Regioselective galloylation of methyl β-D-glucopyranoside by a lipase

Article abstract of DOI:10.1007/s00706-016-1696-8

Chromogenic substrate 4-nitrophenyl gallate was prepared in four steps and used for selection of hydrolases specific to gallic ester hydrolysis from among 22 commercial lipases, proteases, and crude glycanase cocktails. Enzymes displaying galloyl esterase

Full text of DOI:10.1007/s00706-016-1696-8

Monatsh Chem (2016) 147:1137–1142
DOI 10.1007/s00706-016-1696-8
ORIGINAL PAPER
Regioselective galloylation of methyl b-D-glucopyranoside
by a lipase
1
1
Andrej Chyba1 Maria Mastihubova Vladimır Mastihuba
•
•
´
´
´
Received: 8 December 2015 / Accepted: 7 February 2016 / Published online: 1 March 2016
Ó Springer-Verlag Wien 2016
Abstract Chromogenic substrate 4-nitrophenyl gallate
was prepared in four steps and used for selection of
hydrolases specific to gallic ester hydrolysis from among
22 commercial lipases, proteases, and crude glycanase
cocktails. Enzymes displaying galloyl esterase activity
were tested in regioselective galloylation of methyl b-D-
glucopyranoside with vinyl gallate. Lipozyme TL IM in
acetonitrile was found to afford the highest conversion
(37 %). The reaction proceeded with strict regioselectivity
toward the primary hydroxyl of the glucopyranoside ring,
giving in preparative scale 27 % of purified methyl 6-O-
galloyl-b-D-glucopyranoside as a sole product.
Introduction
The structural motif of 6-O-galloyl-b-D-glucopyranos-1-O-
yl (Fig. 1) is an abundant constitutive fragment of bioactive
natural substances. Many of these compounds were iso-
lated from leaves, stems, roots, or seeds of medicinal plants
and possess remarkable biological activities when bearing,
for example, methyl [1], another galloyl [2, 3], p-hydrox-
yphenyl [4], p-hydroxy-3,5-dimethoxyphenyl [5, 6], or
5-hydroxyeugenyl [7, 8] moiety as the aglycone.
Chemical synthesis of such types of molecules is typi-
cally a multistep process which involves several selective
protection and deprotection steps as described for example
in the preparation of 1,6-di-O-galloyl-b-D-glucopyranose
[9]. On the other hand, direct enzymatic regioselective
galloylation of appropriate glucoside may provide a simple,
more straightforward alternative to standard processes.
There are however no literary data providing effective
conditions for enzyme-catalyzed galloylation of either
sugars or generally polyhydroxylated compounds.
Graphical abstract
HO
O
HO
HO
HO
OH
O
Lipozyme TL IM
Acetonitrile
O
O
O
HO
O
HO
HO
HO
+
OMe
HO
HO
OMe
HO
HO
27%
The majority of published reports on enzymatic gal-
loylations concerns preparation of alkyl gallates [10, 11],
with focus on synthesis of industrially exploited antioxi-
dant—propyl gallate—by microbial tannases [12]. Two
main approaches of preparation of propyl gallate are based
either on direct esterification of gallic acid (1) by n-pro-
panol under catalysis of tannases and lipases in organic
media [10, 11, 13–15] or by exploitation of tannic acid as
donor for gallate transfer catalyzed by esterase and depsi-
dase activity of tannases [16–21]. Transesterifications in
optimized conditions may provide chemical yields of pro-
pyl gallate as high as 90 % [17, 19, 20]. This method is,
however, not suitable for preparation of galloyl glycosides
due to complicated separation of chemically similar prod-
ucts from the reaction mixture. Moreover, galloylation of
Keywords Acylation Á Enzymes Á Glycosides Á
Regioselectivity
´
& Vladimır Mastihuba
chemvrma@savba.sk
1
Laboratory of Biocatalysis and Organic Synthesis, Institute of
´
´
Chemistry, Slovak Academy of Sciences, Dubravska cesta 9,
845 38 Bratislava, Slovakia
123

1138
A. Chyba et al.
Screening of galloyl esterases
HO
HO
HO
O
O
The modified photometric method of Haslam and Tanner
[24] was used for routine screening of galloyl esterase
activity among a scale of commercial lipases, proteases,
and crude glycanases either in solid or in liquid form.
Liquid preparations, if positive for galloyl esterase, were
planned to be ultrafiltered and lyophilized in view of their
use as solid catalysts in organic solvents. Measurable
activities were found in three lipase preparations and one
glycanase, all from Thermomyces lanuginosus (orig. Hu-
micola lanuginosa) (Table 1). We expected galloyl
esterase activity also in lipase B from Candida antarctica
due to its wide substrate specificity, including phenolic
acids [27, 28]. The physical properties of its immobilized
preparation Novozyme 435, however, did not allow us to
measure the activity by photometric method—the non-
soluble enzyme particles were freely flowing on the surface
of the buffer solution or adhered on the walls of the reac-
tion vessel above the liquid.
O
HO
HO
O
Aglycon
HO
Fig. 1 6-O-galloyl-b-D-glucopyranos-1-O-yl structural motif
structurally more sophisticated acceptors than n-propanol
is less efficient as proven in direct acylation of catechins
with gallic acid catalyzed by immobilized tannase with
chemical yields not exceeding 6 % [22]. To our best
knowledge, the only successful enzymatic galloylation of
sugars was reported in our recent work [23]. Within our
investigation of substrate specificity and regioselectivity of
Lipolase 100T on wide scale of aromatic donors we carried
out galloylation of methyl a-D-glucopyranoside, achieving
only 12 % yield of 6-O-galloylated product.
The purpose of this study was therefore to find either an
enzyme displaying galloyl esterase (tannase) activity or a
lipase with wider substrate specificity and therefore able to
catalyze more effective galloylation of model glycoside as
a transfer of gallate from its activated donor. At the same
time, the selected enzyme should be active in polar organic
solvents, which are necessary for dissolving the glycosidic
acceptor.
These five enzymes were tested in acylation of methyl
b-D-glucopyranoside (5) with 3 (Scheme 2). It is worth to
point that the conversions after 15 days (Table 2) did not
correspond to galloyl esterase activities displayed by the
respective enzymes as summarized in Table 1. Only two
enzymes, namely Lipozyme TL IM and Lipex 100T, were
moderately effective in the reaction, reaching the respec-
tive conversions 29.8 and 12.1 %. Lipozyme TL IM was
therefore our choice of biocatalyst for further selection of
the reaction conditions.
Results and discussion
Synthesis of substrates
Choice of solvent and excess of galloyl donor
To select the appropriate enzyme and conditions for effec-
tive acylation of the model saccharide with gallic acid (1),
we have prepared two types of gallic esters—4-nitrophenyl
gallate (4-nitrophenyl 3,4,5-trihydroxybenzoate, 2) for gal-
loyl esterase assays and vinyl gallate (vinyl 3,4,5-
trihydroxybenzoate, 3) as the activated galloyl donor for
preparative syntheses. Preparation of the substrate 3 was
executed according to the method published by our group
elsewhere [23]. The first synthesis of 2 as a substrate for
spectrophotometric assay of tannases was realized in 1970
by Haslam and Tanner [24]. 4-Nitrophenyl protocatechu-
ate—another similar chromogenic substrate for tannase
assays was synthetized in 2000 [25]. Both syntheses were
based on protection of phenolic ortho-hydroxyls by
dichlorodiphenylmethane. Our simplified procedure to pre-
pare 2 includes four easily consecutive steps (Scheme 1):
3,4,5-triacetoxybenzoyl chloride (4) was prepared from fully
acetylated 1 [26] and the 4-nitrophenyl 3,4,5-triacetoxy-
benzoate obtained by base catalyzed esterification was
directly deacetylated in acidic conditions.
Enzymatic acylation of saccharides with (poly)phenolic
acids obviously encounters the problem of mutual incom-
patibility of the catalyst, reactants and the reaction
environment. Choice of reaction solvent must respect
hydrophilic character and solubility of reactants, while
lipase-catalyzed esterifications and transesterifications better
proceed in hydrophobic environment with low content of
water. We screened four polar or moderately polar organic
solvents to find the most suitable one for the studied trans-
galloylation. Among them, acetone and acetonitrile (Fig. 2)
allowed us to achieve the highest levels of galloylated
methyl b-D-glucopyranoside (methyl 6-O-(3,4,5-trihydroxy-
benzoyl)-b-D-glucopyranoside, 6). Acetonitrile was therefore
used as reaction solvent in further experiments.
Increased ratio of 3 as a galloyl donor to the acceptor 5
negatively influenced the process of its galloylation (Fig. 3).
Despite of higher reaction rates, the final product concen-
trations decreased and the best results were achieved with
1.5 equivalents of vinyl gallate. The level of galloylation
finished at 37 % after 48 days while reaction with 3 or 5
123

Regioselective galloylation of methyl b-D-glucopyranoside by a lipase
1139
Scheme 1
HO
AcO
AcO
a) Ac2O, H2SO4
b) SOCl2, toluene
O
O
HO
HO
OH
Cl
AcO
4
1
HO
HO
HO
a) p-nitrophenol, Et3N
DMAP, CH2Cl2
b) HCl, acetone
O
O
NO2
2
Table 1 Galloyl esterase activity of selected hydrolases
Enzyme (type)
Origin
Activitya
Lipex 100T (lipase)
Thermomyces lanuginosus
0.05b
9.6b
0.05b
n.d.
0.17b
0b
Pentopan 500BG (xylanase)
Lipozyme TL IM (lipase)
Novozym 435 (lipase)
Lipolase 100T (lipase)
Lipolyve AN (lipase)
Thermomyces lanuginosus
Thermomyces lanuginosus
Candida antarctica
Thermomyces lanuginosus
Aspergillus niger
pig pancreas
PPL (lipase)
0b
Lipase PS (lipase)
Burkholderia cepacia
Pseudomonas fluorescens
bovine pancreas
0b
0b
0b
Lipase AK (lipase)
a-chymotrypsin (protease)
Protease NL (protease)
Liquanase Ultra 2,5L (protease)
Corolase L10 (protease)
Lallzyme Beta (pectinase)
Lallzyme Cuvee Blanc (pectinase)
Cytolase M102 (pectinase)
Rapidase Expression (pectinase)
Ultrazym 100 (pectinase)
Novozym 188 (cellobiase)
Dextrozyme GA 1,5X (glucoamylase)
Celluclast 1.5 L (cellulase)
Carezyme 4500 L (cellulase)
a
Bacillus subtilis
0c
0c
0c
0b
0b
0b
0b
0b
0c
0c
0c
0c
Bacillus subtilis
Carica papaya
Aspergillus niger
Aspergillus niger
Aspergillus niger
Aspergillus niger
Aspergillus niger
Aspergillus niger
Aspergillus niger
Trichoderma reesei
Aspergillus sp.
One unit (IU) corresponds to amount of enzyme forming 1 lmol of p-nitrophenol per 1 min
b
c
IU/g
IU/cm3
equivalents of vinyl gallate reached plateau of 26–27 and
acetonitrile with 1.5 equiv. of vinyl gallate and the reaction
was shaken at 40 °C for 34 days. The reaction mixture
provided the product in amount corresponding to 27 %
chemical yield. Structural analysis of the product by
nuclear magnetic resonance had proven that the reaction
proceeds strictly regioselectively toward the primary
hydroxyl of the acceptor. No formation of digalloyl
derivatives of 5 was observed. Such regioselectivity in
23 % conversion after 22 and 3 days, respectively.
Preparative reaction, isolation and structure
of the product
Preparative galloylation of methyl b-D-glucopyranoside
catalyzed with Lipozyme TL IM was carried out in
123

1140
A. Chyba et al.
Scheme 2
HO
HO
O
O
HO
HO
OH
O
O
Enzyme
Solvent
O
HO
O
HO
HO
HO
+
OMe
HO
HO
OMe
HO
HO
5
6
3
Table 2 Galloylation of 5 catalyzed by hydrolases in acetonitrile
Enzyme
Reaction time/h
Conversion/ %
Pentopan 500BG
Lipolase 100T
Novozym 435
Lipex 100T
360
360
360
360
360
2.1
3.3
3.7
12.1
29.8
Lipozyme TL IM
Fig. 3 Effect of excess of vinyl gallate on galloylation of 5 by
Lipozyme TL IM. Reactions conducted in acetonitrile (filled square
1.5 equiv., filled circle 3 equiv., filled diamond 5 equiv.)
Conclusion
Chromogenic substrate 4-nitrophenyl gallate for assay of
enzymes hydrolysing gallic ester bond was prepared by
new synthetic way in four steps. The photometric assay
identified three lipases and one xylanase (all from Ther-
momyces lanuginosus) comprising gallic ester hydrolase
activity. These enzymes, together with lipase B from
Candida antarctica were tested in galloylations of model
glycoside with vinyl gallate as galloyl donor. The gal-
loylation of methyl b-D-glucopyranoside was achieved in
moderate yield through transesterification catalyzed by
Lipozyme TL IM in acetonitrile. The regioselectivity of
the reaction proceeded exclusively at the primary hydro-
xyl of the glucopyranoside. According to our knowledge,
this is the most effective enzymatic galloylation of sac-
charide reported so far. The product of the reaction is a
natural substance occurring in Sanguisorba officinalis L.
[1]. The reaction presented in this work opens also access
to other biologically active galloylated glycosides of plant
origin.
Fig. 2 Effect of reaction media on galloylation of 5 with 1.5 equiv. of
vinyl gallate (3) catalyzed by Lipozyme TL IM. Reactions conducted
in acetone (empty square), tert-butanol (filled circle), isobutyl methyl
ketone (filled triangle) and acetonitrile (filled square)
acylation of primary hydroxyl of acceptor is consistent
with our experience with another commercial lipase from
Thermomyces lanuginosus (Lipolase 100T). Acylation of
model glycoside with aromatic vinyl esters bearing free
phenolic hydroxyls on aromatic ring (including vinyl gal-
late) proceeded strictly to the primary position in low
yields. On the other hand, the regioselectivity changed if
the aromatic ring was methoxylated or the hydroxyls were
missing. The reactions proceeded faster, with higher yields
and mixtures of monoesters (at the primary hydroxyl) and
corresponding 2,6-diacylated glycoside were formed [23].
123

Regioselective galloylation of methyl b-D-glucopyranoside by a lipase
1141
to 50 % between 12 and 22 min and then back to 0 % in
8 min) was applied. Column thermostat was set up to
30 °C and RI detector to 35 °C. Individual components
were detected at 275 nm and the amount of galloylated
product was calculated from the calibration curve with
linear relation in the interval 0.297–2.97 mM.
Experimental
Enzyme preparations from Novozymes—Lipex 100T,
Pentopan 500 BG, Lipozyme TL IM, Novozym 435,
Lipolase 100T, Liquanase Ultra 2,5L, Corolase L10,
Novozym 188, Dextrozyme GA 1,5X, Celluclast 1,5L and
´
´ˇ
Carezyme 4500L—were gifts from MSc. Marian Illas
(Biotech s.r.o., Slovakia), Lipase AK, Lipase PS and Pro-
tease NL were purchased from Amano Enzyme (USA).
Synthesis of 4-nitrophenyl gallate (2)
To 3.14 g 3,4,5-tri-O-acetoxybenzoyl chloride 4 [26]
(10 mmol) and 1.53 g p-nitrophenol (11 mmol) dissolved
in 50 cm3 dichloromethane, 1.24 cm3 triethylamine
(8.5 mmol) and 0.32 g DMAP (2.5 mmol) were added at
0 °C. The reaction mixture was then stirred under argon
atmosphere at laboratory temperature for 1 h. The reaction
mixture was then diluted with 50 cm3 dichloromethane,
washed with 40 cm3 1 % HCl, 50 cm3 brine, sat. NaHCO3
(2 9 50 cm3) and brine (2 9 50 cm3), dried over Na2SO4,
and concentrated under reduced pressure. Crude honey-like
material (4.4 g) was dissolved in 60 cm3 acetone and
30 cm3 6 M HCl was added. The reaction mixture was
stirred at laboratory temperature for 48 h, then neutralized
by solid Na2CO3 and acetone was eliminated under
reduced pressure. The product was extracted from water
phase by EtOAc (3 9 50 cm3), organic phase was washed
by water (2 9 20 cm3), dried over Na2SO4 and concen-
trated in vacuo to obtain a crude residue. This residue was
purified by flash chromatography (toluene:EtOAc 3:1). The
fractions containing 5 were partly concentrated under
reduced pressure until the moment when the product 5
started to precipitate. The mixture was left in the cold for
several hours, solids were filtered, washed by cold toluene,
and dried. Yield 2.11 g (72 %, two steps); pale yellow
crystals; m.p.: 196–198 °C (CH3OH/CHCl3) (Ref. [24]
197–198 °C); Rf (toluene/EtOAc, 1:1, v/v) = 0.34; 1H
NMR [400 MHz, (CD3)2CO]: d = 8.39 (br s, 3H, OH),
8.35 (d, 2H, J = 9.2 Hz, H-Ar), 7.56 (d, 2H, J = 9.2 Hz,
H-Ar), 7.29 (s, 2H, H-Ar) ppm; 13C NMR (101 MHz,
(CD3)2CO): d = 164.7 (COO), 157.2 (C-Ar), 146.3
(2 9 C–OH), 146.2 (C-Ar), 140.0 (C–OH), 125.9
(2 9 CH-Ar), 123.9 (2 9 CH-Ar), 120.0 (C-Ar), 110.7
(2 9 CH-Ar) ppm.
´
Lallzyme BETA and Lallzyme Cuvee Blanc were pur-
chased from Lallemand (Canada). Lipolyve AN was gift
from Lyven (France). a-Chymotrypsin and pig pancreatic
lipase were from Sigma (St. Louis, MO, USA). Ultrazym
100 was from Ciba Geigy. Rapidase Expression was gift
from O.K.SERVIS BioPro, s.r.o. (Czech Republic, local
supplier of DSM), Cytolase M102 from DSM was ultra-
filtered and lyophilized before use. Gallic acid (1) and
molecular sieves were purchased from Sigma Chemical
(St. Louis, MO, USA), and methyl b-D-glucopyranoside
hemihydrate (2) was purchased from Acro¯s Organics (New
Jersey, USA). The methyl 6-O-galloyl-b-D-glucopyra-
noside (6) used as the standard for HPLC was prepared by
non-optimized enzymatic reaction. Vinyl gallate 3 was
prepared according to the method described by Masti-
´
hubova et al. [23]. Organic solvents for synthesis were
dried and distilled before use. All other chemicals were of
analytical or HPLC grade.
TLC alumina plates Silica gel 60 F254 from Merck
KGaA (Darmstadt, Germany) were used for TLC analysis.
The plates were eluted by ethyl acetate/methanol (9:1, v/v)
and the spots were detected by charring the plates with 5 %
(v/v) ethanolic H2SO4 and heating at ca. 200 °C. High-
performance liquid chromatography was performed on an
Agilent 1200 Series apparatus (Agilent Technologies, Inc.,
Santa Clara, CA, USA) with MZ Aqua Perfect C18 5-lm
reverse-phase column (150 9 4.0 mm) from MZ-Analy-
sentechnik (Mainz, Germany). Spectrophotometric assays
were performed on a UV–visible (UV–Vis) spectropho-
tometer (UV-1800, Shimadzu). Optical rotations were
measured with a digital polarimeter Jasco P-2000 at 20 °C.
1H NMR spectra (400 MHz) and 13C NMR spectra
(101 MHz) were recorded with a Varian 400 MHz spec-
trometer. High resolution mass spectra (HR-MS) were
obtained with the Orbitrap Velos PRO spectrometer from
Thermo Fisher Scientific Inc. (Waltham, Massachusetts,
USA).
Assay of galloyl esterase
Galloyl esterase activity was determined according to the
modified spectrophotometric assay described in the report
of Haslam [24]. The analytical reaction was performed in
10 cm3 DURAN flasks with a sealing lid. Portion of 5 cm3
of freshly prepared 1 mM solution of 4-nitrophenyl gallate
was transferred into each flask and the reaction was placed
on a rotating shaker set up to 250 rpm in thermostat at
constant temperature 40 °C. The reaction was initiated by
adding enzyme preparation (100 mg) except control
Quantitative analysis of reaction mixtures by HPLC
The column was equilibrated/eluted with a mixture of
water/acetic acid/1-butanol (500:1:2.5, v/v/v) with a flow
rate of 1 cm3/min, and a gradient of methanol (ramped up
123

1142
A. Chyba et al.
sample (blank). In 10 min intervals, 1 cm3 aliquots of the
reaction mixture were withdrawn and passed through a
0.22-lm nylon syringe filter. The change of absorbance
was read at 405 nm in 10-mm path length polystyrene
cuvettes. After reading the absorbance, the solutions were
returned back to reaction flasks.
Acknowledgments This work was supported by the Slovak
Research and Development Agency under the contract No. APVV-
0846-12. This publication is also the result of the grant from the
Slovak Grant Agency for Science VEGA under the Project No.
2/0138/12 and the Research and Development Operational Pro-
grammes funded by the ERDF (‘‘Centre of Excellence on Green
Chemistry Methods and Processes’’, CEGreenI, Contract No.
26240120001 as well as ‘‘Amplification of the Centre of Excellence
on Green Chemistry Methods and Processes’’, CEGreenII, Contract
No. 26240120025).
Enzymatic transgalloylations
A typical reaction for screening of reaction conditions was
performed in 10 cm3 DURAN flasks with a sealing lid on a
vibrating shaker set up to 300 rpm in thermostat at constant
temperature 40 °C. The reaction mixture comprised
0.04 M methyl b-D-glucopyranoside 5, 0.06 M vinyl gal-
References
1. Tanaka T, Nonaka G-I, Nishioka I (1984) Chem Pharm Bull
32:117
2. Ishimaru K, Shimomura K (1991) Phytochemistry 30:825
3. Olennikov DN, Chekhirova GV (2013) Chem Nat Compd 49:1
4. Xin-Min C, Yoshida T, Hatano T, Fukushima M, Okuda T (1987)
Phytochemistry 26:515
˚
late 3, 0.4 g of activated molecular sieves 4A, and 0.4 g of
enzyme preparation (except blank) in 5 cm3 of acetonitrile.
To monitor the reaction course, 50-mm3 aliquots of the
reaction mixture were withdrawn at predefined time inter-
vals and analyzed by HPLC.
5. Tao J, Morikawa T, Toguchida I, Ando S, Matsuda H, Yoshikawa
M (2002) Bioorg Med Chem 10:4005
6. Shi C, Xu M-J, Bayer M, Deng Z-W, Kubbutat MHG, Waejen W,
Proksch P, Lin W-H (2010) Phytochemistry 71:435
7. Hussein SAM, Hashim ANM, El-Sharawy RT, Seliem MA,
Linscheid M, Lindequist U, Nawwar MAM (2007) Phytochem-
istry 68:1464
8. Shamaladevi N, Lyn DA, Shaaban KA, Zhang L, Villate S, Rohr
J, Lokeshwar BL (2013) Carcinogenesis 34:1822
9. Li C-W, Dong H-J, Cui C-B (2015) Molecules 20:2034
10. Yu X, Li Y, Wu D (2004) J Mol Catal B-Enzym 30:69
11. Sharma S, Kanwar SS, Dogra P, Chauhan GS (2015) Biotechnol
Prog 31:715
Methyl 6-O-galloyl-b-D-glucopyranoside (6)
The preparative reaction was scaled up 10 times. The
reaction mixture comprised 406 mg of methyl b-D-
glucopyranoside 5 (2 mmol), 588 mg of vinyl gallate 3
˚
(3 mmol), 4 g of activated molecular sieves 4 A, and 4 g
of Lipozyme TL IM in 50 cm3 of acetonitrile. The
reaction mixture was stopped after 34 days by filtration,
the filter cake was washed with acetonitrile and wash-
ings were mixed with the filtrate. Activated charcoal was
added and the suspension was briefly heated. The
mixture was filtered again and solvents from the filtrate
were removed by evaporation. The residue was purified
by chromatography on the column of silica-gel eluted
with EtOAc. Fractions with pure product were collected
and the solvent was removed by evaporation. The honey-
like residue was precipitated from toluene and separated
by filtration to obtain 0.184 g (27 %) of pale yellow
amorphous solid. Rf (EtOAc/methanol, 4:1, v/v) = 0.32;
12. Yao J, Guo GS, Ren GH, Liu YH (2014) J Mol Catal B-Enzym
101:137
13. Yu X-W, Li Y-Q (2005) Appl Biochem Biotechnol 126:177
14. Aithal M, Belur PD (2013) Biotechnol Appl Biochem 60:210
15. Bouaziz A, Horchani H, Salem NB, Chaari A, Chaabouni M,
Gargouri Y, Sayari A (2010) J Mol Catal B Enzym 67:242
16. Gaathon A, Gross Z, Rozhanski M (1989) Enzyme Microb
Technol 11:604
17. Sharma S, Gupta MN (2003) Bioorg Med Chem Lett 13:395
18. Fernandez-Lorente G, Bolivar JM, Rocha-Martin J, Curiel JA,
Mun˜oz R, De Las Rivas B, Carrascosa AV, Guisan JM (2011)
Food Chem 128:214
19. Nie G, Liu H, Chen Z, Wang P, Zhao G, Zheng Z (2012) J Mol
Catal B Enzym 82:102
20. Gonc¸alves HB, Jorge JA, Pessela BC, Lorente GF, Guisan JM,
Guimara˜es LHS (2013) Biotechnol Lett 35:591
21. Nie G, Zheng Z, Yue W, Liu Y, Liu H, Wang P, Zhao G, Cai W,
Xue Z (2014) Process Biochem 49:277
22. Raab T, Bel-Rhlid R, Williamson G, Hansen C-E, Chaillot D
(2007) J Mol Catal B Enzym 44:60
´
23. Mastihubova M, Mastihuba V (2013) Bioorg Med Chem Lett
23:5389
24. Haslam E, Tanner RJN (1970) Phytochemistry 9:2305
25. Iacazio G, Perissol C, Faure B (2000) J Microbiol Meth 42:209
26. Gazit A, Yaish P, Gilon C, Levitzki A (1989) J Med Chem
32:2344
27. Otto RT, Scheib H, Bornscheuer UT, Pleiss J, Syldatk C, Schmid
RD (2000) J Mol Catal B-Enzym 8:201
28. Stamatis H, Sereti V, Kolisis FN (2001) J Mol Catal B-Enzym
11:323
[a]2D0 = -15.2°
(c = 1.0,
CH3OH),
[Ref.
[1]
´
22
½aŠ = -18.6° (c = 1.3, H2O)]; 1H NMR (400 MHz,
D
CD3OD): d = 7.09 (s, 2H, H-Ar), 4.89 (bs, 3H,
3 9 OH), 4.55 (dd, 1H, J = 11.9, 2.1 Hz, H-6a), 4.39
(dd, 1H, J = 11.9, 5.6 Hz, H-6b), 4.22 (d, 1H,
J = 7.8 Hz, H-1), 3.56 (ddq, 1H, J = 7.2, 3.7, 1.9 Hz,
H-5), 3.50 (s, 3H, OCH3), 3.46–3.37 (m, 2H, H-3, H-4),
3.21 (dd, 1H, J = 9.2, 7.9 Hz, H-2) ppm; 13C NMR
(101 MHz, CD3OD): d = 168.3 (COO), 146.5 (2 9 C–
OH), 139.8 (C–OH), 121.4 (C-Ar), 110.1 (2 9 CH-Ar),
105.4 (C-1), 77.9, 71.6 (C-3, C-4), 75.4 (C-5), 75.0 (C-
2), 64.7 (C-6), 57.3 (OCH3) ppm; HRMS (ESI): m/z
calcd for C14H18O10Na ([M ? Na]?) 369.07977, found
369.07911.
´
123