The unexpected kinetic effect of enzyme mixture: The case of enzymatic esterification

Article abstract of DOI:10.1016/j.molcatb.2014.02.020

During the studies towards synthesis of carboxylic acids esters, using ethyl carbonate and carboxylic acid as substrates, we found that different single enzyme systems provide model ethyl 3-phenylpropanoate in very low yield. Systematic studies proved tha

Full text of DOI:10.1016/j.molcatb.2014.02.020

Journal of Molecular Catalysis B: Enzymatic 102 (2014) 225–229
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
The unexpected kinetic effect of enzyme mixture: The case of
enzymatic esterification
Malgorzata Zysk, Anna Zadlo, Anna Brodzka, Catalina Wisniewska, Ryszard Ostaszewski∗
Institute of Organic Chemistry PAS, Kasprzaka Street 44/52, 01-224 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
During the studies towards synthesis of carboxylic acids esters, using ethyl carbonate and carboxylic acid
as substrates, we found that different single enzyme systems provide model ethyl 3-phenylpropanoate
in very low yield. Systematic studies proved that combination of two or more enzymes enhances yield
of reaction. Application of selected enzyme mixture for enzymatic esterification of various carboxylic
acids provided respective esters in excellent yields. Unexpectedly, the same reaction performed with
mixture of five enzymes proceeds in almost quantitative yield. For racemic substrates reaction catalyzed
by enzyme mixture was found to be enantioselective. To the best of our knowledge it is the very first
evidence of cooperation between multiple enzymes in organic solvents.
Received 20 December 2013
Received in revised form 24 February 2014
Accepted 26 February 2014
Available online 12 March 2014
Keywords:
Enzyme catalysis
Multienzymatic system
Esterification
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
with the addition of hydrochloric acid [5]. Catalysis by lewis acids
was modified by Yamamoto and co-workers [6]. They tapped syn-
Esterification is one of the most fundamental reactions in
organic chemistry [1]. Esters are used as fragrances, resins, plas-
ticizers, bulk and fine chemicals in industry. Classic Fischer
esterification requires heating a carboxylic acid and alcohol with an
addition of acid catalyst. It is energy consuming and fully reversible
process, in which water must be removed from reaction mix-
ture. Additionally this reaction usually requires high amount of
catalyst and excess of reagents and it generates a lot of waste,
which is incompatible with rules of green chemistry [2]. Dur-
ing the last few years new, environmentally friendly and efficient
catalysts and conditions for esterification have been established,
however the undesirable presence of heavy metals or other inor-
ganic compounds frequently is found in products [1]. Nowadays
the increasing demands for clean processes have led to the devel-
opment of biodegradable catalysts and mild conditions [2]. Typical
catalysts for this reaction are mineral acids or inorganic compounds
e.g. titanium salts [3]. Of late a great number of papers about
modifications of standard esterification procedure were published.
Ram and Palaniappan used polyaniline salts to direct esterification
of carboxylic acids with alcohols [4], however those experiments
require high temperature. Sobkowicz et al. provided esterification
of carbon and cellulosic nanoparticles, also in high temperature and
ergistic catalytic effect in Zr(IV) and Fe(III), Ga(III) or Sn(IV) binary
metal complexes to catalyze condensation between acid and alco-
hol. They obtained products in high yields, however conditions of
the reaction were harsh.
For the synthesis of esters to be used in pharmaceutical or
food industry special procedures are required that provide prod-
ucts without any traces of inorganic and organic impurities with
special attention has been paid to enzymatic procedures. Enzymes
are specific, efficient and biodegradable biocatalysts [7a]. More-
over they are very substrate selective, which in some instances is a
plus, but in many others unfortunately diminishes their application.
Activity of enzymes could be modulated with organic cosolvents
[7b], immobilization [7c], microwave irradiation [7d]. Esterification
catalyzed by enzymes is reversible reaction, and some modifi-
cations were performed to make this process irreversible. For
example, the molecular sieves are added to absorb the water pro-
duced upon reaction, however this approach is problematic in large
scale syntheses [7a]. Another way to overcome the reversibility
of esterification is performing the reaction with specific alkoxy or
acyl-donors [8–10]. Recently, interesting example of irreversible
esterification of carboxylic acids using carbonates as alkoxy-donors
was reported. The enzymatic process was described for esterifi-
cation of fatty acids with lipase from Mucor miehei, however the
products were obtained in low yields [11]. Carbonates are readily
available and belongs to relatively non-toxic reagents, which are
used as substrates or solvents. Naproxen enzymatic synthesis [12a]
along with preparation of some polyunsaturated fatty acids (PUFA)
∗
Corresponding author. Tel.: +48 22 343 21 20; fax: +48 22 632 66 81.
E-mail addresses: ryszard.ostaszewski@icho.edu.pl, rysza@icho.edu.pl
(R. Ostaszewski).
http://dx.doi.org/10.1016/j.molcatb.2014.02.020
1381-1177/© 2014 Elsevier B.V. All rights reserved.

226
M. Zysk et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 225–229
Table 1
Esterification of 3-phenylpropanoic acid with combinations of enzymes.
Entry
Enzyme
Yield [%]
1
2
3
4
5
6
7
8
9
A, B, C, D, E
A
B
C
D
E
A, B
C, D
B, C
96
<1
<1
<1
<1
<1
<1
<1
5
Scheme 1. Model reaction of phenylpropanoic acid with diethyl carbonate.
esters [12b] with the use of carbonates, has been recently reported.
However, due to very high substrate selectivity of this particu-
lar enzyme, the esterification reactions with carbonates are still
limited. Thus, modulation of enzymatic substrate selectivity is very
important factor.
In our studies we attempted to broaden substrate acceptabil-
ity in irreversible enzymatic esterification methods. Our method
provides compounds, which are commonly used in food and
pharmaceutical industries [13]. Studies on enzymatic reaction of 3-
phenylpropanoic acid and other carboxylic acids with carbonates
are presented, which led to discovery of unique, multi-enzymatic
system for irreversible esterification of diverse carboxylic acids.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
B, D
A, C
A, D
A, E
B, E
C, E
D, E
5
<1
<1
5
14
5
5
A, B, C
A, B, D
A, B, E
A, C, D
A, C, E
A, D, E
B, C, D
B, C, E
B, D, E
C, D, E
A, B, C, D
A, B, C, E
A, B, D, E
A, C, D, E
B, C, D, E
A, B, C, D, F
A, B, C, D, G
A, B, C, D, F, G
F, G
<1
<1
50
<1
13
10
<1
44
42
5
<1
38
66
78
66
<1
<1
10
<1
2. Results and discussion
Initial experiments were focused on irreversible, esterification
method providing esters of carboxylic acids without inorganic
impurities, applying methodology utilizing dialkyl carbonates as
alkoxy group donors. For synthesis of 3-phenylpropanoic acid ethyl
ester diethyl carbonate was used according to the methodology
described by Morrone et al. [12]. In this reaction course diethyl
carbonate serves as alkoxy group donor providing respective ester.
However, there is a need of alcohol additive to begin reaction course
[12]. We proposed a different approach to esterification of car-
boxylic acids with dialkyl carbonates exploiting multienzymatic
catalyst.
[A] papaine, [B] acylase I from Aspergillus melleus, [C] lipase from wheat germ, [D]
Amano lipase AK from Pseudomonas fluorescens, [E] Novozym 435, [F] deactivated
Novozym 435, [G] lipase from Candida antarctica.
2.1. Effect of various biocatalysts
was still much lower in respect to the yield obtained with five-
enzyme mixture. Therefore next set of experiments was performed
with mixture of three different enzymes (Table 1, entries 17–26).
Only one combination of enzymes successfully provided prod-
uct in 50% yield (Table 1, entry 19). Two other enzyme mixture
provided product in 44% and 42% (Table 1, entries 24 and 25).
The presence of two enzymes: Novozym 435 and acylase I from
A. melleus was found to be crucial for yield enhancement. The
same phenomenon was observed in reactions catalyzed by mix-
ture of four enzymes (Table 1, entries 27–31) and the highest yield
of reaction was 78% (Table 1, entry 30). It is interesting to note
that the absence of Novozym 435 in enzyme mixture substantially
decreased the yield of reaction (Table 1, entry 26). This observation
indicates that Novozym 435 is crucial for the reaction. However,
reaction catalyzed by initial five-enzyme mixture provided prod-
uct in substantially higher yield (Table 1, entry 1). The course of
reaction was also studied by GC method. It turned out that only
small amount of ethanol (less than 1%) was present in reaction
mixture with carboxylic acid. Without carboxylic acid formation
of ethanol was not observed, what is in opposition to mechanism
proposed previously [12a]. In order to get inside into catalytic
effect of lipase from Candida antarctica present in Novozym 435,
thermally deactivated Novozym 435 was used together with four
other enzymes. In this experiment only traces of product 1 were
obtained (Table 1, entry 32). Changing deactivated Novozym 435
to native lipase from C. antarctica in enzyme mixture the same
result was obtained (Table 1, entry 33). However, when reaction
was conducted with those two biocatalysts together with four other
enzymes, the product was obtained in 10% yield (Table 1, entry
Screening of biocatalysts was performed using over 45 commer-
cially available enzymes in the model reaction conducted in toluene
at 40 ◦C (Scheme 1).
In order to speed up screening process 45 enzymes were divided
into nine reaction mixtures (vials). Each group consisted of five dif-
ferent enzymes. It was observed that only one group consisted of:
lipase from wheat germ, acylase I from Aspergillus melleus, papain
crude powder, Amano lipase AK from Pseudomonas fluorescens, and
Novozym 435 catalyzed model reaction efficiently. Results are pre-
sented in Table 1.
Preparative scale reaction provided ester 1 (model compound)
with above mentioned enzyme mixture in 96% isolated yield
(Table 1, entry 1). In other groups formation of ester 1 was not
observed. To determine which of enzymes is responsible for the
formation of ester 1, the series of experiments with single enzyme
from the active group were performed: lipase from wheat germ,
acylase I from A. melleus, papain crude powder, Amano lipase AK
from P. fluorescens, and Novozym 435 (Table 1, entries 2–6). Surpris-
ingly in all reactions with a single enzyme, no significant formation
of 1 was observed (Table 1, entries 1–5). This somehow confusing
observation led to the conclusion that two or more enzymes are
responsible for observed catalytic effect. To identify the catalytic
effect of enzymes pairs, second set of experiments was performed
(Table 1, entries 7–16). According to the data included in Table 1
under standard conditions product 1 was obtained for some of
these two-enzyme mixtures (Table 1, entries 9, 10, 13–16). Only
one combination of two enzymes gave the product in substan-
tially higher yield 14% (Table 1, entry 14). However, this yield

M. Zysk et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 225–229
227
Scheme 3. Esterification of carboxylic acids with dialkyl carbonates.
of carbon dioxide molecule. The release of carbon dioxide is a driv-
ing force of the whole process, shifting equilibriums towards ester
product.
2.2. Effect of substrate
Since observation that five-enzyme catalytic system was very
efficient for the synthesis of ester 1 its catalytic ability to esterify
other carboxylic acids was tested. The series of experiments with
different carboxylic acids were performed (Scheme 3).
Scheme 2. Proposed mechanism of enzymatic esterification with dialkyl carbonate.
Obtained results are shown in Table 2. In order to get inside
into reaction kinetics in all cases reaction was stopped after 24 and
48 h. The highest yields were obtained in esterification of aromatic
carboxylic acids without substituent (Table 2, entries 3–6). It was
observed that the yield is increasing, when the alkyl chain between
carboxylic group and aromatic ring is longer (Table 2, entries 1–4).
Reactions with substrates possessing electron withdrawing sub-
stituents (Table 2, entries 11–14) proceeded slightly slower than
these with substrates possessing electron donating substituents
(Table 2, entries 8–10) within 24 h. There are two exceptions in
reaction with 3-(o-methoxyphenyl)-propanoic acid and (Table 2,
entry 7) p-methoxybenzoic acid (Table 2, entries 1 and 2), where
very low yield of reactions can be caused by the steric hindrance in
ortho position.
The influence of organic solvent on reaction course was
verified and reactions in dialkyl carbonates as solvents for 3-
phenylpropanoic acid were performed (Table 2, entries 5 and 6). It
provided product in similar, 78% and 86% yield respectively, which
indicates that use of solvent can be omitted (Table 2, entries 5 and
6). In majority of cases reaction was completed within 48 h. Ester-
ification of 2-phenyletanoic acid and 3-phenylpropanoic acid was
almost completed within 24 h (Table 2, entries 3–6). Altogether,
these observations show that the presented enzymatic system is
34). Deactivated Novozym 435 and native lipase from C. antarctica
without four other enzymes provided only trace amount of prod-
uct (Table 1, entry 35). These results demonstrate strong influence
of solid support on the catalytic effect present in enzyme mixture.
Additional experiments were performed to identify possible sta-
bilizing effect of having co-proteins present in assay mixture that
contains Novozym 435. During control experiment using an unre-
lated protein, bovine serum albumin in combination with Novozym
435 only traces of product 1 were formed. The same observation
was noticed during experiments with the same amount of only
Novozym 435 (20 mg) as amount of mixture of five enzymes (A, B,
C, D, E). Therefore the increase of protein in reaction mixture is not
responsible for catalytic effect. According to our results the additive
of ethanol is not required to begin the reaction course. Therefore
different mechanism shown in Scheme 2 is proposed.
In the first step free hydroxyl group of enzyme reacts with
dialkyl carbonate providing enzyme-alkyl carbonate (EAC) and
alcohol molecule (which is similar to acyl-enzyme formation). It
is followed by nucleophile attack (carboxylic acid). This reaction
results in formation of the intermediate product A, which degra-
dation proceeds via enzyme-hydrogen carbonate with dissociation
Table 2
Esterification of carboxylic acids with dialkyl carbonates (R1OCO2R1).
Entry
Substrate
R1 (2)
Prod.
Yield [%]a
Yield [%]b
91
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
p-methoxybenzoic acid
p-methoxybenzoic acid
2-phenyletanoic acid
2-phenyletanoic acid
3-phenylpropanoic acid
3-phenylpropanoic acid
3-(o-methoxyphenyl)-propanoic acid
3-(o-methoxyphenyl)-propanoic acid
3-(p-methoxyphenyl)-propanoic acid
3-(p-methoxyphenyl)-propanoic acid
3-(p-chlorophenyl)-propanoic acid
3-(p-chlorophenyl)-propanoic acid
3-(p-bromophenyl)-propanoic acid
3-(p-bromophenyl)-propanoic acid
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
2a
2b
2c
2d
2e
1
35
46
87
98
93
100
100
89 (78)c
100 (100)c
100 (100)c
89
96 (86)c
2f
26
75
71
81
67
73
70
72
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2r
100
100
100
100
100
100
100
(
(
(
(
)-2-phenylpropanoic acid
)-2-phenylpropanoic acid
)-3-phenyl-2-methylpropanoic acid
)-3-phenyl-2-methylpropanoic acid
49 (94%ee)d
71 (ee nd)
34 (ee nd)
64 (55%ee)d
96 (ee nd)
100 (ee nd)
92 (ee nd)
100 (ee nd)
Me
Et
nd: not determined.
a
Reaction conducted 24 h.
Reaction conducted 48 h.
Reaction conducted in dialkyl carbonate.
Determined with HPLC analysis (chiral column Chiralcel OD-H).
b
c
d

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M. Zysk et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 225–229
able to transform very wide spectrum of substrates, which is very
rarely observed in regard to enzymatic transformations. Usually,
wide substrate acceptance of enzymes results in diminishing stere-
oselectivity of biotransformations. The reactions catalyzed by the
five-enzyme system with model chiral substrates were performed.
For esters obtained from ( )-2-phenylpropanoic acid and ( )-3-
phenyl-2-methylpropanoic acid (Table 2, entries 15 and 18) HPLC
analyses with chiral column were performed. Ester of carboxylic
acid 2n was obtained in 49% yield and 94% enantiomeric excess
(Table 2, entry 15, E = 101), whereas ester of carboxylic acid 2r
was obtained in 64% yield and 55% enantiomeric excess (Table 2,
entry 18, E = 14). These results indicate that, despite wide spectrum
of transformed substrates, the studied enzymatic system resem-
bles its stereoselectivity. This methodology can be extended for
synthesisoptically pureesters, what is of greatimportance forphar-
maceutical industry. Significantly, all reactions were performed in
40 ◦C. It is important to note, that leading the reactions in low tem-
perature vastly reduces the loss of energy, which is necessary in
standard conditions of esterification.
P. fluorescens was purchased from Sigma, Novozym 435 (Novo
sp 435A) was purchased from Novo Industri A/S, papain crude
powder was purchased from Merck.
4.2. Chemicals
All the chemicals were obtained from commercial chemical
sources (Sigma Aldrich, Poland). The solvents were of analytical
grade.
4.3. Experimental setup
To the solution of acid (1 mmol) in toluene (1 ml), the corre-
sponding dialkyl carbonate (2 mmol) and enzymes (per 4 mg of
each enzyme) were added in 5 ml vial. The reaction mixture was
stirring for 24 or 48 h at 40 ◦C in vortex mixture (Heidolph Promax
1020) equipped with incubator (Heidolph Inkubator 1000). After
cooling, the mixture was washed with NaHCO3 (3×) and brine (1×).
Organic solvent was removed under vacuum to obtain the product
2a–2r.
3. Conclusions
4.4. Analysis
In summary, the first multi-enzymatic system for efficient
irreversible esterification of carboxylic acids is presented. Exper-
iments with single and multi-enzymatic system proved that the
most efficient catalytic effect was observed in the mixture of five
enzymes. Discovered enzymatic system irreversibly transforms
selected carboxylic acids, utilizing commonly available dialkyl
carbonates. At the same time, the catalyzed reactions are still ste-
reoselective. The method is environmentally friendly and efficient.
Altogether, the presented studies describes innovative and efficient
enzymatic tool for synthesis of esters, which significantly expand
the spectrum of enzyme applications.
All reactions were monitored by TLC on Merck silica gel Plates
60 F254, detector UV–Vis, mobile phase: hexan/ethyl acetate
(9/1 = v/v). The NMR analyses were performed on Varian Gemini
200 MHz. HPLC analyses were performed on Varian ProStar appa-
ratus with UV Varian ProStar 330 detector and Chromatopac C-R6A
analyzer equipped with Chiralcel OD-H column (not thermostated),
flow rate: 1 ml/min, mobile phase: hexan/i-propanol (9/1 = v/v),
wavelength of detector 232 nm.
Methyl p-methoxybenzoate (2a) [14]. 1H NMR (CDCl3, 200 MHz)
ε 3.81 (s, 3H), 3.85 (s, 3H), 7.80–7.84 (m, 2H), 7.96–8.00 (m, 2H).
13C NMR (CDCl3, 200 MHz) ␧ 51.6, 55.5, 113.0, 122.1, 131.8, 163.0,
166.7.
4. Experimental
Ethyl p-methoxybenzoate (2b) [14]. 1H NMR (CDCl3, 200 MHz) ε
1.29–1.36 (t, J = 7.0 Hz, 3H), 3.86 (s, 3H), 4.21–4.32 (q, J = 7.0 Hz, 2H),
7.78–7.85 (m, 2H), 7.99–8.09 (m, 2H). 13C NMR (CDCl3, 200 MHz) ε
14.4, 55.3, 60.6, 113.3, 122.6, 131.3, 163.1, 166.7.
4.1. Enzymes
Enzymes used in screening:
Methyl 2-phenyletanoate (2c) [15]. 1H NMR (CDCl3, 200 MHz)
ε 3.69 (s, 2H), 3.78 (s, 3H), 7.24–7.41 (m, 5H). 13C NMR (CDCl3,
200 MHz) ε 41.3, 52.5, 127.1, 128.5, 129.5, 134.1, 171.9.
Ethyl 2-phenyletanoate (2d) [16]. 1H NMR (CDCl3, 200 MHz) ε
1.19–1.30 (t, J = 7.0 Hz, 3H), 3.60 (s, 2H), 4.09–4.21 (q, J = 7.0 Hz, 2H),
7.22–7.36 (m, 5H). 13C NMR (CDCl3, 200 MHz) ε 14.4, 41.6, 61.0,
127.2, 128.7, 129.1, 134.3, 171.4.
1. Lipase from Mucor javanicus (Sigma), Amano Lipase PS from
Burkholderia cepacia (Sigma), lipase from Rhizopus arrhizus
(Sigma), Lipoprotein lipase from Pseudomonas sp. (Sigma), Pro-
tease from Aspergillus oryzae (Sigma).
2. Lipase from Penicillium roqueforti (Sigma), lipase from Chro-
mobacterium viscosum (Sigma), lipase from Candida lipolytica
(Sigma), protease from Bacillus amyloliquefaciens (Sigma), cel-
lulase type VI from Trichoderma viride (Sigma).
3. Lipase from Candida rugosa (Sigma), lipase from M. miehei
(Sigma), RLAP, driselase from Basidiomycetes (Sigma), protease
from Bacillus licheniformis (Sigma).
4. Lipase from Rhizopus oryzae (Sigma), lipase type II from porcine
pancreas (Sigma), PLE, BLAP, pectinase from Rhizopus sp. (Sigma).
5. Lipase from Pseudomonas sp. (Sigma), lipase from P. fluorescens,
CLAP, BKAP, pectinase from Aspergillus niger (Serva).
Methyl 3-phenylpropanoate (2e) [17]. 1H NMR (CDCl3,
200 MHz) ε 2.61–2.69 (t, 2H, J = 7.0 Hz), 2.93–3.01 (t, 2H, J = 7.0 Hz),
3.69 (s, 3H), 7.20–7.39 (m, 5H). 13C NMR (CDCl3, 200 MHz) ε 31.3,
36.0, 51.9, 126.6, 128.6, 128.8, 140.5, 173.3.
Ethyl 3-phenylpropanoate (1) [18]. 1H NMR (CDCl3, 200 MHz) ε
1.19–1.27 (t, 3H, J = 7.2 Hz), 2.57–2.66 (t, 2H, J = 7.9 Hz), 2.91–2.99
(t, 2H, J = 7.9 Hz), 4.07–4.18 (q, 2H, J = 7.2 Hz), 7.18–7.35 (m, 5H). 13
C
NMR (CDCl3, 200 MHz) ε 14.5, 31.2, 36.3, 60.8, 126.6, 128.7, 130.2,
140.8, 173.0.
6. Lipase B C. antarctica recomb. A. oryzae (Sigma), Lipozyme imm.
from M. miehei (Sigma), DLAP, JLAP, aldolase from Rabbit muscle
(Sigma).
7. Lipase from Rhizopus niveus (Sigma), lipase imm. in Sol–Gel-
AK from hog pancreas (Fluka), lipase from Candida cylindracea,
WLAP, esterase from horse liver (Sigma).
8. Lipase from hog pancreas (Sigma), lipase imm. in Sol-Gel-AK
from M. miehei (Fluka), lipase from Aspergillus sp., GLAP, PLAP,
Mixture A, B, C, D, E: The lipase from wheat germ, acylase I from
A. melleus were purchased from Fluka, the Amano lipase AK from
Methyl 3-(o-methoxyphenyl)-propanoate (2f) [19]. 1H NMR
(CDCl3, 200 MHz) ε 2.55–2.64 (t, 2H, J = 8.2 Hz), 2.90–2.98 (t, 2H,
J = 8.2 Hz), 3.63 (s, 3H), 3.81 (s, 3H), 6.82–7.30 (m, 4H). 13C NMR
(CDCl3, 200 MHz) ε 26.3, 34.5, 55.3, 58.5, 110.4, 120.6, 127.6 128.8,
130.1, 157.7, 173.6.
Ethyl 3-(o-methoxyphenyl)-propanoate (2g) [19]. 1H NMR
(CDCl3, 200 MHz) ε 1.16–1.25 (t, 3H, J = 7.0 Hz), 2.51–2.63 (t, 2H,
J = 8.2 Hz), 2.83–2.90 (t, 2H, J = 8.2 Hz), 3.78 (s, 3H), 4.07–4.17 (q,
2H, J = 7.0 Hz), 6.85–7.15 (m, 4H). 13C NMR (CDCl3, 200 MHz) ε 14.5,
26.1, 34.7, 53.0, 61.0, 111.0, 120.2, 127.8, 128.5, 131.1, 158.0, 173.5.

M. Zysk et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 225–229
229
Methyl 3-(p-methoxyphenyl)-propanoate (2h) [20]. 1H NMR
(CDCl3, 200 MHz) ε 2.48–2.56 (t, 2H, J = 8.1 Hz), 2.80–2.84 (t, 2H,
J = 8.1 Hz), 3.60 (s, 3H), 3.78 (s, 3H), 6.62–7.01 (m, 4H). 13C NMR
(CDCl3, 200 MHz) ε 32.0, 36.2, 54.0, 58.1, 128.3, 129.1, 132.6, 140.1,
173.2.
analysis,
Chiralcel
OD-H
column,
hexan/i-PrOH = 9/1,
RT1 = 4.362 min, RT2 = 5.150 min; [␣]D = −12.3 (ca. 1.0, CHCl3,
20 ◦C) [24b].
Acknowledgement
Ethyl 3-(p-methoxyphenyl)-propanoate (2i) [18]. 1H NMR
(CDCl3, 200 MHz) ε 1.19–1.27 (t, 3H, J = 7.0 Hz), 2.51–2.62 (t, 2H,
J = 8.2 Hz), 2.85–2.91 (t, 2H, J = 8.2 Hz), 3.79 (s, 3H), 4.05–4.19 (q,
2H, J = 7.0 Hz), 6.80–7.14 (m, 4H). 13C NMR (CDCl3, 200 MHz) ε 14.7,
31.8, 36.0, 53.0, 61.0, 128.5, 129.3, 132.3, 140.0, 173.4.
Methyl 3-(p-chlorophenyl)-propanoate (2j) [17]. 1H NMR
(CDCl3, 200 MHz) ε 2.57–2.62 (t, 2H, J = 7.7 Hz), 2.87–2.95 (t,
2H, J = 7.7 Hz), 3.66 (s, 3H), 7.12–7.30 (m, 4H). 13C NMR (CDCl3,
200 MHz) ␧ 30.2, 35.6, 52.0, 128.4, 129.3, 132.0, 139.0, 173.2.
Ethyl 3-(p-chlorophenyl)-propanoate (2k) [18]. 1H NMR (CDCl3,
200 MHz) ε 1.19–1.27 (t, 3H, J = 7.2 Hz), 2.55–2.63 (t, 2H, J = 7.9 Hz),
2.88–2.95 (t, 2H, J = 7.9 Hz), 4.07–4.17 (q, 2H, J = 7.2 Hz), 7.11–7.27
(m, 4H). 13C NMR (CDCl3, 200 MHz) ε 14.8, 31.6, 36.0, 60.8, 128.1,
129.0, 132.1, 140.0, 173.0.
This work was supported by project “Biotransformations for
pharmaceutical and cosmetics industry” No. POIG.01.03.01-00-
158/09-01 part-financed by the European Union within the
European Regional Development Fund.
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(CDCl3, 200 MHz) ε 1.19–1.26 (t, 3H, J = 7.2 Hz), 2.55–2.63 (t, 2H,
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(
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3.67–3.72 (m, 1H), 4.10–4.19 (q, J = 7.0 Hz, 2H), 7.21–7.25 (m, 5H).
13C NMR (CDCl3, 200 MHz) ε 14.1, 18.8, 45.6, 61.3, 127.1, 127.6,
128.1, 140.3, 174.6.
(
)-Methyl 3-phenyl-2-methylpropanoate (2p) [23]. 1H NMR
(CDCl3, 200 MHz) ε 1.06–1.08 (d, 3H, J = 7.5), 2.45–2.65 (m, 2H),
2.90–2.98 (m, 1H), 3.55 (s, 3H), 7.10–7.24 (m, 5H). 13C NMR (CDCl3,
200 MHz) ε 17.0, 40.0, 41.7, 51.9, 126.6, 128.6, 128.7, 129.2, 129.3,
139.6, 176.9.
(
)-Ethyl 3-phenyl-2-methylpropanoate (2r) [24a]. 1H NMR
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3H), 2.45–2.65 (m, 2H), 2.90–2.98 (m, 1H), 4.05–4.20 (q, J = 7.0 Hz,
2H), 7.10–7.24 (m, 5H). 13C NMR (CDCl3, 200 MHz) ␧ 14.6, 16.9,
40.2, 41.5, 61.2, 126.3, 128.6, 128.9, 129.1, 129.6, 139.2, 176.7; HPLC
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