Regioselective alcoholysis of silybin A and B acetates with lipases

Article abstract of DOI:10.1016/j.molcatb.2011.04.007

Large screening identified lipase AK to be capable of the selective deacetylation of pentaacetyl silybins A and B to yield 3,5,20,23-tetra-O-acetyl- silybins A and B, and 3,20,23-tri-O-acetyl-silybins A and B, respectively. Deacetylation occurred at phenolic OH groups, only. These new compounds prepared from the optically pure silybins can serve as new stereochemically pure synthons for selective silybin modifications.

Full text of DOI:10.1016/j.molcatb.2011.04.007

Journal of Molecular Catalysis B: Enzymatic 71 (2011) 119–123
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Regioselective alcoholysis of silybin A and B acetates with lipases
Katerˇina Purchartováa,b , Petr Marhola , Radek Gazˇáka , Daniela Montic , Sergio Rivac , Marek Kuzmaa ,
Vladimír Krˇena,∗
a Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenˇská 1083, CZ-142 20 Prague, Czech Republic
b Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12840 Praha 2, Czech Republic
c Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco 9, I-20131 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Large screening identified lipase AK to be capable of the selective deacetylation of pentaacetyl sily-
bins A and B to yield 3,5,20,23-tetra-O-acetyl-silybins A and B, and 3,20,23-tri-O-acetyl-silybins A and
B, respectively. Deacetylation occurred at phenolic OH groups, only. These new compounds prepared
from the optically pure silybins can serve as new stereochemically pure synthons for selective silybin
modifications.
Received 25 February 2011
Received in revised form 30 March 2011
Accepted 6 April 2011
Available online 13 April 2011
© 2011 Elsevier B.V. All rights reserved.
Keywords:
Silymarin
Silibinin
Silybin B
Lipase
Deacylation
1. Introduction
Studies dealing with selective lipase-catalyzed deacylations are
scarce and are mostly limited to quercetin, catechin and morin.
The chemistry of flavonoids, analogously to that of other poly-
ols like carbohydrates, is complicated by the need for complex
protection/deprotection strategies. Further problems stem from
the high sensitivity of flavonoids towards oxidative agents and
alkaline conditions, and their tendency to form complexes with
some metal cations. This strongly limits the use of certain protec-
tion/deprotection strategies. The use of enzymes can circumvent
these limitations, both in terms of selectivity and mild conditions;
and is also compatible with food and drug applications.
Lambusta et al. [7] has demonstrated partial deacetylation of
quercetin and catechin pentaacetates with three lipases (from
Pseudomonas cepacia, Mucor miehei and Candida rugosa). Another
study dealing with lipase-catalyzed (P. cepacia lipase) deacetylation
of luteolin, kaempferol, kaempferide, and quercetin peracetates,
found 7- and 4ꢀ-acetates to be preferentially removed in these types
of compounds [10].
However, the use of lipases for the modification of flavonoids
is complicated by technical limitations, e.g. low solubility in
water (but also in highly nonpolar solvents) that is circumvented
by the use of suitable organic solvents and by another, often
neglected fact, that many flavonoids are potent inhibitors of lipases.
This effect has been demonstrated e.g. with 3-hydroxyflavone, 5-
hydroxyflavone, catechin or kaempferol that even display their
potential as inhibitors of lipases [11], or some flavonoids contained
in citruses [12] and in medicinal plants [13].
The flavonolignan silybin (1) (CAS No. 22888-70-6) is the major
component of the silymarin complex extracted from the seeds
of Silybum marianum (L.) Gaertn. (Carduus marianus L., Aster-
aceae) (milk thistle) [14] occurring as an equimolar mixture of two
diastereomers, silybin A (1a), and silybin B (1b) (Fig. 1). We have
recently developed a preparatory-scale chemoenzymatic separa-
tion method [15–17], which made it possible to study biological
activities with optically pure diastereomers of silybin and also to
perform chemical transformations with optically pure and well
defined compounds.
Lipases, which are obvious candidates for the selective
derivatization of flavonoids, have been often used for selective
modifications of various natural compounds [1,2].
Most of the so far described lipase-catalyzed acylations were
focused on flavonoid glycoside acylations where the acyl prefer-
entially substitutes the primary OH group of the glycone (typically
glucose) [3–6]; other positions at the flavonoid moiety are seldom
hit [5,7]. This was also demonstrated by molecular modeling using
the CalB (Candida antarctica lipase B) and series of quercetin gly-
cosides (e.g. rutin, isoquercitrin) [8] showing that the only position
susceptible to enzymatic acylation on the aglycone part (quercetin)
is its 3ꢀ-OH. A comprehensive review of enzymatic acylations of
flavonoids has been recently published by Chebil et al. [9].
∗
Corresponding author. Tel.: +420 296 442 510; fax: +420 296 442 509.
E-mail address: kren@biomed.cas.cz (V. Krˇen).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.04.007

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K. Purchartová et al. / Journal of Molecular Catalysis B: Enzymatic 71 (2011) 119–123
Fig. 1. Diastereomeric silybins (1a and 1b).
Silybin is currently advocated for the treatment of cirrho-
2.2. HPLC
Chromatographic analyses were performed on a Shimadzu
Prominence system (Kyoto, JP) consisting of a DGU-20A mobile
phase degasser, two LC-20AD solvent delivery units, a SIL-20AC
cooling autosampler, CTO-10AS column oven and SPD-M20A diode
array detector with semi-micro cell volume (2.5 ␮l). Shimadzu LC
solution software (Kyoto, JP) was used to collect and process chro-
matographic data.
sis, chronic hepatitis, and liver diseases associated with alcohol
consumption and environmental toxin exposure [18], as it is an
efficient antioxidant and chemoprotectant. Silybin is considered
to be very safe and no serious adverse effects have been reported
[19]. Other beneficial effects of silybin include chemopreventive as
well as hypocholesterolemic, cardioprotective, and neuroprotec-
tive effects [20,21]. For example, silybin is in phase-II clinical trials
in the US for the treatment of prostate adenocarcinoma [22]. Sily-
bin activities are linked to a large number of effects at the cellular
and molecular level, such as estrogenic activity, the modulation
of drug transporters (P-glycoprotein) [23] and a specific action on
DNA expression via the suppression of nuclear factor-␬B [20,24].
The importance of silybin is demonstrated, for example, by the high
frequency of relevant papers (ca 200 per year).
Silybin has a unique flavonoid structure, differing from most
known flavonoids so far functionalized with lipases. It does not have
a double bond at position 2,3 (like quercetin or rutin), which makes
it very sensitive towards oxidation even under weak alkaline condi-
tions [25]. Being a flavonolignan, it possesses a lignan moiety with
a primary alcoholic 23-OH group. These structural features intrin-
sically create four stereocenters and this makes silybin chemistry
much more complicated than in other more simple flavonoids.
The importance of regioselectively acylated derivatives of
silybin (prepared mostly by chemical synthesis) has been well
established. Several acyl derivatives of silybin have significantly
better biological activity than silybin [26–28], but the synthesis of
derivatives other than the 23-O-acyl is quite problematic due to
poor regioselectivity and low yields [26].
A
Chromolith Performance RP-18e monolithic column
(100 mm × 3 mm i.d., Merck, DE) was used with solvent system
A: MeCN/MeOH/H2O/HCO2H (2/37/61/0.1, v/v/v/v) and B: 100%
MeCN. The optimal gradient was 0–4 min 0% B, 4–25 min 0–40% B,
25–26 min 40% B, 26–28 min 40–0% B; flow rate 1.2 ml/min. The
column oven temperature was set to 25 ◦C and the samples were
kept at 20 ◦C in the autosampler. The PDA data was acquired in the
200–350 nm range and the 285 nm signal was extracted.
2.3. Chemistry
2.3.1. 3,5,7,20,23-Penta-O-acetyl-silybin (2)
Natural silybin (mixture 1a and 1b, 1:1) (1, 500 mg, 1.04 mmol)
was acetylated using the standard acetylation procedure
(Ac2O/pyridine 4 ml, 1:1, v/v, overnight) [29], which yielded
705 mg (98.2%) of the title compound 2 as a yellow amorphous
solid. Acetylations of pure silybins A (1a) and B (1b), prepared as
described in [17], were performed analogously yielding perac-
etates 2a and 2b, respectively. The 1H and 13C NMR data for 2a
and 2b, which was compatible with their structures, is given in
supplementary material.
In this work, we report the screening of a library of com-
mercially available hydrolases (lipases, proteases, and acylases)
for the regioselective deacylation of peracetylated silybins. An
optimized protocol for obtaining optically pure 3,5,20,23-tetra-
O-acetyl-silybins and 3,20,23-tri-O-acetyl-silybins using lipase AK
from Pseudomonas sp. is described.
2.3.2. Screening of enzymatic alcoholysis of
3,5,7,20,23-penta-O-acetyl-silybin (2)
The screening of lipases for the alcoholysis of silybin peracetate
2 was accomplished by dissolving 2 (5 mg, 7.2 ␮mol) in a mixture of
tert-butyl methyl ether (MTBE) or toluene/n-butanol (1.1 ml, 10:1,
v/v) containing the respective lipase (5 mg; see Table 1). The reac-
tion mixture was incubated at 45 ◦C and 450 rpm in a Thermomixer
(Eppendorf, DE), reaction progress was monitored by TLC (mobile
phase: CHCl3/toluene/acetone/HCO2H—80:10:5:1) and HPLC. No
diastereomeric selectivity, e.g. discrimination between 2a and 2b,
was observed in any enzymatic deacetylation.
2. Experimental
2.1. General methods
NMR spectra were recorded on a Bruker Avance III 600 MHz
spectrometer (600.23 MHz for 1H, 150.93 MHz for 13C at 30 ◦C) in
DMSO-d6 (99.9 at.% D, Sigma–Aldrich, Steinheim, DE). The resid-
ual signal of the solvent was used as an internal standard (ıH
2.500 ppm, ıC 39.60 ppm). NMR experiments: COSY, HSQC, and
HMBC were performed using the manufacturer’s software. 1H NMR
and 13C NMR spectra were zero-filled to fourfold data points and
multiplied by a window function prior to Fourier transformation.
A two-parameter double-exponential Lorentz–Gauss function was
applied for 1H to improve resolution and line broadening (1 Hz)
was applied to obtain a better 13C signal-to-noise ratio. Chemical
shifts are given on a ␦-scale with digital resolution justifying the
reported values to three (ıH) or two (ıC) decimal places.
2.3.3. Preparative reactions catalyzed by lipase AK
3,5,7,20,23-Penta-O-acetyl-silybin (2, 100 mg, 0.144 mmol) was
dissolved in a mixture of MTBE/n-butanol (22 ml, 10:1, v/v) with the
addition of lipase AK from Pseudomonas sp. (Amano, JP) (100 mg).
The reaction mixture was incubated in a 50 ml Falcon tube at 45 ◦C
under shaking (450 rpm) for 48 h. The residue after filtration and
evaporation was purified by column chromatography on silica gel
(mobile phase: CHCl3/acetone/toluene/HCO2H—95:5:5:1) yielding
3,20,23-tri-O-acetyl-silybin (3, 9 mg, 15.3%) and 3,5,20,23-tetra-
O-acetyl-silybin (4, 42 mg, 77.9%), both as yellowish amorphous

K. Purchartová et al. / Journal of Molecular Catalysis B: Enzymatic 71 (2011) 119–123
121
Table 1
Hydrolases tested for silybin peracetate (2) alcoholysis.a
Enzyme
Source
Producer
MTBE
toluene
Lipase AK
Lipase PS
Lipase D
Lipase L
Pseudomonas sp.
Amano
Amano
Amano
Amano
᭹
᭹
᭹
᭹
Pseudomonas cepacia
Rhizopus delemar
Candida lipolytica
Mucor javanicus
Lipase M
Amano
᭹
Lipase F-AP15
Lipase N
Lipase R
Lipase CV
Rhizopus japonicus Lipase
Lipase CE
Lipase A
Lipase GC
Candida rugosa Lipase
PPL
Novozym 435
CAL-A
Rhizopus oryzae
Rhizopus niveus
Amano
Amano
Amano
Amano
Biocatalysts Ltd.
Amano
Amano
Amano
Sigma
᭹
᭹
Penicillium roquefortii
Chromobacterium viscosum
Rhizopus japonicus
Humicola lanuginosa
Aspergillus niger
᭹
᭹
Geotrichum candidum
Candida rugosa
porcine pancreas
Candida antarctica (Lip. B)
Candida antarctica (Lip. A)
Candida antarctica (Lip. B)
Bacillus subtilis
Sigma
Novozymes
Novozymes
Novozymes
Sigma
᭹
᭹
CAL-B
Subtilisin
᭹
᭹
᭹
Protease N
Proleather
Protease P
Acid Protease II
Acylase Amano 3000
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Rhizopus niveus
Amano
Amano
Amano
Amano
Aspergillus sp.
Amano
᭹
᭹
a
Reaction conditions: MTBE (48 h), toluene (150 h); ᭹—conversion > 2%.
solids; some unreacted 2 was recovered (yields are given in relation
to the substrate consumed). 3: MALDI MS (m/z) 631 [M+Na]+ (calc.
631.1), 4: 673 [M+Na]+ (calc. 673.2). Optically pure 3,20,23-tri-O-
acetyl-silybin A (3a, 8 mg, 21.5%) and 3,20,23-tri-O-acetyl-silybin
B (3b, 20 mg, 28.9%), 3,5,20,23-tetra-O-acetyl-silybin A (4a, 24 mg,
70.5%) and 3,5,20,23-tetra-O-acetyl-silybin B (4b, 40 mg, 63.1%)
were prepared analogously as above. 1H and 13C NMR data for
compounds 3, 3a, 3b, and 4, 4a, 4b are given in supplementary
material.
H-3 or H-23 and the acetyl carbonyl is a direct proof of acetylation
at these positions. The acetylation of hydroxyls at C-5 and C-7 is
manifested by the chemical shift changes of C-5, C-6, C-7, C-8, and
C-4a. Acetylation of hydroxyl at C-20 causes substantial changes in
the carbon chemical shifts at C-19, C-20, and C-21. Furthermore,
it was also possible to detect a four-bond heteronuclear correla-
tion between the methyl protons of the acetyl group and acetylated
carbon, which is direct evidence for its substitution.
Kinetic studies (Fig. 2) have proved that the products are formed
sequentially, the first product being tetraacetate 4b. Careful moni-
toring of the reaction and appropriate termination enables to obtain
tetraacetate 4b in ca 50% purity or triacetate 3b in ca 90% purity,
which facilitates purification. The time course of the reactions with
the respective pure diastereomers does not differ substantially.
Time profile of the reaction may slightly vary with different batches
of Lipase AK.
Phenolic esters are readily cleaved by basic hydrolysis, in general
they are more easily hydrolyzed than those of aliphatic alcohols.
Basic hydrolysis of peracetylated silybin is, however, non-selective,
yielding a complex mixture. Moreover, the presence of a base
leads to fast decomposition of the silybin molecule. Therefore, basic
hydrolysis is impracticable with silybin. Quantitative hydrolysis of
silybin esters is usually achieved by refluxing the corresponding
3. Results and discussion
The aim of this study was to investigate selective deprotection
of silybin peracetate with the possibility of preparing synthons
for further silybin derivatization. The field of silybin chemistry
is experiencing dynamic progress triggered by recent biological
discoveries [21] as well as the availability of optically pure sily-
bins [16,17] and their congeners. For the chemical synthesis of
selectively protected derivatives of silybin, at least two types of
(orthogonal) protecting groups need to be used.
The acetate group has been chosen for this acylation mainly
for its high yields and feasibility—acylation with longer acyls gives
considerably lower yields [16,26]. Large screening of lipases and
proteases in the presence of peracetylated silybin (2) under various
reaction conditions showed that only a limited array of enzy-
matic activities accept this compound as a substrate. From the
24 hydrolases tested, only eleven were active, with very low (ca
2%) conversion in most cases. Better results were achieved with
the lipases PS, subtilisin and Acylase Amano 3000, where the con-
version ranged from 5% to 10%. Only lipase AK gave good enough
conversion rates (40%) to be used in preparatory-scale reactions.
All active lipases yielded the same major products, i.e., 3,20,23-
tri-O-acetyl-silybin (3) and 3,5,20,23-tetra-O-acetyl-silybin (4)
(Scheme 1), whose structures were determined mainly by NMR
spectroscopy. As all OH signals are visible in the 1H NMR spec-
tra of acetyl silybins measured in DMSO-d6 and their experimental
assignment by HMBC is available, the absence of some of them
(compared to the parent compound) provides the first hint of
the acetylation site. The downfield acylation shift is observable
with the C-3 or C-23 protons. The heteronuclear coupling between
Fig. 2. Time course study of hydrolysis of 2b (ꢀ) at 45 ◦C in MTBE/n-butanol by lipase
AK, products 3b (ꢁ) and 4b (᭹) were obtained.

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K. Purchartová et al. / Journal of Molecular Catalysis B: Enzymatic 71 (2011) 119–123
Scheme 1.
ester in an ethanolic solution of conc. HCl. This method is, how-
ever, again non-selective and naturally cannot be used for selective
hydrolysis. Better selectivity has been observed during attempts to
remove the TBDMS group (at 23-OH) in the presence of acetates
at the remaining positions of the silybin molecule [25]. The appli-
cation of either an ion exchanger (Dowex 50W-X8) in MeOH or a
Lewis acid (BF3.Et2O) in CHCl3 led to selective hydrolysis of the
TBDMS group together with the acetates at the 5- and 20-OH
groups of the silybin molecule. This observation hints that these
two positions are the most sensitive to acidic hydrolysis. In con-
trast to these chemical methods, the biotransformation method
we have developed selectively cleaves the acetate at 7-OH (par-
tially also at 5-OH). The difference between the result of the acidic
and the enzymatic hydrolysis of silybin acetates is probably caused
by a better (sterical) accessibility of the acetate at C-7 for the
enzyme compared to other positions. Accordingly, existing stud-
ies of regioselective deacylation of simpler flavonoids (quercetin,
morin) reported regioselective deacylation of the 7-OH position of
the flavonoid skeleton [7,10,30].
Czech Republic (OC09045, ME10027), Czech Science Foundation
(P301/11/0767), and Academy of Sciences of the Czech Republic
(AV0Z50200510). Dr. Ladislav Cvak from Galena Teva Co. (Opava,
Czech Republic) is thanked for his kind provision of silybin.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcatb.2011.04.007.
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