Highly regioselective synthesis of novel aromatic esters of arbutin catalyzed by immobilized lipase from Penicillium expansum

Article abstract of DOI:10.1016/j.molcatb.2010.07.003

Ester derivatives of phenolic glycosides have attracted more attention in food, cosmetic and pharmaceutical industries, due to their potent biological activities. In the present study, a group of novel aromatic esters of arbutin were successfully synthesized by using immobilized lipase from Penicillium expansum with excellent 6′-regioselectivities (>99%) and moderate to excellent isolated yields (68-93%), except for 6′-O-vanilloyl-arbutin and 6′-O-(p-hydroxycinnamoyl)-arbutin with 28% and 34% yields, respectively. Among all the acyl donors tested, the lipase was most active towards vinyl 3-phenylpropionate, while remarkable decrease in the activity was recorded when the phenyl of acyl donors carried the hydroxyl and/or methoxyl.

Full text of DOI:10.1016/j.molcatb.2010.07.003

Journal of Molecular Catalysis B: Enzymatic 67 (2010) 41–44
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Highly regioselective synthesis of novel aromatic esters of arbutin catalyzed by
immobilized lipase from Penicillium expansum
Rong-Ling Yanga, Ning Lia, Min Yeb, Min-Hua Zonga,∗
a State Key Laboratory of Pulp and Paper Engineering, College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China
b School of Biosciences and Bioengineering, South China University of Technology, Guangzhou 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Ester derivatives of phenolic glycosides have attracted more attention in food, cosmetic and pharmaceu-
tical industries, due to their potent biological activities. In the present study, a group of novel aromatic
esters of arbutin were successfully synthesized by using immobilized lipase from Penicillium expansum
with excellent 6ꢀ-regioselectivities (>99%) and moderate to excellent isolated yields (68–93%), except for
6ꢀ-O-vanilloyl-arbutin and 6ꢀ-O-(p-hydroxycinnamoyl)-arbutin with 28% and 34% yields, respectively.
Among all the acyl donors tested, the lipase was most active towards vinyl 3-phenylpropionate, while
remarkable decrease in the activity was recorded when the phenyl of acyl donors carried the hydroxyl
and/or methoxyl.
Received 18 March 2010
Received in revised form 17 June 2010
Accepted 4 July 2010
Available online 15 July 2010
Keywords:
Enzyme catalysis
Lipase from Penicillium expansum
Arbutin
© 2010 Published by Elsevier B.V.
Regioselective acylation
Aromatic esters
1. Introduction
and tyrosinase inhibitory activities [9]. Huang et al. reported that
methyl trans-cinnamate had more potent tyrosinase inhibitory
Arbutin, p-hydroxylphenyl ␤-d-glucopyranoside, is attracting
increasing attention due to a variety of biological activities such
as antioxidative activity, lowing blood glucose, protecting the
membrane from the freeze–thaw damage [1,2]. In particular, this
phenolic glycoside acts as a well-known inhibitor against tyrosi-
nase which catalyzes two distinct reactions of melanin synthesis
and has been used as a skin whitening agent in cosmetics [3]. For
centuries, arbutin-containing plant extracts have been used widely
as the diuretic and urinary anti-infective agent in folk medicine. It
was reported that fatty acid esters of arbutin displayed promoting
biological activities compared with the parent compound [4–7]. For
example, Tokiwa et al. reported that arbutin undecylenic acid ester
was able to inhibit the activity of tyrosinase more efficiently than
arbutin [4]. Arbutin laurate was more effective than the unmodi-
fied phenolic glycoside in preventing the oxidation of linoleic acid
[6].
effect than cinnamic acid [10]. Recent study showed that chem-
ical or enzymatic modification of phenolic acids could result in
multifunctional amphiphilic antioxidants, which displayed higher
biological activities than the precursors [11]. For example, arbutin
ferulate inhibited the oxidation of low-density lipoproteins more
efficiently than arbutin and exhibited a higher antiradical activity
than ferulic acid, a natural antioxidant [12]. Hence, it is expected
that the mutual derivatives of arbutin and aromatic acids, dual
prodrugs, have more potent biological activities than the corre-
sponding precursors.
Enzymes have been widely applied in the modification of
polyhydroxylated compounds [13–16], due to the mild reaction
conditions, excellent selectivity, simplicity and being environ-
mentally friendly. Nevertheless, there are only several reports on
enzymatic synthesis of ester derivatives of arbutin [4–7,12,17].
In addition, the product yields were unsatisfactory. For example,
arbutin cinnamate was formed in a yield of 28% via an enzymatic
route [17].
Lipase from Penicillium expansum is a commercial enzyme with
molecular mass, optimal pH and temperature being 28 kDa, 9.5 and
34 ◦C, respectively. It was reported that the enzyme had a low pro-
tein sequence homology compared with other typical lipases and
had a high percentage of hydrophobic residues (51.4%) in the N-
terminal region [18]. As a result, it is expected that the lipase will
displayuniquecatalytic properties. Previously, we successfully syn-
thesized biodiesel and a series of fatty acid esters of arbutin by using
Arylaliphatic acids, distributed widely in nature, possess var-
ious interesting biological properties such as antioxidative, free
radical scavenger, and antimicrobial activities, as well as antitu-
mor activities. For example, benzoic acid has a long history of
use as an effective preservative in the food industry. In addition,
cinnamic acid and its derivatives have antioxidative activities [8]
∗
Corresponding author. Tel.: +86 20 8711 1452; fax: +86 20 2223 6669.
E-mail address: btmhzong@scut.edu.cn (M.-H. Zong).
1381-1177/$ – see front matter © 2010 Published by Elsevier B.V.
doi:10.1016/j.molcatb.2010.07.003

42
R.-L. Yang et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 41–44
Fig. 1. Lipase-catalyzed synthesis of ester derivatives of arbutin.
immobilized lipase from P. expansum [19–21]. In the present work,
we continued to extend its application potential to the synthesis of
a group of novel aromatic esters of arbutin 3a–j with highly potent
interests (Fig. 1).
2f, 45%; vinyl p-coumarate 2h, 53%; vinyl p-methoxycinnamate 2i,
61%; vinyl 3,4-dimethoxycinnamate 2j, 56%.
The NMR data of the vinyl esters 2f–j is available as
supplementary information.
2.3. Enzymatic acylation of arbutin
2. Materials and methods
In
a typical experiment, a reaction mixture of arbutin
2.1. Biological and chemical materials
(0.04 mmol), 5 equivalents of vinyl ester in anhydrous THF (2 ml)
was added to a sealed-cap vial (10 ml) containing immobilized
enzyme (1.7 U) and incubated at 50 ◦C, 200 rpm. Aliquots were
withdrawn from the reaction mixture at specified time intervals,
and then diluted by 25-fold with the corresponding mobile phase
prior to HPLC analysis. All the experiments were conducted in trip-
licate.
Crude powder of lipase from P. expansum (3000 U/g of hydrolytic
activity) was kindly donated by Leveking Bioengineering Co.,
Ltd., Shenzhen, China. The macroporous adsorbent resin D4020
with the specific surface area of 540–580 m2/g and average pore
diameter of 100–105 Å, which was composed of the crosslinked-
polystyrene, was from Chemical Co. of Nankai University, Tianjin,
China. The enzyme was immobilized on resin D4020 according
to the method described by Li et al. [19]. The immobilization
method and determination of lipase transesterification activity
are available as supplementary information. The immobilized
lipase showed a transesterification activity of 32 U/g resin (one
unit of lipase activity was defined as the amount of enzyme
required for producing 1 ␮mol 6ꢀ-O-butyryl-arbutin from arbutin
and vinyl butyrate in THF per minute at 35 ◦C). Arbutin was from
Sigma–Aldrich, USA. Phenylacetic acid, 3-phenylpropionic acid, 4-
phenylbutyric acid, 5-phenylvaleric acid, vanillic acid, p-coumaric
acid, p-methoxycinnamic acid and 3,4-dimethoxycinnamic acid
were from Darui Chemicals Co., Ltd., Shanghai, China. Vinyl cin-
namate and vinyl benzoate were purchased from Alfa Aesar, USA.
All other chemicals were also from commercial sources and were
of high purity available.
2.4. HPLC analysis
HPLC analysis was carried out on an Agilent 1100 chromato-
graph (Agilent Technologies Co., Ltd., USA) with a UV detector
at 282 nm using Zorbax SB-C18 column (4.6 mm × 250 mm,
5 ␮m, Agilent Technologies Co., Ltd., USA).
A gradient elu-
tion at 1.0 ml/min with water/methanol (40:60, v/v) from 0
to 3.0 min, and then water/methanol (20:80 v/v) from 5.0 min
was used. The retention times of the parent compound
and the esters were 2.35 min (arbutin 1), 3.48 min (6ꢀ-O-
benzoyl-arbutin, 3a), 3.50 min (6ꢀ-O-phenylacetyl-arbutin, 3b),
4.35 min [6ꢀ-O-(3-phenylpropionyl)-arbutin, 3c], 5.59 min [6ꢀ-O-
(4-phenylbutyryl)-arbutin, 3d], 7.10 min [6ꢀ-O-(5-phenylvaleryl)-
arbutin, 3e], 2.80 min (6ꢀ-O-vanilloyl-arbutin, 3f), 4.58 min (6ꢀ-
O-cinnamoyl-arbutin, 3 g), 2.94 min (6ꢀ-O-p-hydroxycinnamoyl-
arbutin, 3h), 4.65 min (6ꢀ-O-p-methoxycinnamoyl-arbutin, 3i),
4.80 min [6ꢀ-O-(3,4-dimethoxycinnamoyl)-arbutin, 3j], respec-
tively.
2.2. Synthesis of vinyl esters
Vinyl esters were synthesized from aromatic acid as described
by Swern and Jordan [22]. Upon completion of the reaction, the
reaction mixture was filtered. The solvent was removed by vac-
uum distillation, and the crude product was purified through
silica gel column chromatography using petroleum ether/ethyl
acetate as the eluent. The vinyl esters of phenylacetic acid
(2b), 3-phenylpropionic acid (2c), 4-phenylbutyric acid (2d), 5-
phenylvaleric acid (2e) are colorless oil. The vinyl esters of vanillic
acid (2f), p-coumaric acid (2h), p-methoxycinnamic acid (2i) and
3,4-dimethoxycinnamic acid (2j) are white powder. The NMR data
of the vinyl esters 2b–e is the same as that reported previously
[23]. Isolated yields for vinyl esters were as follows: vinyl vanillate
2.5. Synthesis, purification and structure characterization of the
desired ester derivatives
THF (20 ml) containing arbutin (0.4 mmol, 989 mg),
5 equivalents of vinyl ester and immobilized enzyme (16.7 U)
was incubated in a 100 ml Erlenmeyer shaking flask capped with a
septum at 200 rpm and 50 ◦C. Upon completion of the reaction, the
enzyme was filtered off, and the filtrate was concentrated in vacuo.
The residue was separated and purified through silica gel column
chromatography using petroleum ether/ethyl acetate as the eluent.

R.-L. Yang et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 41–44
43
Table 1
5-phenyvalerylation after 5.5 h (entries 9 and 10). It might derive
from the larger steric hindrance of the longer acyl chain. These
results demonstrate that the lipase is more specific toward vinyl 3-
phenylpropionate 2c and differs in substrate specificity from lipase
from Burkholderia cepacia, which is more specific toward vinyl 4-
phenybutyrate 2d in the synthesis of aromatic esters of floxuridine
[23].
It was worth noting that the enzymatic cinnamoylation of
arbutin was much slower than 3-phenylpropionation (entries 6 and
13) in spite of the fact that cinnamoyl is similar in chain length to the
3-phenylpropionyl. It could come from the unfavorable resonance
Enzymatic synthesis of aromatic esters of arbutin.
Entry
Acyl donor
Time (h)
Conversion (%)
6ꢀ-Regioselectivity (%)
1
2
3
4
5
6
7
8
2a
1
72
1
72
1
4
1
5
1
5.5
96
1
68
96
1
80
1
80
13
97
13
99
81
99
77
99
70
99
30
22
99
36
10
80
9
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
2b
2c
2d
2e
9
10
11
12
13
14
15
16
17
18
effect caused by the conjugated system between the C C and C
O
2f
2g
double bonds. In addition, as compared to 3-phenylpropionate, cin-
namate is more rigid due to the presence of unsaturated C C, thus
resulting in steric strain. Similar results were also reported by other
researchers with different enzymes [26,27]. Encouragingly, the iso-
lated yield of 6ꢀ-O-cinnamoyl-arbutin 3g achieved in this work was
much higher than that of the previous report (88% vs. 28%), demon-
strating the potential of the enzyme in the preparation of arbutin
esters.
2h
2i
2j
70
The reaction conditions: arbutin (0.04 mmol), 5 equivalents of vinyl ester, immobi-
lized enzyme (1.7 U) in anhydrous THF (2 ml) at 50 ◦C, 200 rpm.
It was clear from Table 1 that substituents present in the phenyl
moiety of the acyl donor exerted a negative impact on the reac-
tion. For example, the replacement of vinyl benzoate 2a with vinyl
vanillate 2f as the acyl donor substantially lowered the reaction
rate of the enzymatic acylation, as indicated by the low conversion
(30%) after a reaction time of 96 h (entry 11). The unfavorable steric
hindrance of the substituent(s) might contribute to this. Addition-
ally, different substituents affected the enzymatic reaction quite
differently (entries 14–18). The enzyme was slightly more active in
the p-methoxylcinnamoylation (entry 16, 80% after 80 h) than that
in the 3,4-dimethoxycinnamoylation (entry 18, 80% after 70 h). It
might be attributed to the steric strain from extra 3-methoxy in 3,4-
dimethoxycinnamoyl compared with the p-methoxylcinnamoyl.
And the reaction rate and conversion were much higher in the p-
methoxylcinnamoylation (entries 15 and 16, 80% after 80 h) than
those in the p-hydroxylcinnamoylation (entry 14, 36% after 96 h).
Although the hydroxyl is less steric than the methoxyl, the former
is more polar than the later and thus may have more significant
negative effect on enzyme performances owing to the non-polar
environment of the acyl-binding pocket in the enzyme active site
[24,26–28].
The structures of aromatic esters of arbutin were characterized
by 13C and 1H NMR (Bruker AVANCE Digital 400 MHz NMR spec-
trometer, Germany) at 100 and 400 MHz, respectively. All ester
derivatives of arbutin are white powder. Isolated yields of aromatic
esters of arbutin were as follows: 6ꢀ-O-benzoyl-arbutin 3a, 86%; 6ꢀ-
O-phenylacetyl-arbutin 3b, 87%; 6ꢀ-O-(3-phenylpropionyl)-arbutin
3c, 93%; 6ꢀ-O-(4-phenylbutyryl)-arbutin 3d, 91%; 6ꢀ-O-(5-
phenylvaleryl)-arbutin 3e, 90%; 6ꢀ-O-vanilloyl-arbutin 3f, 28%;
6ꢀ-O-cinnamoyl-arbutin 3g, 88%; 6ꢀ-O-(p-hydroxycinnamoyl)-
arbutin 3h, 34%; 6ꢀ-O-(p-methoxycinnamoyl)-arbutin 3i, 78%;
6ꢀ-O-(3,4-dimethoxycinnamoyl)-arbutin 3j, 68%.
For NMR data, see supplementary information.
3. Results and discussion
The immobilized enzyme-mediated acylation of arbutin was
conducted with aromatic acid vinyl esters as acyl donors (Table 1).
Interestingly, in all cases, only the 6ꢀ-ester of arbutin was detected
by NMR and HPLC, indicating that the immobilized enzyme
displayed almost absolute 6ꢀ-regioselectivity (>99%). This is in
agreement with our recent report [21].
4. Conclusions
As shown in Table 1, the reaction rate was greatly dependent
on the chain length of acyl donors 2a–e (entries 1–10). For exam-
ple, the lipase displayed the lowest activity in the benzoylation
(entries 1 and 2), which might stem from the unfavorable reso-
nance effect and the steric hindrance of the phenyl ring present in
the acyl moiety [24,25]. According to the classical valence–bond
theories, delocalization of the electrons occurs in the conjugated
system where the C O of the carboxyl is conjugated with the aro-
matic phenyl, thus resulting in the increase of the electron density
of the carbon of the C O [25]. Consequently, it is unfavorable for
the nucleophilic attack of the alcohol on the acyl–enzyme interme-
diate. In addition, the bulky and rigid phenyl ring would result in
a serious steric strain in enzymatic benzoylation [24]. The reac-
tion rate and the maximum substrate conversion of enzymatic
phenylacetylation (entries 3 and 4) were similar to those of benzoy-
lation. Interestingly, the reaction was markedly accelerated when
vinyl phenylacetate 2b was replaced by vinyl 3-phenylpropionate
2c, and the enzymatic acylation afforded a substrate conversion
of 99% in 4 h (entries 5 and 6). This could be accounted for by
the significantly reduced steric strain of the acyl group. However,
the reaction rate decreased slightly with further increment of the
chain length (entries 7–10). For example, a 99% conversion was
achieved with 4-phenylbutanoylation after 5 h (entries 7 and 8) and
Immobilized lipase from P. expansum is an excellent biocatalyst
for the synthesis of the mutual derivatives of arbutin and aro-
matic acids. Some interesting information about the interactions
between the enzyme and acyl groups was obtained via the study of
enzyme acyl recognition. These findings may help to tailor the cat-
alytic performances of the synthetically useful enzyme by chemical
modification and protein engineering approaches.
Acknowledgments
We thank the National Natural Science Foundation of China
(20876059 and 20906032), and Major State Basic Research Devel-
opment Program ‘973’ (2010CB732201) for financial support.
Appendix A. Supplementary data
The methods of preparing immobilized enzyme, assaying lipase
transesterification activity, NMR data and spectra of vinyl esters
(2f–j) and aromatic esters of arbutin (3a–j) are available as supple-
mentary information.

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R.-L. Yang et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 41–44
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