Regioselective enzymatic acylation of troxerutin in nonaqueous medium

Article abstract of DOI:10.1016/j.cclet.2009.08.017

A series of monosubstituted troxerutin esters have been synthesized by enzyme-catalyzed regioselective acylation of troxerutin in nonaqueous medium. Using divinyl dicarboxylates (CH₂{double bond, long}CH{single bond}OOC{single bond}(CH₂)_n{single bond}COO{single bond}CH{double bond, long}CH₂, n = 2, 3, 4, 7, 8, 11) featuring different chain length as acyl donors and alkaline protease from Bacillus subtilis as catalyst, troxerutin was regioselective acylated at B' ethoxyl group. The results indicated that the regioselectivity of the enzyme-catalyzed acylation was not affected by the chain length of the acyl donor.

Full text of DOI:10.1016/j.cclet.2009.08.017

Available online at www.sciencedirect.com
Chinese Chemical Letters 21 (2010) 59–62
www.elsevier.com/locate/cclet
Regioselective enzymatic acylation of troxerutin in
nonaqueous medium
a
a
a
b
Yong Mei Xiao a, , Pu Mao , Zhen Zhao , Liang Ru Yang , Xian Fu Lin
*
a College of Chemistry & Chemical Engineering, Henan University of Technology, Zhengzhou 450001, China
b Department of Chemistry, Zhejiang University, Hangzhou 310027, China
Received 8 June 2008
Abstract
A series of monosubstituted troxerutin esters have been synthesized by enzyme-catalyzed regioselective acylation of troxerutin
in nonaqueous medium. Using divinyl dicarboxylates (CH2 CH–OOC–(CH2)n–COO–CH CH2, n = 2, 3, 4, 7, 8, 11) featuring
different chain length as acyl donors and alkaline protease from Bacillus subtilis as catalyst, troxerutin was regioselective acylated
at B’ ethoxyl group. The results indicated that the regioselectivity of the enzyme-catalyzed acylation was not affected by the chain
length of the acyl donor.
# 2009 Yong Mei Xiao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Troxerutin; Acylation; Enzymatic synthesis; Regioselectivity
Flavonoids are widely used in food, cosmetics, medicines and various commodity preparations [1] due to their
antioxidant, antimicrobial, anti-inflammatory, anti-tumor and anti-angiogenic activities [2,3], while their application
is still limited by their low stability and solubility in lipophilic or hydrophilic media [4]. In order to improve their
stability or modify the solubility, many groups have studied the derivatization of flavonoids by chemical, enzymatic
and chemoenzymatic methods. Biocatalysts with high specificity and selectivity have gained recognition as favorable
alternatives to catalyze the acylation of flavonoids [5–7]. It has been reported that regioselective acylation of
flavonoids enhanced their antioxidant and antimicrobial activities [8].
Enzymatic acylation of flavonoids provides a relatively efficient synthetic approach to the availability of a large
range of flavonoids derivatives, which would facilitate the investigation of their biological properties. In the paper, we
investigated the regioselective enzymatic acylation of troxerutin and synthesized a series of monosubstituted
troxerutin esters. The obtained troxerutin derivative containing a vinyl group may be further utilized as precursor of
functional material or monomer of polymeric drug.
1. Experimental
Alkaline protease from Bacillus subtilis was from Wuxi Enzyme Co. Ltd. (4500 U/mg, P. R. China). Troxerutin was
provided by Sichuan Yabao Guangtai Pharmaceutical Co. Ltd. (P. R. China). All other chemicals were of the highest
purity commercially available. Divinyl carboxylates were prepared and purified as described by the patent [9].
* Corresponding author.
E-mail address: henangongda@yahoo.com (Y.M. Xiao).
1001-8417/$ – see front matter # 2009 Yong Mei Xiao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2009.08.017

60
Y.M. Xiao et al. / Chinese Chemical Letters 21 (2010) 59–62
Scheme 1. Enzymatic synthesis of vinyl troxerutin esters.
The process of the reaction was monitored by TLC. Infrared spectra were measured with a Nicolet Nexus FTIR 670.
The acylation position of the product was established by 1H NMR and 13C NMR (Bruker Advance DMX 500). Mass
spectra were obtained on a Bruker Esquire-LC instrument (methanol; negative mode).
To initiate the reaction, 2.5 g alkaline protease from B. subtilis was added to the mixture of troxerutin (2.8 g,
0.00377 mol) and divinyl dicarboxylate (0.015 mol) in pyridine(100 mL). The suspensions were shaken at250 rpmfor5
days at 50 8C. The reaction was followed by TLC (eluent, ethyl acetate/methanol/H2O, 15:3.6:1, v/v) and terminated by
filtering off the enzymes. The filtrates were concentrated under reduced pressure and the products were obtained by
column chromatography purification. Enzymatic synthesis of troxerutin esters 2a–2f was shown in Scheme 1.
2. Results and discussion
Transesterification of troexrutin with divinyl dicarboxylates in the presence of alkaline protease from B. subtilis,
afforded monosubstituted troxerutin derivatives 2 (Scheme 1). The products obtained were characterized by ESI-MS,
1
FT-IR, H NMR and 13C NMR.
In the 13C NMR spectrum of 2c (Table 1), the signal of C-A0 upfielded from 70.78 to 67.41 ppm, and the signal of C-
B0 downfielded from 60.03 to 62.98 ppm, while the chemical shifts for C-A and C-B did not change obviously,
indicating that the structure of the esterification product should be 2 [10]. 13C NMR spectra analysis of 2a–2f indicated
that the transesterifications all selectively occurred at the B0 primary hydroxyl position, not B primary hydroxyl
Table 1
13C NMR data of troxerutin and vinyl derivatives (DMSO-d6, d).
Carbon number
Troxerutin
2a
2b
2c
2d
2e
2f
C-4
C-7
C-9
C-5
C-2
C-40
C-30
C-3
C-10
C-60
C-50
C-20
C-1000
C-1
C-1000
C-6
C-8
177.97
165.15
161.34
157.05
156.95
151.37
148.02
134.16
123.05
122.88
114.90
113.29
105.55
101.78
101.41
98.87
177.95
165.13
161.30
156.94
156.88
151.54
148.15
134.24
123.81
123.04
113.89
113.58
105.53
101.81
101.36
98.85
177.94
165.11
161.27
156.90
156.10
150.73
148.06
134.19
123.37
122.99
114.90
113.50
105.52
101.70
101.38
98.85
178.11
165.29
161.46
157.09
157.04
151.66
148.29
134.40
123.85
123.01
115.66
113.72
105.69
101.91
101.46
99.01
177.92
165.11
161.27
156.89
151.45
148.06
147.49
134.21
122.80
122.70
115.34
115.13
105.48
101.85
101.37
98.84
177.96
165.14
161.31
156.93
156.88
151.53
148.14
134.26
123.72
123.06
115.55
113.86
105.54
101.88
101.37
98.86
177.93
165.11
161.27
156.89
151.47
150.78
148.07
134.22
123.35
123.00
116.18
115.44
105.48
101.86
101.37
98.84
93.39
93.36
93.33
93.51
93.32
93.36
93.32

Y.M. Xiao et al. / Chinese Chemical Letters 21 (2010) 59–62
61
Table 1 (Continued )
Carbon number
Troxerutin
2a
76.84
2b
76.79
2c
76.99
2d
76.79
2e
76.85
2f
C-300
C-500
C-200
C-4000
C-3000
C-2000
C-400
C-A0
C-A0
C-A
C-5000
C-600
C-B0
C-B0
C-B
76.86
76.45
74.65
72.23
71.08
71.08
70.99
70.82
70.82
70.67
68.76
67.62
60.03
60.03
59.79
18.18
76.80
76.38
74.59
72.17
71.04
71.03
70.94
70.77
67.45
70.59
68.71
67.60
59.97
62.89
59.72
18.14
173.41
170.87
141.67
98.42
33.85
33.46
29.37
29.32
29.30
29.18
29.09
28.91
28.78
24.86
22.48
76.40
74.61
72.21
71.13
71.07
70.86
70.78
67.24
70.60
68.70
67.57
60.00
63.24
59.75
18.13
173.34
170.01
141.61
98.67
28.79
76.36
74.60
72.15
71.03
71.03
70.94
70.77
67.40
70.58
68.40
67.16
59.96
62.89
59.72
18.15
172.88
170.49
141.64
98.57
33.36
32.77
32.50
76.57
74.76
72.35
71.29
71.11
71.00
70.94
67.41
70.78
68.87
67.60
60.12
62.98
59.91
18.30
173.34
170.87
141.83
98.65
33.61
33.28
24.30
76.38
74.59
72.15
71.05
71.04
70.94
70.77
67.42
70.59
68.70
67.22
59.98
62.76
59.72
18.14
173.40
170.86
141.64
98.42
33.80
33.42
28.75
28.61
24.77
24.42
76.42
74.61
72.20
71.13
71.07
70.96
70.80
67.28
70.63
68.72
67.59
60.01
62.77
59.75
18.15
173.40
170.87
141.70
98.43
33.88
28.97
24.81
C-6000
C
O
OCH CH2
OCH CH2
(CH2)n
position. There are three primary hydroxyl groups in the troxerutin derivative and the DS (Degree of Substitution) of
1
1
the products was determined by H NMR and MS. In the H NMR spectrum of 2c, the ratio of proton integral for
(CH2)n groups and troxerutin showed the product to be monosubstituted ester. The ESI-MS analyses were in
1
accordance with the H NMR results. Similar results were observed in 2a–2f.
Enzyme is an important factor for selectivity and activity. Six other commercially available enzymes (CCL,
Lipozyme, PPL, Novozym 435, HPL and Alkaline protease from Bacillus licheniformis) were chosen for the
transesterification. The results showed that CCL, Lipozyme, PPL and HPL did not show any reactivity in pyridine even
after 7 days, while alkaline protease from Bacillus licheniformis showed similarly catalytic activity as alkaline
protease from Bacillus subtilisin.
The obtained vinyl troxerutin esters have high application potential as precursor of functional material in
pharmaceutical and material chemistry. Studies on the reaction of vinyl troxerutin and metformin are being in
progress.
Acknowledgments
We thank the Science and Technology hall of Henan Province (No. 072300420070; No. 82102330014), China, for
financially supported to this work.
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