Highly enantioselective acylation of rac-alkyl lactates using Candida antarctica lipase B

Article abstract of DOI:10.1021/op0498722

By using Candida antarctica lipase B under mild conditions, the highly enantioselective acylation of alkyl (R)-lactate from racemic mixture with vinyl alkanoate has been accomplished. In this research effects of the organic solvent, the alkyl chain length of the alkyl lactates and of the vinyl alkanoates, and the reaction temperature on the enantiomeric excess as well as the reaction rate, were investigated. In all cases, only alkyl (R)-lactate was stereoselectively acylated at >99.5% ee. The lipase-catalyzed acylation rate of the alkyl lactates was affected by the nature of the organic solvents, but showed no correlation to log P of the solvent. The lipase-catalyzed acylation rate of the alkyl lactates was enhanced by increasing the chain length of the vinyl alkanoate from acetyl to butanoyl and by raising the reaction temperature to 65°C. Finally, the lipase-catalyzed acylation and subsequent vacuum distillation successfully provided both butyl (R)-O-butanoyllactate and butyl (S)-lactate in excellent yields (48%) and enantioselectivities (>99.5% ee) on a large scale. It is expected that the present method will prove to be more efficient in achieving the chiral resolution of racemic alkyl lactate than other conventional methods in terms of environmental friendliness and simplicity.

Full text of DOI:10.1021/op0498722

Organic Process Research & Development 2004, 8, 948−951
Highly Enantioselective Acylation of rac-Alkyl Lactates Using Candida
antarctica Lipase B
Yeon Soo Lee,* Joo Hee Hong, Nan Young Jeon, Keehoon Won, and Bum Tae Kim
Korea Research Institute of Chemical Technology, 100 Jangdong, Yuseonggu, Daejon 305-343, South Korea
Abstract:
derivatives with lipases have been reported,5,6 but the
substrate concentration and purifying process were not good
enough for industrial applications.
By using Candida antarctica lipase B under mild conditions,
the highly enantioselective acylation of alkyl (R)-lactate from
racemic mixture with vinyl alkanoate has been accomplished.
In this research effects of the organic solvent, the alkyl chain
length of the alkyl lactates and of the vinyl alkanoates, and the
reaction temperature on the enantiomeric excess as well as the
reaction rate, were investigated. In all cases, only alkyl (R)-
lactate was stereoselectively acylated at >99.5% ee. The lipase-
catalyzed acylation rate of the alkyl lactates was affected by
the nature of the organic solvents, but showed no correlation
to log P of the solvent. The lipase-catalyzed acylation rate of
the alkyl lactates was enhanced by increasing the chain length
of the vinyl alkanoate from acetyl to butanoyl and by raising
the reaction temperature to 65 °C. Finally, the lipase-catalyzed
acylation and subsequent vacuum distillation successfully
provided both butyl (R)-O-butanoyllactate and butyl (S)-lactate
in excellent yields (48%) and enantioselectivities (>99.5% ee)
on a large scale. It is expected that the present method will
prove to be more efficient in achieving the chiral resolution of
racemic alkyl lactate than other conventional methods in terms
of environmental friendliness and simplicity.
In the present research, we describe the enantioselective
acylation of racemic alkyl lactates with vinyl alkanoates using
Novozym 435, which is an immobilized form of lipase B
from Candida antarctica (CALB) (Scheme 1). CALB was
used for the enantioselective acylation of lactic acid with
vinyl acetate in tert-butyl methyl ether but was found not to
be efficient in resolving lactic acid.5 Instead of lactic acid,
we used alkyl lactates as an acyl acceptor. The effects of
the organic solvent, alkyl chain length of the alkyl lactates
and vinyl alkanoates, and the reaction temperature were
investigated briefly, and the large-scale enzymatic stereo-
selective acylation of butyl lactate was achieved without the
use of organic solvents.
Results and Discussion
First, as it is well-known that enzyme properties such as
activity and enantioselectivity can depend on the nature of
organic solvent in which the reaction is carried out, the effects
of the organic solvent were investigated. Among the many
physical parameters of organic solvents, log P value, which
is the logarithm of the partition coefficient of the solvent
between octanol and water, has been used for correlating
enzyme properties of a solvent nature.7,8 The lipase-catalyzed
acylation of ethyl lactate (R1 ) ethyl) with vinyl propionate
or butanoate (R2 ) ethyl or propyl) was conducted in a
variety of organic solvents at 25 °C. The conversion and
enantiomeric excess of (S)-1 and (R)-2 are shown at a
reaction time of 26 h, in Table 1. Log P values of the organic
solvents used, which were calculated using a hydrophobic
fragmental method developed by Rekker and de Kort,9 are
also described in ascending order in Table 1. Only the ethyl
(R)-lactates were stereoselectively acylated by the Novozym
435 in all the tested solvents. This is in accord with the
empirical rule established by Kazlauskas et al..6 This
empirical rule predicts which enantiomer will react the fastest
by comparing the size of the substituents at the chiral center
of secondary alcohols. The enantioselectivity was found to
Introduction
Lactic acid (2-hydroxypropionic acid) is a naturally
occurring organic acid and one of the simplest optically
active compounds having (R)- and (S)-optical isomers as
enantiomers. Optically pure (R)- and (S)-lactic acid deriva-
tives are used as a starting material for chiral drugs1 and
chiral polylactic acids.2 Whereas (S)-lactic acid is com-
mercially produced today through the microbial fermentation
of glucose, (R)-lactic acid is difficult to obtain. There are
two major methods to produce (R)-lactic acid production:
kinetic resolution of a racemic mixture3 and the direct
cultivation of microorganisms producing (R)-lactic acid.4
Due to their excellent chiral recognition, lipases have long
been used to achieve kinetic resolution of chiral alcohol or
carboxylic acid. Lactic acid possesses both a chiral alcohol
and a chiral carboxylic acid group, which can be resolved
by lipases. Several attempts to resolve racemic lactic acid
(5) Adam, W.; Lazarus, M.; Schmerder, A.; Humpf, H.-U.; Saha-Mo¨ller, C.
R.; Schreier, P. Eur. J. Org. Chem. 1998, 2013.
(6) Kazlauskas, R. J.; Weissfloch, A. N. E.; Rappaport, A. T.; Cuccia, L. A.
J. Org. Chem. 1991, 56, 2656.
(7) Chaudhary, A. K.; Kamat, S. V.; Beckman, E. J.; Nurok, D.; Kleyle, R.
M.; Hajdu, P.; Russell, A. J. J. Am. Chem. Soc. 1996, 118, 12891.
(8) Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Biotechnol. Bioeng. 1987, 30,
81.
* Corresponding author. Telephone: +82-42-860-7154. Fax: +82-42-861-
0307. E-mail: yslee@krict.re.kr.
(1) Kitazaki, T.; Tasaka, A.; Hosono, H.; Matsushita, Y.; Itoh, K. Chem. Pharm.
Bull. 1999, 47, 360.
(2) Murdoch, J. R.; Loomis, G. L. U.S. Patent 4,719,246, 1988.
(3) Hsieh, C.-L.; Houng, J.-Y. U.S. Patent 5,605,833, 1997.
(4) Cooper, B.; Kuesters, W.; Martin, C.; Siegel, H. U.S. Patent 4,769,329,
1988.
(9) Rekker, R. F.; de Kort, H. M. Eur. J. Med. Chem. 1979, 14, 479.
948
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Vol. 8, No. 6, 2004 / Organic Process Research & Development
10.1021/op0498722 CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/17/2004

Scheme 1
Table 1. Effects of the organic solvent on the conversion and
enantiomeric excess
stability in polar organic solvents as well as nonpolar
ones.11-13 The reaction conversion rate in hydrophilic 1,4-
dioxane (log P ) -1.1) was as high as in hydrophobic
hexane (log P ) 3.5), and no correlation was found between
log P and the reaction conversion rate. It was revealed that,
in all the solvents used, when vinyl butanoate was employed
as the acyl donor, higher reaction conversion was achieved
than when vinyl propionate was used. The effects of the alkyl
chain length of the vinyl alkanoate will be examined and
explained in detail below.
We also investigated the effects of the alkyl chain length
in the alkyl lactates and vinyl alkanoates and the reaction
temperature. Alkyl lactates with three different chain lengths
(methyl, ethyl, or butyl) were enzymatically acylated with
vinyl acetate, propionate, or butanoate at various temperatures
in diisopropyl ether. Table 2 shows that ee values of (S)-1
and (R)-2 were >99.5% ee in all cases. The structure of
substrates (acyl donor and acyl acceptor) and reaction
temperature can generally affect the enantioselectivity. E can
be higher for the longer acyl groups, larger substituent of
sec-alcohol, and lower temperature.13 However, in the present
tested range, the alkyl chain length of the alkyl lactates and
vinyl alkanoates, and the reaction temperature had little effect
on the enantioselectivity. By contrast, the reaction rate was
dependent on the chain length of the vinyl alkanoate and
the reaction temperature. Figure 1 demonstrates the effect
of the length of the alkyl lactate and vinyl alkanoate on the
reaction time at the point of 50% conversion at 50 °C.
Increasing the length of R1 in the alkyl lactate had a little
effect on the reaction rate, but increasing the length of R2 in
the vinyl alkanoate significantly increased the reaction rate.
This may be due to the substrate specificity of CALB.
Although CALB has a much broader specificity towards
straight-chain fatty acids than other lipases, the conversion
of fatty acid esterification with octanol in hexane increased
when the chain length of the fatty acid was increased to the
level of a butanoic acid.14 A study on CALB specificity using
several ethyl esters of different acyl chain lengths in
cyclohexane also showed that lipase activity increased with
increasing the length from C2 to C6.15 Since the elevation of
the reaction temperature results in an increase in the reaction
rate, the reaction temperature should be as high as possible
ee (%)
conversionb
organic solvent
1,4-dioxane
log Pa
R2
(%)
(S)-1 (R)-2
-1.1 ethyl
propyl
-0.33 ethyl
43.5
45.7
37.6
47.0
38.8
44.2
46.7
50.0
39.3
47.8
30.2
44.3
47.0
50.0
45.0
49.9
39.9
48.7
43.2
49.9
77 >99.5
84 >99.5
60 >99.5
89 >99.5
63 >99.5
79 >99.5
88 >99.5
100 >99.5
65 >99.5
92 >99.5
43 >99.5
80 >99.5
89 >99.5
100 >99.5
82 >99.5
100 >99.5
66 >99.5
95 >99.5
76 >99.5
99 >99.5
acetonitrile
propyl
tetrahydrofuran
ethyl ether
0.49 ethyl
propyl
0.85 ethyl
propyl
tert-butyl methyl ether 1.4 ethyl
propyl
dichloromethane
isopropyl ether
toluene
1.5 ethyl
propyl
1.9 ethyl
propyl
2.5 ethyl
propyl
3.0 ethyl
propyl
carbon tetrachloride
hexane
3.5 ethyl
propyl
a Log P values were calculated using a hydrophobic fragmental method
developed by Rekker and de Kort. b Conversion of the acylation reaction of ethyl
lactate at 25 °C for 26 h.
be extremely high E (>200)10 irrespective of the nature of
the organic solvent and of the acyl donor.
The general guidelines for the use of enzymes in organic
solvents usually recommend using nonpolar (log P > 2)
solvents in order to ensure high activity and stability.8
However, CALB displays a rather unique activity and
(10) Enatiomeric ratio (E) ) ln[1 - c(1 + eep)]/ln[1 - c(1 - eep)] where c is
conversion and eep is enantiomeric excess of product. In all cases, (S)-2
peaks were not detected by GC (eep ) 1) such that E becomes infinity.
However, it should be pointed out that values of E > 200 cannot be
accurately determined due to the inaccuracies emerging from the determi-
nation of the enantiomeric excess by GC or HPLC because in this range
even a very small variation of ee causes a significant change in the
numerical value of E. In this sense, we expressed E > 200 when eep was
>99.5% ee.
(11) Anderson, E. M.; Larsson, K. M.; Kirk, O. Biocatal. Biotransform. 1998,
16, 181.
(12) Kirk, O.; Christensen, M. W. Org. Process Res. DeV. 2002, 6, 446.
(13) Rotticci, D.; Ottosson, J.; Norin, T.; Hult, K. In Enzymes in Nonaqueous
SolVents; Vulfson, E. N., Halling, P. J., Holland, H. L., Eds.; Humana
Press: Totowa, New Jersey, 2001; pp 261-276.
(14) Kirk, O.; Bjo¨rkling, F.; Godtfredsen, S. E.; Larsen, T. O. Biocatalysis 1992,
6, 127.
(15) Garcia-Alles, L. F.; Gotor, V. Biotechnol. Bioeng. 1998, 59, 163.
Vol. 8, No. 6, 2004 / Organic Process Research & Development
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949

Table 2. Effects of the alkyl chain length in alkyl lactate and
vinyl alkanoate and of reaction temperature on the reaction
rate and enantioselectivity
Table 3. Effects of concentration on the reaction rate and
enantioselectivitya
butyl
vinyl
ee (%)
lactate butanoate Novozym 435 solvent timeb
% ee
(mmol) (mmol)
(mg)
(mL)
(h) (S)-1 (R)-2
R1
R2
temp (°C)
timea (h)
(S)-1
(R)-2
1
5
5
2
10
10
20
2
10
10
20
3
2
1
0
25 >99.5 >99.5
14 >99.5 >99.5
10 >99.5 >99.5
methyl
methyl
methyl
ethyl
methyl
ethyl
50
65
50
65
50
65
25
35
50
65
25
35
50
65
25
35
50
65
50
65
50
65
50
65
8
4
6
3
3
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
10
9
>99.5 >99.5
propyl
methyl
a The lipase-catalyzed acylation reaction was carried out in isopropyl ether
2
at 65 °C. b The time required to reach 50% conversion.
72
32
15
8
32
20
8
Even though an enzymatic catalysis in nonaqueous
solvents has many advantages, it would be more technologi-
cally attractive to perform enzymatic reactions in a mixture
of the substrates themselves without the use of bulk
solvents.16 This approach, if feasible, can combine the
advantages of nonaqueous enzymology with high levels of
productivity. Vinyl alkanoate is often used as both a solvent
and an acylating agent in enzymatic esterification reactions.17
In the present study, a solvent-free reaction system was
attempted. Increasing the substrate concentration and de-
creasing the amount of solvent, we conducted the lipase-
catalyzed acylation reactions at 65 °C, when the ratio of butyl
lactate (mmol) and vinyl butanoate (mmol) to the enzyme
(mg) was fixed at 1:2:2. In Table 3, the reaction time at the
point of 50% conversion and the ee values of (S)-1 and (R)-2
are shown. As shown in Table 3, the more concentrated the
substrate was, the more accelerated the enzymatic acylation
rate was without any stereoselectivity loss. This result means
that a significant amount of racemic alkyl lactate can be
resolved effectively in a small reactor with >99.5% optical
purity even without the use of bulk organic solvents.
ethyl
ethyl
ethyl
6
propyl
24
12
4
3
14
5
6
4
4
3
butyl
butyl
butyl
methyl
ethyl
propyl
a The time required to reach 50% conversion.
Last, we attempted a scale-up experiment in order to
explore the possibility of the enzymatic chiral resolution of
racemic alkyl lactate. The enantioselective butanoylation of
racemic butyl lactate with vinyl butanoate was carried out
in the presence of Novozym 435 at 65 °C on a large scale.
Following the disappearance of the butyl (R)-lactate (19 h)
in the GC, filtration and vacuum evaporation were used to
remove the enzyme and remaining vinyl butanoate from the
reaction medium, respectively. A subsequent vacuum distil-
lation of the resultant reaction mixture offered butyl (S)-
lactate and butyl (R)-O-butanoyllactate.18 The yields and
enantiomeric purities of the these compounds were 48% and
>99.5% ee, respectively. The used Novozym 435 was reused
three times with less than 10% loss of activity per cycle while
Figure 1. Effect of the chain length in the alkyl lactate and
vinyl alkanoate on the reaction time at 50 °C. The data in figure
are the part of those shown in Table 2.
(16) (a) Vulfson, E. N.; Gill, I.; Sarney, D. In Enzymatic Reactions in Organic
Media; Koskinen, A. M. P., Klibanov, A. M., Eds.; Blackie Academic &
Professional: London, 1996; pp 244-265. (b) Won, K.; Lee, S. B.
Biotechnol. Prog. 2001, 17, 258.
to ensure the proper reaction rate. In this respect, Novozym
435 is very appropriate, because immobilized CALB is
known to be highly thermostable and can be used in
continuous operation at 60-80 °C without any significant
loss in activity for extended periods of time.11 We also found
that an increase in reaction temperature to 65 °C decreased
the reaction time required to reach 50% conversion without
any activity loss (Table 2). Raising the temperature by 10
°C reduced the reaction time by approximately half.
(17) Wang, Y. F.; Lalonde, J. J.; Momongan, M.; Bergbreiter, D. E.; Wong,
C.-H. J. Am. Chem. Soc. 1988, 110, 7200.
(18) Butyl (S)-lactate: Retention time 10.51 min; [R]25 ) -8.2° (c ) 0.02,
D
chloroform); 1H NMR (CDCl3, 300 MHz) δ 4.29-4.24 (m, 1H), 4.18 (dt,
2H, J ) 3.0 Hz, J ) 6.7 Hz), 2.80 (d, 1H, J ) 5.3 Hz), 1.70-1.60 (d, 2H),
1.44 (d, 3H, J ) 6.6 Hz), 1.41-1.26 (m, 2H), 0.95 (t, 3H, J ) 7.5 Hz).
Butyl (R)-butanoyllactate: Retention time 21.29 min; [R]25D ) +40.2° (c
) 0.02, chloroform); 1H NMR (CDCl3, 300 MHz) δ 5.08 (q, 1H, J ) 7.2
Hz), 4.15 (dt, 2H, J ) 1.9 Hz, J ) 6.7 Hz), 2.37 (dt, 1H, J ) 2.7 Hz, J )
7.5 Hz), 1.73-1.60 (m, 4H), 1.48 (d, 3H, J ) 6.9 Hz), 1.39-1.35 (m,
2H), 0.98 (t, 3H, J ) 7.2 Hz), 0.93 (t, 3H, J ) 7.5 Hz).
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enantioselectivity was not influenced. In this manner, the
enantioselective acylation of alkyl (R)-lactate from racemic
alkyl lactate was successfully performed on a large scale
without the use of any organic solvents.
Autopol III (Rudolph Research). Enantiomeric excess and
the conversion of alkyl lactate were determined by GC
analysis on a CycloSil-B chiral column (Agilent, Palo Alto,
CA) with a flame ionization detector. The column temper-
ature was maintained at 100 °C for 5 min and then increased
to 200 °C at 10 °C/min. The injector and detector temper-
atures were both set at 230 °C. Nitrogen was supplied as a
carrier gas.
Typical Procedure for the Enzymatic Acylation of
Racemic Alkyl Lactates with Vinyl Alkanoates. Unless
otherwise mentioned, the reaction was carried out as fol-
lows: A solution of racemic alkyl lactates (0.3 mmol) and
vinyl alkanoates (0.6 mmol) in organic solvents (3 mL) was
incubated at a reaction temperature. The reaction was started
by the addition of Novozym 435 (10 mg). The resultant
reaction mixture was shaken at 200 rpm. The progress of
the reaction was monitored by GC analysis.
Enzymatic Resolution of rac-Butyl Lactate on a Large
Scale. To the solution of racemic butyl lactate (73.1 g, 0.5
mol) and vinyl butanoate (114.1 g, 1 mol), 200 mg of
Novozym 435 were added at 65 °C. The resultant reaction
mixture was agitated with a mechanical stirrer at 50 rpm,
and then the acetaldehyde was distilled out. The progress of
the reaction was monitored by GC analysis. After complete
butanoylation of butyl (R)-lactate, Novozym 435 was re-
moved by filtration and the excess of vinyl butanoate was
evaporated under reduced pressure. Vacuum distillation of
the residue at 20 mmHg gave 35.1 g (yield 48%) of the butyl
(S)-lactate (80-82 °C) and the 51.9 g (yield 48%) of the
butyl (R)-O-butanoyllactate (114-117 °C).18
Conclusion
This experiment marks for the first time that the enzymatic
kinetic resolution of racemic alkyl lactate for the production
of alkyl (S)-lactate and alkyl ester of (R)-lactyl alkanoate
has been successfully performed through enantioselective
acylation of alkyl lactate with vinyl alkanoate using CALB.
The effects of the organic solvent, alkyl chain length of the
alkyl lactates and vinyl alkanoates, and the temperature on
the enantiomeric excess and reaction rate were all investi-
gated. In all cases, only the alkyl (R)-lactate was highly
stereoselectively acylated at >99.5% ee (E > 200). Organic
solvents affected the reaction rate, which showed no cor-
relation with log P of the organic solvent. Increasing the
chain length of the alkyl lactate had little effect on the
reaction rate, but changing the acyl group of the vinyl
alkanoate from acetyl to butanoyl increased the reaction rate
significantly. Raising the reaction temperature to 65 °C also
reduced the reaction time. The reaction proceeded efficiently
even without the use of bulk organic solvents. Finally, a
scale-up experiment revealed that butyl (R)-O-butanoyllactate
and butyl (S)-lactate were successfully obtained in excellent
yields (48%s) and enantiomeric purities (>99.5% ee). It is
expected that the present method will prove to be more
efficient in achieving the chiral resolution of racemic alkyl
lactate than other conventional methods in terms of envi-
ronmental friendliness and simplicity of process.
Acknowledgment
This work was financially supported by grants-in-aid from
the KOCI (Korea Research Council for Industrial Science
and Technology).
Experimental Section
All chemicals and solvents were purchased from com-
mercial suppliers and used without prior purification. No-
vozym 435 is a generous gift from Novozymes A/S,
Received for review June 29, 2004.
OP0498722
1
Denmark. H NMR spectra were recorded on a Bruker 300
spectrometer. The optical rotation was determined with
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