comparison with the specific
rotation of the known TBS-protected (S)-hydroxy derivative
4. Experimental
4.1. Materials and analytical methods
Cyclohexane-1,3-diol, TBSOTf, 2,6-lutidine, sodium dichromate,
sulfuric acid, diethyl ether, ethanol, MTBE, and vinyl acetate were
purchased from Sigma–Aldrich and Acros organics. Various lipases
namely, Candida antarctica B (CAL-B, Novozyme 435, Novo Nordisk
A/S LC200015), C. antarctica A (CAL-A, Chirazyme L5, Roche Diag-
nostics), Candida rugosa (CRL, Sigma L1754), Pseudomonas fluores-
cens (PFL, Fluka 62312), Pseudomonas stutzeri (Meito Sangyo Co.
½
a 2D5
ꢂ
¼ ꢀ5:6 (c 1.05, CHCl3).17 The absolute configuration of the
Table 1
Kinetic resolution of 3-hydroxycyclohexanone using different lipases
O
O
O
O
Lipase
25 °C
O
+
+
O
OH
OH
O
2
4
6
5
Entry
Enzyme
Time (h)
Acetate 5 yielda (%)
Acetate 5 eeb (%)
Ec
Enantio preference
Alcohol 6 eed (%)
1
2
3
4
5
6
7
8
PFL
PCL
PPL-II
CRL
Amano PSD-I
Pseudomonas stutzeri
Alcaligenes sp.
CAL-B
15
15
15
15
15
15
15
15
15
4
57.64
39.81
30.43
94.55
92.02
93.80
90.84
94.61
47.68
70.20
81.72
66.85
89.37
46.69
52.20
75.93
91.16
0.32
3.03
0.58
0.62
0.75
4.32
41.68
10.94
23.65
6.58
7
12
32
ndd
2
nd
nd
nd
nd
10
2
(R)
(R)
(R)
nd
(R)
nd
nd
nd
nd
(R)
(R)
(R)
(R)
nd
71.02
50.22
39.87
5.55
34.93
8.77
6.14
13.16
3.93
98.18
48.90
47.69
55.32
59.55
9
CAL-A
10
11
12
13
14
Amano PSD-I
Pseudomonas stutzeri
Alcaligenes sp.
CAL-B
4
4
4
4
2
2
nd
CAL-A
0.68
a
b
c
Yield of (R)-3-oxocyclohexyl acetate 5 was determined by GC analysis using a CP WAX 52 CB column (see Section 4.1).
Ee of (R)-3-oxocyclohexyl acetate 5 was determined by GC using chiradex GTA column (see Section 4.1).
ln½1ꢀcð1þeep Þꢂ
E was calculated by using the equation E ¼
; nd: not determined.
ln½1ꢀcð1ꢀeep Þꢂ
d
ees
Ee of the alcohol was calculated by using equation Conversion ¼
.
eesþeep

1738
S. K. Karmee et al. / Tetrahedron: Asymmetry 22 (2011) 1736–1739
Ltd, Lot No.TH8901) Alcaligenes sp. (Meito Sangyo Co. Ltd, Lot No.
B3102), Pseudomonas cepacia (PCL, Sigma 62309), Amano lipase
PSD-I (P. cepacia immobilized on diatomite, Aldrich 534870), and
Porcine pancreatic lipase-Type II (PPL, Sigma L3126) were pur-
chased or obtained as a gift.
4.4. Screening of lipases for the kinetic resolution of rac-3-
hydroxycyclohexanone 2
rac-3-Hydroxycyclohexanone 2 (50 mg, 0.43 mmol), freshly dis-
tilled vinyl acetate 4 (2 mL) and the required lipase (450 U) were
added to screw capped vials (5 mL). Then these vials were con-
stantly stirred in a shaker at 25 °C for 15 h. From the reaction mix-
1H and 13C NMR spectra were recorded on a Bruker Avance 400
(400 MHz and 100 MHz, respectively) instrument. Chemical shifts
(d) are given as parts per million relative to the residual solvent
peak and coupling constants (J) are in Hertz. The splitting pattern
abbreviations are as follows: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet, and br, broad peak.
ture a 50
l
L sample was centrifuged to separate the lipase. From
this 30 L of the reaction mixture was added to 970
l
l
L of ethyl
acetate. All of the obtained samples were analyzed by GC to deter-
mine the conversion and ee.
Column chromatography was performed using Silica Gel 60
(70–230 mesh) and the specified eluants. Optical rotations were
recorded using a Perkin–Elmer 241 polarimeter (sodium D line at
25 °C). The yields of the 3-oxocyclohexyl acetate and 3-hydroxycy-
clohexanone were quantified by a Shimadzu type GC2014 Direct
4.5. Procedure for the kinetic resolution of rac-3-
hydroxycyclohexanone 2 using PPL
rac-3-Hydroxycyclohexanone 2 (1.60 g, 14 mmol), freshly dis-
tilled vinyl acetate 4 (15 mL) and the PPL-II (14400 U) were added
to a 100 mL round-bottomed flask. Then the reaction mixture was
constantly stirred at 25 °C for 15 h. The ee (94%) of the acetate 5
was determined using a chiradex GTA column (50 m ꢃ 0.25 mm ꢃ
GC and CP WAX 52 CB column (50 m ꢃ 0.53 mm ꢃ 2
lm). The fol-
lowing conditions were used for the nonchiral separation: injector
250 °C, detector (FID) 270 °C, carrier gas N2, FID hydrogen 30 oxy-
gen 300, column flow 20 mL/min, maximum temp: 255 °C, temper-
ature program: start 165 °C hold time 11 min, rate 30 °C/min to
250 °C hold time 1 min. The quantification of 3-oxocyclohexyl ace-
tate and 3-hydroxycyclohexanone was done by using calibration
curves. The retention times of 3-oxocyclohexyl acetate and 3-
hydroxycyclohexanone were 4.8 and 8.6 min.
0.12 lm) as described above. After the reaction the enantiomeri-
cally enriched 3-hydroxycyclohexanone 6 and (R)-3-oxocyclohexyl
acetate 5 were separated by silica gel chromatography using ethyl
acetate/petroleum ether (30:70). Yield of the isolated (R)-3-oxocy-
clohexyl acetate 5 was 25% (265 mg).
The enantiomeric excess (ee) of the enantiomerically enriched
3-oxocyclohexyl acetate 5 was determined by a Shimadzu type
GC2010 split/less GC, chiradex GTA column (50 m ꢃ 0.25mm ꢃ
4.6. Procedure for the ethanolysis of (R)-3-oxocyclohexyl
acetate 5 using Novozym 435
0.12lm), and FID detector. The following conditions were used
for the chiral separation: injector 200 °C, detector 220 °C, split
60, carrier gas Helium, FID H2 30 O2 300, column flow: 0.49 mL/
min, maximum temperature 175 °C, and temperature program:
start 150 °C, hold time 10 min, rate 25 °C/min to 170 °C hold time
1.20 min. Retention times of (R)- and (S)-3-oxocyclohexyl acetate
were found to be 6.3 and 6.9 min.
A mixture of (R)-3-oxocyclohexylacetate 5 (234 mg, 1.5 mmol),
ethanol (1 mL), MTBE (1 mL), and CAL-B (500 U) was stirred con-
stantly at 25 °C for 30 h. The enzyme was filtered off and the sol-
vent was evaporated to yield (R)-3-hydroxycyclohexanone
7
(114 mg, 66% yield).
Activities of the lipases were determined according to the re-
ported procedure.24
4.7. Synthesis of enantiomerically enriched 3-(tert-
butyldimethylsiloxy) cyclohexanone 8
4.2. Synthesis of rac-3-hydroxycyclohexanone 2
At first, TBSOTf (487
enantiomerically enriched 3-hydroxycyclohexanone 7 (114 mg,
1 mmol) and 2,6-lutidine (410 L, 3.54 mmol) in dry dichloro-
methane (5 mL) at 0 °C under argon. The reaction mixture was stir-
red for 4 h and then the reaction was quenched by addition of
lL, 2.12 mmol) was added to a mixture of
A solution of Na2Cr2O7ꢁ2H2O (3.49 g, 11.72 mmol), concentrated
H2SO4 (2 mL), and H2O (14 mL) was added dropwise to a stirred
solution of cyclohexane-1,3-diol 1 (4 g, 34.44 mmol) in Et2O
(14 mL). The reaction was stirred for 3.5 h at 25 °C, and then the
reaction mixture was extracted with Et2O (100 mL ꢃ 3). Afterward,
the organic layers obtained were combined, dried over Na2SO4 and
concentrated in vacuo. The crude product was purified by column
chromatography using petroleum ether/EtOAc (7:3) as an eluent.
The 3-hydroxycyclohexanone 2 (1.60 g, 40% yield) was obtained
as a colorless oil. 1H NMR (400 MHz, CDCl3) d 1.69–1.79 (2H, m),
1.99–2.08 (2H, m), 2.28–2.32 (2H, dd, J = 7.0, 6.2 Hz), 2.37–2.42
(1H, dd, J = 14.0, 7.6), 2.61–2.66 (1H, dd, J = 14.0, 4.1 Hz), 4.17–
4.19 (1H, m); 13C NMR (100 MHz, CDCl3) d 20.64, 32.46, 40.88,
50.21, 69.41, 211.23.
l
water (500 lL). After the extraction with dichloromethane
(5 mL ꢃ 3) the organic phase was washed with brine, and dried
over magnesium sulfate. After removal of the solvent the residue
obtained was purified by column chromatography (ethyl acetate/
petroleum ether, 1:9). The enantiomerically enriched 3-(tert-butyl-
dimethylsiloxy) cyclohexanone 8 was obtained as a colorless oil
(126 mg, 55% yield). ½a D25
ꢂ
¼ þ4:2 (c 0.96, CHCl3); {lit.17 (S)-3-(tert-
butyldimethylsiloxy) cyclohexanone ½a D25
¼ ꢀ5:6 (c 1.05,CHCl3)};
ꢂ
1H NMR (400 MHz, CDCl3) d 0.03 (6H, s), 0.85 (9H, s), 1.65–1.70
(2H, m), 1.80–1.89 (1H, m), 2.03–2.10 (1H, m), 2.20–2.30 (2H, m),
2.37–2.38 (1H, dd, J = 13.7, 6.5 Hz), 2.49–2.53 (1H, dddd, J = 13.9,
3.6, 1.1, 1.1 Hz), 4.15–4.17 (1H, m); 13C NMR (100 MHz, CDCl3) d
ꢀ4.88, ꢀ3.59, 17.97, 20.49, 25.68, 33.31, 41.06, 50.82, 70.19,
210.00.
4.3. Acylation of rac-3-hydroxycyclohexanone 2
Pyridine (2.0 mL) and acetic anhydride (108
l
g, 1.06 mol)
l
were added to 3-hydroxycyclohexanone 2 (40
lg, 0.34 lmol).
The reaction mixture was stirred at room temperature. After
Acknowledgments
12 h,
a quantitative yield of 3-oxocyclohexylacetate 3 was
achieved. 1H NMR (400 MHz, CDCl3) d 1.68–1.79 (m, 4H), 1.95 (s,
3H), 2.28 (t, 2H), 2.36–2.41 (dd, 1H, J = 6.3, 14.8), 2.50–2.55 (dd,
1H, J = 4.3, 14.8), 5.11–5.15 (m, 1H); 13C NMR (100 MHz, CDCl3) d
20.67, 21.15, 29.27, 40.90, 46.51, 71.52, 170.12, 208.37.
A senior research fellowship of the Technische Universiteit Delft
to S.K.K. is gratefully acknowledged. The authors would like to
thank Dr. J. Jin, P. C. Oskam and M. Gorseling for the fruitful
collaboration.

S. K. Karmee et al. / Tetrahedron: Asymmetry 22 (2011) 1736–1739
1739
13. Sabitha, G.; Padmaja, P.; Reddy, K. B.; Yadav, J. S. Tetrahedron Lett. 2008, 49,
919–922.
References
14. Chakraborty, T. K.; Das, S. Chem. Lett. 2000, 80–81.
15. Vicario, J. L.; Badía, D.; Carrillo, L. Synthesis 2007, 2065–2092.
16. Ramachary, D. B.; Mondal, R. Tetrahedron Lett. 2006, 47, 7689–7693.
17. Arai, M. A.; Tsutsumi, R.; Hara, H.; Chen, T. C.; Sakaki, T.; Urushino, N.; Inouye,
K.; Kittaka, A. Heterocycles 2005, 66, 469–479.
18. Feng, X.; Yun, J. Chem. Commun. 2009, 6577–6579.
19. Gärtner, P.; Hofbauer, K.; Reichel, C.; Geisendorfer, T.; Gmeiner, G. J. Mass
Spectrom. 2008, 43, 958–964.
1. Mestres, R. Green Chem. 2004, 6, 583–603.
2. Sukumaran, J.; Hanefeld, U. Chem. Soc. Rev. 2005, 34, 530–542.
3. Clapés, P.; Fessner, W.-D.; Sprenger, G. A.; Samland, A. K. Curr. Opin. Chem. Biol.
2010, 14, 154–167.
4. Hertweck, C. Angew. Chem. 2009, 121, 4782–4811. Angew. Chem., Int. Ed. 2009,
48, 4688-4716.
5. Brovetto, M.; Gamenara, D.; Saenz Mendez, P.; Seoane, G. A. Chem. Rev. 2011,
111, 4346–4403.
6. Karmee, S. K.; Hanefeld, H. ChemSusChem 2011, 4, 1118–1123.
7. Haddad, M.; Dorbasis, J.; Larchevêque, M. Tetrahedron Lett. 1997, 38, 5981–
5984.
8. Acetti, D.; Brenna, E.; Fuganti, C.; Gatti, F. G.; Serra, S. Eur. J. Org. Chem. 2010,
142–151.
9. Narasaka, K.; Pai, H. C. Tetrahedron 1984, 40, 2233–2238.
10. Narasaka, K.; Pai, H. C. Chem. Lett. 1980, 1415–1418.
11. Keck, G. E.; Wager, C. A.; Sell, T.; Wager, T. T. J. Org. Chem. 1999, 64, 2172–
2173.
20. Paravidino, M.; Hanefeld, U. Green Chem. 2011, 13, 2651–2657.
21. Gardossi, L.; Poulsen, P. B.; Ballesteros, A.; Hult, K.; Svedas, V. K.; Vasic-Racki,
´
D.; Carrea, G.; Magnusson, A.; Schmid, A.; Wohlgemuth, R.; Halling, P. J. Trends
Biotechnol. 2010, 28, 171–180.
22. Jin, J.; Oskam, P. C.; Karmee, S. K.; Straathof, A. J. J.; Hanefeld, U. Chem. Commun.
2010, 46, 8588–8590.
23. Jin, J.; Straathof, A. J. J.; Pinkse, M. W. H.; Hanefeld, U. Appl. Microbiol. Biotechnol.
2011, 89, 1831–1840.
24. Veum, L.; Kuster, M.; Telalovic, S.; Hanefeld, U.; Maschmeyer, T. Eur. J. Org.
Chem. 2002, 1516–1522.
12. Ikeda, N.; Omori, K.; Yamamoto, H. Tetrahedron Lett. 1986, 27, 1175–1178.