Novozyme 435 asymmetric hydrolysis of enol ester with series acid moiety

Article abstract of DOI:10.14233/ajchem.2014.16200

(R)-2-pentylcyclopentanone can be synthesized by the asymmetric hydrolysis of enol esters, catalyzed by immobilized candida antarctica (novozyme 435) lipase. Different acid moieties influence the stereoselectivity of lipase. Enol esters can be prepared fr

Full text of DOI:10.14233/ajchem.2014.16200

Asian Journal of Chemistry; Vol. 26, No. 2 (2014), 607-610
http://dx.doi.org/10.14233/ajchem.2014.16200
Novozyme 435 Asymmetric Hydrolysis of Enol Ester with Series Acid Moiety
*
QUAN LI, WEIMIN JIA , ZHIJIAN WANG and XIAODAN GUO
School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai, P.R. China
*Corresponding author: Tel: +86 13817705124; E-mail: sying5@126.com
Received: 17 September 2013;
Accepted: 21 October 2013;
Published online: 15 January 2014;
AJC-14597
(R)-2-pentylcyclopentanone can be synthesized by the asymmetric hydrolysis of enol esters, catalyzed by immobilized candida antarctica
(novozyme 435) lipase. Different acid moieties influence the stereoselectivity of lipase. Enol esters can be prepared from anhydrides and
2-pentylcyclopentanone. When introducing optical (S)-(+)-2-methyl-butyric acid, the hydrolysis of optical enol ester showed great
enhancement of the specific rotation compared to the racemic enol ester, the specific rotation of product raise from [α]25D-10° (c 0.1,
CH3OH) to [α]25D-72° (c 0.1, CH3OH). However, when bringing chiral acid moity, the specific rotation still can not catch up with the value
compared to the isobutyric moiety. The specific rotation of (R)-2-pentylcyclopentanone is [α]25D-102.20° (c 0.1, CH3OH), the optimum
temperature and pH were 30 °C and 6.5, respectively. Then 81.06 % ee of (R)-δ-decalactone was prepared by the Baeyer-Villiger oxidation
of (R)-2-pentylcyclopentanone.
Keywords: Asymmetric hydrolysis, Specific rotation, Enol ester, (R)-2-Pentylcyclopentanone, Chiral acid moiety.
Literature14-16 has decleared a way to produce chiral
α-substituted cyclohexanone through the hydrosis of enol
esters. Referring to the former methods, it is easy to prepare
(R)-δ-decalactone by the Baeyer-Villiger oxidation17-19 of (R)-
(2)-pentylcyclopentanone which can be obtained by asymmetric
INTRODUCTION
Recently, the research of optically active aroma chemicals
has been reported1-3. Stereoisomers show different physiolo-
gical activity4 as the receptor protein can discriminate two
enantiomers (R) and (S) of a chiral odorant molecule, there-
hydrolysis of enol esters. The general formula of synthesis
fore optically active aroma chemicals show a powerful chiral
odorant whereas the antipode is weak odorless5. The perfume
has been described in the hydrolysis of enol acetates15 and
and hydrolysis have been shown in Fig. 1. The influence of R2
δ-decalactone is found naturally in coconut and raspberry and
the (R)-enantiomer is main ingredients6. It is racemic to
99 % ee 2-substituted cyclohexanone can be obtained.
The influence of R116 also can be seen in the enantioselective
synthesis perfume by ordinary chemical methods, chiral catalyst
hydrolysis of 1-acetoxy-2-methylcyclohexene, the highest
is usually expensive for example the reduction of α-substi-
ee % was 90 %.
tuted cyclopentanone catalyzed by BINAP-Ru(II) show high
However, few papers concern about the effect on the alchol
moiety in the hydrolysis of enol ester with chiral carboxylic
optical purity7,8, but the application is severe restricted to the
high price of catalyst.
acid moiety. The objective of the present work is to explore
Applications of biological catalysts are low cost and have
the optimum anhydride in its optimum pH and temperature.
And the specific rotation of hydrolyzate changes with the
been carried out by employing the whole cell or enzyme9,10. It
prefer to use single purified enzyme as it can avoid side reac-
appearance of chiral enol ester. The result was carefully
tions of irrelevant enzymes exist in the cell. (R)-δ-decalactone
can be obtained according to the reduction of keto acid and
Massoia lactone: Literature11,12 reported the baker yeast reduce
discussed base on the structure of lipase.
EXPERIMENTAL
the massioa lactone to δ-decalactone, in the process of reduction,
excess concentration of substrate and product poison the baker
yeast. Similar status exist in the reduction of carbonyl acid to
hydroxy acid which go against to baker yeast13. Thus it shows
great superiority to employ single purified enzyme in the
optimum condition of pH and temperature.
Conversion was detected by GC-1690 gas chromatograph.
The specific rotation determined by Autopal IV polarimeter.
The specific rotation of (R)-2-pentylcyclopentanone has not
been reported before, but the absolute configuration was
assigned based on the lactone synthesized by Baeyer-Villiger
oxidation, the specific rotation of (R)-δ-decalactone is reported

608 Li et al.
Asian J. Chem.
to be [α]25D + 55.82° (c 1.93, CH3OH)20. The ee % was determined
by the HPLC (high performance liquid chromatography).
Reagents and chemicals were purchased from commercial
resource and used without any handling.
Baeyer-Villiger
Synthesis of enol esters: The synthesis of enol esters
catalyzed by toluene-4-sulfonic acid monohydrate was
chemically synthesized following the method16 reported,
anhydride can be obtained referring patent21. Tables 1 and 2
show the preparation of series enol esters with different R1 and
R2 group and the general formula of synthesis and hydrolysis
have been shown in Fig. 1.
Fig. 2. Preparation of (R)-δ-decalactone by the Baeyer-Villiger oxidation
of (R)-2-alkylcyclopentanone with m-CPBA at room temperature
RESULTS AND DISCUSSION
Table-2 shows the effect on conversion yield and specific
rotation in the pH of 6.5 and the temperature is 30 °C. The
enol esters were obtained by various anhydrides reacted with
2-substituted cyclopentanone, then the enol ester were hydro-
lyzed, thus the optimum substrate was determined by type of
anhydride. The conversion yield of (1) and (2) were 100 %,
but the reaction time increase rapidly, which indicate that the
reaction rate decreases rapidly. The product was confirmed to
have R configuration by the sign of [α]25D-82° (c 0.1, CH3OH)
and [α]25D-102° (c 0.1, CH3OH) corresponding to acetic
anhydride and isobutyric anhydride, respectively. While the
specific rotation of product with 2-methylbutyric anhydride
and isovaleric anhydride decrease to -12 and -11°. And it took
longer time in the hydrolysis of (4) and (5), the comparation
indicated that the hydrolysis of enol ester is limited to the
substrate, especially the acid moiety.
TABLE 2
HYDROLYSIS OF SERIES ENOL ESTERS
Substrate
React time
H
Conversion yielda
%
Specific
rotationb
2
100
100
61
-82.03
-102.20
-80.65
-12.28
-11.64
-78
(1)
(2)
(3)
(4)
(5)
(6)
(7)
24
150
150
150
2
67
73
100
24
100
-10
aA conversion was determined by gas chromatography
25
b[α]D was determined by Autopal IV polarimeter set at 589 nm
wavelength, sample resolved in methanol was kept at 25 °C, and the
configuration can be determined to be R after the baeyer-villiger
oxidation and determined the specific rotation, the value is plus
Besides, when (S)-(+)-2-methylbutyric acid was intro-
duced in the enol ester, the specific rotation of product exhibited
enormous enhancement from [α]25D-12° (c 0.1, CH3OH) to
[α]25D-80° (c 0.1, CH3OH) compared to the corresponding
racemic enol ester. The result proved that Novozyme 435 prefer
to show a better asymmetric hydrolysis to the (S)-enol ester.
In addition, comparing to 2-pentylcyclopentanone, 2-
heptylcyclopentanone moiety shows a greater effect in the
hydrolysis of enol ester. (1) and (6), (2) and (7) are prepared
from same anhydride respectively, the hydrolysis of (7) shows
quite low specific rotation compared to (2), while (1) and (6)
have a relative approximate result.
Fig. 1. Synthesis of enol ester and preparation of (R)-2-alkylcyclo-
pentanone
Asymmetric hydrolysis of enol esters: Enol ester
hydrolyzed with Novozyme 435 lipase (10 % w/w substrate).
The reaction was conducted at a constant temperature and the
pH of solution was maintained to constant according to the
auto temperature control and auto dropwise 5 % NaHCO3.
Gas chromatographic track analyze to ensure the hydrolysis
complete or cease.
Table-2 indicates the influence of acid moiety in the asym-
metric hydrolysis, the highest specific roation were obtained
with isobutyric anhydride, the former two iterms have a 100 %
conversion yield while the esters with longer chain acid moiety
can not hydrolyze completely and the specific rotation of product
2-pentylcyclopentanone decrease.
Baeyer-Villiger oxidation of (R)-2-pentylcyclopentanone:
The reaction formula can be seen from Fig. 2. Baeyer-Villiger
oxidation is known for the retention of configuration, the
configuration of 2-pentylcyclopentanone could be detected
after the Baeyer-Villiger oxidation17-19, which was performed
following the method8 and the retention of configuration is
about 90 %.
It can be explained by the stereo specificity theory, whose
model described the structure of lipase containing active
centre22. There are two grooves present on the surface of the
active site in the structure of lipase detected by the X-ray diffrac-
tion23, one accept acid moiety, the other one accept the alchol
TABLE-1
PREPARATION OF ENOL ESTERS WITH DIFFERENT ANHYDRIDE AND 2-ALKYLCYCLOPENTANONE
Enol esters with 2-pentylcyclopentanone
1-Acetoxy-2-pentylcyclopentene (1)
Enol esters with 2-heptylcyclopentanone
Acetyl anhydride
1-Acetoxy-2-heptylcyclopentene (6)
Isobutyl anhydride
1-Isobutyryloxy-2-heptylcyclopentene (7)
a
1-Isobutyryloxy-2-pentylcyclopentene (2)
(S)-(+)-2-methyl butyl anhydride
2-Methyl butyl anhydride
3-Methyl-butyl anhydride
-
1-[(S)-(+)-2-methyl]-butyryloxy-2-pentylcyclopentene (3)
1-[( )-2-methyl]-butyryloxy-2-pentylcyclopentene (4)
Isovaleryloxy-2-pentylcyclopentene (5)
a
-
a
-
aIt was difficult to obtain enol esters with corresponding anhydride and 2-pentylcyclopentanone.

Vol. 26, No. 2 (2014)
Novozyme 435 Asymmetric Hydrolysis of Enol Ester with Series Acid Moiety 609
moiety, the groove of acid moiety is broader than another24,
which means that the impact of alchol moiety is greater than
acid moiety, thus the hydrolyzed result of (7) can be explained.
The (S)-enantisomer of ester react faster than the (R)-
enantisomer as the larger moiety is placed into the stereo speci-
ficity pocket. Thus a lower level of asymmetric hydrolysis may
be contributed to the acid moiety is too small, while the results
of (4) and (5) may be caused by the large acid moiety.
The different specific rotation of the hydrolyzate of (3)
and (4) were obviously caused by the chiral acid moiety in the
enol ester, the introduction of chiral acid moiety affect the
stereoselectivity of product, as has been described that the
groove of the active centre combine with acid moiety was
affected by the chiral acid moiety. Kazlauskas et al.25 declared
that in the condition of α-substituted of carboxylic acid,
lipases prefer to react with (S)-enantiomers. The chiral acid
moiety result in severe steric crowding based on the size of
the substi-tuents at the stereocenter. Besides Kazlauskas
predicted that enantiomer of chiral carboxylic acid esters react
faster than racemic carboxylic acid esters through the X-ray
crystallography. The chiral centre of 2-methylbutyric acid is
positioned at the a-carbon with regard to the carboxyl moiety
changes.
Temperature (°C)
Fig. 4. Effect of temperature against the specific rotation of (R)-2-
pentylcyclopentanone at the pH of 6.5
Generally, the enantioselectivity of lipase is controlled
by the stereo recognition of the active site. Figs. 3 and 4 have
demonstrated that the Novozyme 435 is rather sensitive to pH
and temperature. The effect of pH and temperature may due
to the deformation of Novozyme 435.
Temperature, as well as pH, could has significant effects on
the reaction rate, this is due to the fact that enzymes are highly
sensitive towards any slight temperature and pH changes and will
denature when it beyond the optimum temperature or pH.
Fig. 3 shows the effect of pH against the specific rotation
of (R)-2-pentylcyclopentanone prepared by the hydrolysis of
(2) in 30 °C. In this investigation, the specific rotation of
product change with the increasing of pH from 5.5 to 6.5, the
product exhibited the highest specific rotation when pH is 6.5.
However, it occurs a sudden change within the addition of 0.5
pH, the peak demonstrated that the Novozyme 435 is sensi-
tive to the pH, especially the pH exceed to the 6.5.
Fig. 5 shows that the hydrolyzing process of (3) and (4)
in the condition of pH = 6.5 and 30 °C in the whole reaction
time. The rate of hydrolysis react decreases with the reaction
conduct and the hydrolysis ceased at about 100 h. Apparently
the hydrolyzing degree of (4) is greater than (3). However, the
most important result is that the yield and the specific rotation
of hydrolyzate show enormous difference, both results
demonstrate that the influence to alcohol moiety of (S)-2-
methylbutyric moiety in enol ester is positive. The introduction
of chiral acid moiety fortifies the stereoselective in the asym-
metric hydrolysis.
Fig. 3. Effect of pH against the specific rotation of (R)-2-pentylcyclo-
pentanone at 30 °C
Fig. 4 shows that the temperature affect the specific
rotation of product at the pH of 6.5. It changes small from
20 °C to 30 °C, however, decrease rapidly when the temperature
exceed to 30 °C.
Time (h)
Fig. 5. Conversion yield of novozyme 435 catalyze of (3) and (4) at the
pH = 6.5 and the temperature 30 °C

610 Li et al.
Asian J. Chem.
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methylbutyric acid moiety compared to racemic 2-methyl-
butyric acid, while further increase depends on variety of
anhydride. The optimum anhydride is isobutyric anhydride
and enantioselectivity was strongly affected by the changes in
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The authors thank Shanghai Institute of Technology for
funding the current study and also thank to Dr. Rong Shaofeng
for his advices throughout this research work. Thanks to Shi
Zhangping and Yu Jinsheng for their constant help in the de-
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