Convenient double-enzymatic synthesis of both enantiomers of 6-methyl-ε-caprolactone

Article abstract of DOI:10.1016/j.tetasy.2007.03.016

Both enantiomers of 6-methyl-ε-caprolactone (6-MeCL) are obtained in high enantiomeric excess by the combination of an enzymatic ring opening of racemic 6-methyl-ε-caprolactone and subsequent enzymatic ring closure. Immobilized Candida antarctica lipase B (Novozym 435) was selected as the biocatalyst for both the ring-opening and the ring-closing reaction. This route provides ready access to enantiopure (S)-6-MeCL (ee = 99.6%) and (R)-6-MeCL (ee = 98.8%).

Full text of DOI:10.1016/j.tetasy.2007.03.016

Tetrahedron: Asymmetry 18 (2007) 787–790
Convenient double-enzymatic synthesis of both
enantiomers of 6-methyl-e-caprolactone
Bart A. C. van As, Dah-Kee Chan, Patrick J. J. Kivit, Anja R. A. Palmans* and E. W. Meijer*
Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of Technology, PO Box 513,
5600 MB Eindhoven, The Netherlands
Received 19 February 2007; accepted 16 March 2007
Abstract—Both enantiomers of 6-methyl-e-caprolactone (6-MeCL) are obtained in high enantiomeric excess by the combination of an
enzymatic ring opening of racemic 6-methyl-e-caprolactone and subsequent enzymatic ring closure. Immobilized Candida antarctica
lipase B (Novozym 435) was selected as the biocatalyst for both the ring-opening and the ring-closing reaction. This route provides ready
access to enantiopure (S)-6-MeCL (ee = 99.6%) and (R)-6-MeCL (ee = 98.8%).
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
(S)-6-MeCL is through either an elaborate, multi-step pro-
cedure or by an enantioselective Bayer–Villiger oxidation
using cyclohexanone monooxygenases.6 Although the lat-
ter method affords (S)-6-MeCL in high enantiomeric excess
(E >200), such biochemical oxidations require the use of
whole cells of engineered Escherichia coli strains, which
are not readily available in every chemical laboratory.
Over the last decade, enantioselective (also referred to as
stereoselective) polymerizations of racemic lactones have
shown promise as a method for preparing optically active
polymers. Lipases, for example, were found to be efficient
catalysts for the enantioselective ring-opening polymeriza-
tion of lactones.1 The recent use of Novozym 435 (Candida
antarctica lipase B immobilized on an acrylic resin) in
the polymerization of 4-methyl-e-caprolactone (4-MeCL)
and 4-ethyl-e-caprolactone (4-EtCL) led to the formation
of enantioenriched poly-(S)-4-MeCL and poly-(S)-4-Et-
CL.1e,2 The combination of the enantioselective poly-
merization of 4-MeCL with the controlled radical
polymerization, such as an atom transfer radical poly-
merization or nitroxide-mediated radical polymerization,
afforded chiral block copolymers through a one-pot
approach.2,3
Recently, we observed that the enzymatic ring opening of
6-MeCL by an equimolar amount of a nucleophile is a
reversible reaction and suffers from a rather unfavorable
equilibrium. Therefore, we anticipated that it should be
possible to isolate the ring-opening product and perform,
quantitatively, an enzymatic ring closure by distilling off
the regained cyclic structures. Herein, we report on a
two-step enzymatic synthesis of both enantiomers of
6-MeCL using (1) the well-established selectivity of Nov-
ozym 435 to ring open the (S)-enantiomer and isolate the
unreacted (R)-lactone and (2) enzymatic ring closure of
the resulting ring-opening product yielding the (S)-lactone.
This affords a straightforward and accessible approach that
allows for the synthesis of both (R)- and (S)-6-MeCL
(Scheme 1).
Recently, we have become interested in (R)- and (S)-6-e-
methylcaprolactone (6-MeCL) as building blocks for chiral
poly-6-MeCL.4 For a thorough study of this system, we
considered it desirable to have both enantiomers of
6-MeCL at our disposal. Previous research has shown that
(R)-6-MeCL can be easily obtained through kinetic resolu-
tion of 6-MeCL using lipases, since it is the slower reacting
enantiomer.5 However, to date the only method to obtain
2. Results and discussion
Previous research has shown that the kinetic resolution of
6-MeCL by butanolysis using Novozym 435 shows (S)-
selectivity, although with low enantioselectivity (E-values
*
Corresponding authors. Tel.: +31 40 2472655; fax: +31 40 2371036;
e-mail addresses: a.palmans@tue.nl; e.w.meijer@tue.nl
0957-4166/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.03.016

788
B. A. C. van As et al. / Tetrahedron: Asymmetry 18 (2007) 787–790
OH
O
O
O
O
OH
O
Novozym 435
O
+
toluene
r.t.
rac-6-MeCL
1
(R)-6-MeCL
O
O
Novozym 435
T = 125°C
OH
OH
O
O
+
1
(S)-6-MeCL
Scheme 1.
of 3–7 were reported).7 We adapted this procedure and per-
formed the enzymatic ring opening of 6-MeCL in toluene
at room temperature. 4-tert-Butylbenzyl alcohol (TBBA)
was selected as the nucleophile, providing ring-opening
product 1, which has a sufficiently high boiling point for
convenient removal by distillation of the remaining (R)-6-
MeCL (Scheme 1). The addition of 1,2,3,4-tetramethylbenz-
ene as an internal standard to the reaction mixture means
that the reaction kinetics could be easily monitored by chi-
ral GC. Fitting the enantiomeric excess (ee) of (R)-6-MeCL
as a function of 6-MeCL conversion according to the
method of Chen et al., resulted in an enantiomeric ratio
(E ratio) of 11.8
meric excess of (R)-6-MeCL was further increased by enzy-
matic hydrolysis, which also occurs with preference for the
(S)-enantiomer (Scheme 2).5b The reaction was carried out
in a toluene/water biphasic system at room temperature.
The addition of K2CO3 to the aqueous phase ensured that
the hydroxyacid product 2 was extracted from the organic
phase, shifting the equilibrium completely to the product
side. The reaction was monitored by chiral GC, and, after
complete hydrolysis of (S)-6-MeCL, (R)-6-MeCL, was iso-
lated from the organic phase with an ee of 98.8% in an
overall yield of 25% (see Table 1, entry 2).
Since enzymatic reactions are equilibria, 1 can be subjected
to enzymatic ring closure, yielding enantiomerically
enriched 6-MeCL and the starting alcohol TBBA (Scheme
1). By distilling off 6-MeCL and TBBA, the equilibrium is
shifted completely to the lactone side. Since the sense of
chirality for both the forward reaction as well as the reverse
reaction is by definition the same, enzymatic ring closure
will also occur with enantiopreference for the (S)-enantio-
mer of 6-MeCL.8 Using this procedure, the enzymatic
ring-closure distillation was performed under reduced pres-
sure (0.05 mmHg) and at 125 °C. The absence of a non-
enzymatic ring closure was confirmed in a blank reaction.
After column chromatography to remove TBBA, (S)-6-
MeCL was isolated with an ee of 95.2% and a yield of
30% (see Table 1, entry 3). This ring closure demonstrates
that, as expected, the reverse reaction is (S)-selective, as the
ee increased from 62.0% in 1 to 95.2% in (S)-6-MeCL. The
remaining (R)-6-MeCL was recovered as oligomeric species
in the residual fraction after distillation. After removal of
Using this approach, 1 was isolated with an ee of 62%
(yield 47%).9 Distillation and subsequent work-up of the
remaining lactone afforded (R)-6-MeCL with an ee of
84.2% in a yield of 38% (see Table 1, entry 1). The enantio-
Table 1. Results of the enzymatic ring opening and closing of 6-MeCL
a
Entry
Compound
½aꢀD
ee (%)
Yieldb (%)
1
2
3
4
(R)-6-MeCL
(R)-6-MeCL
(S)-6-MeCL
(S)-6-MeCL
n/dc
+24.8
n/dc
ꢁ25.9
84.2
98.8
95.2
99.6
38
25d
30
13d
a c = 13 g/100 mL in CHCl3.
b Maximum attainable yield for each enantiomer = 50%.
c n/d = not determined.
d Overall yield.
O
O
O
Novozym 435
O
O
OH
+
HO
toluene/K2CO3(aq)
r.t.
rac-6-MeCL
(R)-6-MeCL
2
Scheme 2.

B. A. C. van As et al. / Tetrahedron: Asymmetry 18 (2007) 787–790
789
trace amounts of water by storage on molar sieves, (S)-6-
MeCL crystallized as transparent needles (melting point
31 °C).
sured at ambient temperature in CHCl3 on a Jasco DIP-
370 digital polarimeter. TLC staining was performed with
1% p-methoxybenzaldehyde in a mixture of ethanol, acetic
acid, and H2SO4 90/5/5 v/v and subsequent heating with a
heat gun.
To further increase the enantiomeric excess, (S)-6-MeCL
with an ee of 95% was subjected to a second cycle of enzy-
matic ring opening and ring closure. This yielded nearly
enantiomerically pure (S)-6-MeCL with an ee of 99.6% in
an overall yield after two cycles of 13% (see Table 1, entry
4). Characterization of the enantiomers by specific rotation
values showed that the [a]D of (R)-6-MeCL was +24.8
(entry 2) and [a]D of (S)-6-MeCL was ꢁ25.9 (entry 4). This
is in good agreement with the previously reported values of
+25.0 for (R)-6-MeCL an ꢁ25.1 for (S)-6-MeCL.6b
4.3. Synthesis and isolation of (R)- and (S)-6-MeCL
4.3.1. Synthesis of compound 1 and isolation of (R)-6-
MeCL. Novozym 435 (6.0 g) was added to a 10 mL sam-
ple vial. The vial was placed overnight in a vacuum oven
(10 mmHg) at 50 °C in the presence of P2O5. The oven
was backfilled with nitrogen and the vial removed
from the oven. 4-tert-Butylbenzyl alcohol (TBBA, 14.7 g;
89.6 mmol), 6-MeCL (20.2 g; 157.6 mmol), 1,2,3,4-tetra-
methylbenzene (0.5 g; 4.2 mmol), and toluene (25 mL) were
added to a 100 mL round-bottomed flask. The dried Nov-
ozym 435 was added to the flask, representing the start of
reaction. The mixture was stirred at room temperature for
3 h. During the reaction, samples (ꢂ0.02 mL) were with-
drawn from the reaction mixture using a glass Pasteur pip-
ette. The sample was diluted with dichloromethane and the
enzyme removed from the sample by filtration over cotton
wool. The samples were analyzed by chiral GC for con-
version of TBBA and both enantiomers of 6-MeCL. At
92% (S)-6-MeCL conversion, the enzymatic reaction was
stopped by filtration using a class 3 glass filter. The residual
enzyme was flushed three times with dichloromethane. The
filtrate was concentrated and the remaining 6-MeCL and
TBBA were removed by distillation using a Kugelrohr
apparatus (115 °C, 0.05 mmHg), yielding 1 (24.4 g; 47%).
1H NMR of 1: d (ppm) 7.3–7.4 (m, Ar–H), 5.1 (Ar–CH2–
O), 3.8 (m, CH(CH3)OH), 2.4 (m, benzyl–CH2–OCOCH2),
1.70–1.35 (m, OCOCH2(CH2)3), 1.3 (Ar–C(CH3)3), 1.15 (d,
CH3).
3. Conclusions
In conclusion, a double kinetic resolution involving
enzymatic ring opening and ring closure was developed
to obtain both enantiomers of 6-MeCL. Both (R)- and
(S)-6-MeCL were obtained in excellent enantiomeric excess
of 99% and in acceptable overall yields of 25% and 13%,
respectively. This convenient route provides easy access
to these enantiomers without the need for multi-step proce-
dures or biochemical oxidations.
4. Experimental
4.1. Materials
6-Methyl-e-caprolactone (6-MeCL) was synthesized by
a Baeyer–Villiger oxidation of 2-methylcyclohexanone
following a reported procedure.2 2-Methylcyclohexanone
was purchased from Fluka and used as received. Novozym
435 was obtained from Novozymes A/S. All other chemi-
cals were purchased from Aldrich and used as received
unless otherwise noted.
The mixture of TBBA, 1,2,3,4-tetramethylbenzene, and
(R)-6-MeCL, was further purified by column chromatogra-
phy over neutral aluminum oxide using dichloromethane/
acetic acid 99/1 v/v as the eluent. Two fractions were
obtained: fraction 1 (Rf = 0.7, (R)-6-MeCL, 7.75 g (38%),
ee 84.2%, chemical purity 94%, impurities are 2-methyl-e-
caprolactone (5%) and 1,2,3,4-tetramethylbenzene (1%))
and fraction 2 (Rf = 0.4, TBBA, 1.3 g).
4.2. Analytical methods
1H and 13C NMR spectra were measured using a Varian
Mercury Vx 400 spectrometer (400 MHz) in CDCl3. Chiral
gas chromatography (GC) was performed on a Shimadzu
6C-17A GC equipped with an FID employing a Chrom-
pack Chirasil-DEX CB (DF = 0.25) column. Injection
and detection temperatures were set at 250 and 300 °C,
respectively. Separations were carried out with the column
temperature programmed to increase in temperature from
125 to 225 °C, which afforded in all cases baseline separa-
tion of the enantiomers of 6-MeCL. The internal standard
method using 1,2,3,4-tetramethylbenzene as the internal
standard was employed to determine the lactone conver-
sions. The enantiomeric excess (ee) was calculated as
follows: ee = (R ꢁ S)/(R + S) where R and S represent
the surfaces of the GC peaks of the (R)- and (S)-enantio-
mer, respectively. All samples were measured using a
Shimadzu AOC-20i autosampler. The yields of the (R)-
and (S)-enantiomers were calculated with the assumption
that 50% is the maximum attainable yield in a kinetic res-
olution for each enantiomer. Optical rotations were mea-
4.3.2. Enzymatic ring closure of 1. Novozym 435 (2.0 g)
was added to a 5 mL sample vial. The vial was placed over-
night in a vacuum oven (10 mmHg) at 50 °C in the presence
of P2O5. The oven was backfilled with nitrogen and the vial
removed from the oven. Compound 1 (24.4 g; 83.6 mmol)
and the dried Novozym 435 were added to a Kugelrohr
flask. The system was heated to 125 °C. The lactone
and TBBA were distilled off under reduced pressure
(0.05 mmHg). The mixture of TBBA and (S)-6-MeCL
was separated by column chromatography over neutral
aluminum oxide using dichloromethane/acetic acid 99/1
v/v as the eluent. Two fractions were obtained: fraction 1
(Rf = 0.7, (S)-6-MeCL) and fraction 2 (Rf = 0.4, TBBA,
˚
8.9 g). After removal of trace water by storage on 3 A
molecular sieves, 5.7 g (S)-6-MeCL (30%) was obtained

790
B. A. C. van As et al. / Tetrahedron: Asymmetry 18 (2007) 787–790
as transparent needle crystals (ee 95.2%, chemical purity
Acknowledgments
99%, both determined by chiral GC). Mp = 31 °C.
The authors would like to thank NRSC-C and NWO for
financial support.
4.3.3. Enzymatic hydrolysis of remaining (S)-6-MeCL in
(R)-6-MeCL. (R)-6-MeCL (6.6 g; 51.5 mmol) with an ee
of 84.2% was dissolved in 6 mL toluene and transferred
to a 50 mL round-bottomed flask. Novozym 435 (0.4 g)
and 3 mL of a 1 M K2CO3 solution in water were added
to the mixture. The mixture was stirred vigorously at room
temperature. During the reaction, samples (ꢂ0.02 mL)
were withdrawn from the reaction mixture using a glass
Pasteur pipette. The sample was diluted with dichlorometh-
ane and the enzyme was removed from the sample by filtra-
tion over MgSO4 on cotton wool. The samples were
analyzed by chiral GC for conversion of both enantiomers
of 6-MeCL. After all (S)-6-MeCL had been consumed, the
enzymatic reaction was stopped by filtration using a class 3
glass filter. The residual enzyme was flushed three times
with dichloromethane. All solvents were removed in vacuo
and the crude product was dissolved in dichloromethane.
The dissolved product was washed twice with water and
once with brine. The organic layer was concentrated, yield-
ing 4.3 g of (R)-6-MeCL (65%, ee 98.8%, chemical purity
94%, impurities are 2-methyl-e-caprolactone (5%) and
1,2,3,4-tetramethylbenzene (1%)). [a]D = +24.8 {lit.6b
[a]D = +25.0 (c 1.8, CHCl3)}.
References
1. (a) Svirkin, Y. Y.; Xu, J.; Gross, R. A.; Kaplan, D. L.; Swift,
G. Macromolecules 1996, 29, 4591–4597; (b) Xie, W.; Li, J.;
Chen, D.; Wang, P. G. Macromolecules 1997, 30, 6997–6998;
(c) Kikuchi, H.; Uyama, H.; Kobayashi, S. Macromolecules
2000, 33, 8971–8975; (d) Runge, M.; O’Hagan, D.; Haufe, G.
J. Polym. Sci. A: Polym. Chem. 2000, 38, 2004–2012; (e)
Al-Azemi, T. F.; Kondaveti, L.; Bisht, K. S. Macromolecules
2002, 35, 3380–3386; (f) Kikuchi, H.; Uyama, H.; Kobayashi,
S. Polym. J. 2002, 34, 835–840.
2. Peeters, J.; Veld, M.; Scheijen, F.; Heise, A.; Palmans, A. R.
A.; Meijer, E. W. Biomacromolecules 2004, 5, 1862–1868.
3. Van As, B. A. C.; Thomassen, P.; Kalra, B.; Gross, R. A.;
Meijer, E. W.; Palmans, A. R. A.; Heise, A. Macromolecules
2004, 37, 8973–8977.
4. (a) Van As, B. A. C.; Van Buijtenen, J.; Broxterman, Q. B.;
Verzijl, G. K. M.; Heise, A.; Palmans, A. R. A.; Meijer, E. W.
J. Am. Chem. Soc. 2005, 28, 9964–9965; (b) Van Buijtenen, J.;
Van As, B. A. C.; Meuldijk, J.; Palmans, A. R. A.; Vekemans,
J. A. J. M.; Hulshof, L. A.; Meijer, E. W. Chem. Commun.
2006, 30, 3169–3171.
4.3.4. Enzymatic ring opening and ring closure of (S)-6-
MeCL. (S)-6-MeCL (4.46 g; 34.8 mmol, ee = 95.2%) was
subjected to enzymatic ring opening by TBBA and subse-
quent enzymatic ring closure analogous to the procedure
described above, using 10.7 g TBBA (65.2 mmol) and 3.0 g
Novozym 435 for the ring-opening and 2.0 g Novozym 435
for the ring-closure procedure. The mixture of TBBA and
(S)-6-MeCL was separated by column chromatography
over neutral aluminum oxide using dichloromethane/
acetic acid 99/1 v/v as the eluent. Then, 1.89 g (S)-6-MeCL
(42%) was obtained with an optical purity of 99.6% and a
chemical purity of 99% (both determined by chiral GC).
[a]D = ꢁ25.9 {lit.6b [a]D = ꢁ25.1 (c 1.7, CHCl3)}.
5. (a) Fellous, R.; Lizzani-Cuvelier, L.; Loiseau, M. A.; Sassy, E.
Tetrahedron: Asymmetry 1994, 5, 343–346; (b) Shioji, K.;
Matsuo, A.; Okuma, K.; Nakamura, K.; Ohno, A. Tetra-
hedron Lett. 2000, 41, 8799–8802; (c) Fouque, E.; Rousseau, G.
Synthesis 1989, 661–666.
6. (a) Kyte, B. G.; Rouviere, P.; Cheng, Q.; Stewart, J. D. J. Org.
Chem. 2004, 69, 12–17; (b) Pirkle, W. H.; Adams, P. E. J. Org.
Chem. 1979, 13, 2169–2175.
7. Kondaveti, L.; Al-Azemi, T. F.; Bisht, K. S. Tetrahedron:
Asymmetry 2002, 13, 129–135.
8. Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am.
Chem. Soc. 1982, 25, 7294–7299.
9. The ee of the product was calculated based on conversions of
both enantiomers of 6-MeCL (see Ref. 1e for details).