Cis-trans isomerization of silybins A and B

Article abstract of DOI:10.3762/bjoc.10.105

Methods were developed and optimized for the preparation of the 2,3-cis- and the 10,11-cis-isomers of silybin by the Lewis acid catalyzed (BF ₃-OEt₂) isomerization of silybins A (1a) and B (1b) (trans-isomers). The absolute configuration of all optically pure compounds was determined by using NMR and comparing their electronic circular dichroism data with model compounds of known absolute configurations. Mechanisms for cis - trans-isomerization of silybin are proposed and supported by quantum mechanical calculations.

Full text of DOI:10.3762/bjoc.10.105

cis–trans Isomerization of silybins A and B
Michaela Novotná‡1, Radek Gažák‡1,2, David Biedermann1,
Florent Di Meo3,4, Petr Marhol1, Marek Kuzma1, Lucie Bednárová5,
Kateřina Fuksová1, Patrick Trouillas3,6,7 and Vladimír Křen*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 1047–1063.
1Institute of Microbiology, v.v.i. AS CR, Vídeňská 1083, Prague 4,
CZ-14220, Czech Republic, 2Department of Biochemistry, Faculty of
Science, Charles University in Prague, Hlavova 8, CZ-12840 Prague
2, Czech Republic, 3Inserm UMR-S850, Faculté de Pharmacie,
Université de Limoges, 2 Rue du Docteur Marcland, F-87025
Limoges, France, 4Present address: Department of Physics,
Chemistry and Biology (IFM), Linköping University, SE-58183,
Linköping, Sweden, 5Institute of Organic Chemistry and Biochemistry,
v.v.i. AS CR, Flemingovo náměstí 2, Prague 6, CZ-16610, Czech
Republic, 6Department of Physical Chemistry, University of Olomouc,
tř. 17. listopadu 12, CZ-77146 Olomouc, Czech Republic and
7Laboratoire de Chimie des Matériaux Nouveaux, Université de Mons,
Place du Parc 20, B-7000 Mons, Belgium
doi:10.3762/bjoc.10.105
Received: 04 December 2013
Accepted: 04 April 2014
Published: 08 May 2014
Associate Editor: S. Bräse
© 2014 Novotná et al; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Vladimír Křen* - kren@biomed.cas.cz
* Corresponding author ‡ Equal contributors
Keywords:
2,3-cis-silybin; 10,11-cis-silybin; isomerization; silibinin; silybin;
silymarin
Abstract
Methods were developed and optimized for the preparation of the 2,3-cis- and the 10,11-cis-isomers of silybin by the Lewis acid
catalyzed (BF3∙OEt2) isomerization of silybins A (1a) and B (1b) (trans-isomers). The absolute configuration of all optically pure
compounds was determined by using NMR and comparing their electronic circular dichroism data with model compounds of
known absolute configurations. Mechanisms for cis–trans-isomerization of silybin are proposed and supported by quantum
mechanical calculations.
Introduction
The flavonolignan silybin (alternative name silibinin), occur- isomers as well as other flavonolignans from silymarin (crude
ring in the fruits of Silybum marianum (milk thistle), consists of defatted extract from the fruits of S. marianum) are products of
two stereoisomers – silybin A (1a) and B (1b) – in a ca. 1:1 a phenolic oxidative coupling of the flavonoid taxifolin and the
ratio (Figure 1). Their absolute configuration is known [1,2] and lignan coniferyl alcohol. The mechanism of this coupling reac-
their separation was accomplished recently [3-5]. Both silybin tion was described [6] and implied a 10,11-trans relative con-
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Beilstein J. Org. Chem. 2014, 10, 1047–1063.
figuration, as in all the major components of silymarin. Little is The content of silybin cis-isomers in silymarin is presumably
known about the structures of the minor components of sily- very low. The composition of silymarin strongly depends upon
marin. It was speculated that some of them were 10,11-cis- its source, which is influenced by the variety of S. marianum
analogues of the major silymarin constituents [7], but these and by the cultivation, harvest and processing conditions. Varia-
minor components were never isolated in sufficient amount and tions of the minority content (not only silybin cis-isomers) is
purity to enable such unwarranted hypotheses to be verified even more pronounced. Therefore, their identification in a par-
[8,9]. Another study focusing on the minor components of sily- ticular silymarin preparation would have only limited informa-
marin identified two new compounds isosilybin C (3) and D (4), tion value.
however these were shown to be regioisomers of isosilybins A
(5) and B (6) [10] (Figure 2).
Silybin is an important pharmaceutical commodity and a
complete understanding of its composition is needed in order to
Other authors reported 2,3-cis-isomers of silybin [11,12], the understand its pharmaceutical properties. Therefore, the detailed
relative configuration of which were corroborated by 1H NMR structural knowledge and availability of (potential) minor impu-
coupling constants, i.e., J2,3 of ca. 11 Hz in the trans-isomers rities is a fundamental requisite, e.g., for master file assembly.
and 2–3 Hz in the cis-isomers. Nevertheless, their absolute
configurations remained unknown. The origin of the cis-isomers The aim of this work was to prepare stereochemically pure 2,3-
either as biosynthetic side products or as artifacts formed during cis- and 10,11-cis-isomers of silybin A (1a) and B (1b) by a
their isolation is also unknown.
Lewis acid catalyzed isomerization to determine their absolute
configuration and to propose a mechanism for the cis–trans-
The isolation of naturally occurring cis-isomers in the pure form isomerization processes.
and their complete structure identification would be the only
Results and Discussion
unambiguous way how to prove identity of natural and
synthetic cis-isomers. Unfortunately, the conventional identifi- Chemistry
cation by LC–MS or UV–vis scanning is inadequate as the In our previous work on the enzymatic kinetic resolution of
major and minor compounds exhibit the same MS and UV silybin [4,5], BF3∙OEt2 in EtOAc was found to catalyze the
profiles.
transesterification of silybin to yield not only 23-O-
Figure 1: Selected naturally occurring trans-silybins and their acetates.
Figure 2: Isosilybins occurring as minor components of silymarin.
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Beilstein J. Org. Chem. 2014, 10, 1047–1063.
acetylsilybin (2, ca. 90%), but two novel compounds with UV CH3CN and DMSO silybin decomposed, and in CHCl3 the
spectra similar to that of silybin. Enzymatic alcoholysis reaction failed. In EtOAc, the reaction was accompanied by
(n-butanol) of this mixture by Novozym 435 led, after a C-23 O-acetylation, which complicated the reaction mixture
prolonged reaction time, to the removal of the acetyl groups analysis. DMF at 50 °C was found to be the most suitable
from trans-isomers 2a and 2b to give 1a and 1b (Figure 1), solvent for the isomerization and was used for further Lewis
while the two minor isomers remained acetylated. Separation of acid screening. Eventually, BF3·OEt2 proved to be the most
the resulting mixture by silica gel column chromatography suitable Lewis acid for silybin isomerization (Table 1). Other
yielded an inseparable mixture of the two new compounds. In Lewis acids either gave lower isomerization yields (SnCl4), did
HPLC they were assigned to two separate peaks with the same not work at all, or even caused decomposition. Toluene-4-
molecular mass (m/z 524). 1H NMR spectra indicated that the sulfonic acid, as a representative protic acid, gave no reaction
two compounds differ only slightly from the silybin derivatives under the same conditions.
2a and 2b, respectively. The unknown compounds exhibit a
J10,11 of ca. 3 Hz, whereas natural silybin (1) has a J10,11 of ca. In this work, the preparative reactions with the two solvents
8 Hz. Based on this evidence, the unknown compounds from DMF and EtOAc and with stereochemically pure silybin A (1a)
the acylation reaction were proposed to be a diastereoisomeric and B (1b) (Figure 1) exhibited a strong dependence on the
mixture of 23-O-acetyl-10,11-cis-silybin A (7) and B (8) solvent. In DMF (BF3·OEt2, 50 °C), the reaction led to a single
(Figure 3) in a ca. 1:1 ratio.
type of C-2, C-3-isomerization yielding the corresponding 2,3-
cis-10,11-trans-silybin A (9) and 2,3-cis-10,11-trans-silybin B
As the yields of the two 10,11-cis-silybin isomers were low (10) (Scheme 1), respectively. No C-10, C-11-isomerization
(ca. 10%), we optimized the reaction conditions for better was observed in this case. In EtOAc, various cis-isomers were
yields. In the initial screening, aimed at finding the most suit- obtained, depending on the reaction time and also on the type of
able solvent and Lewis acid (Table 1), we used natural silybin, starting material (1a or 1b). A detailed analysis of silybin B
i.e., ca. 1:1 mixture of 1a and 1b.
(1b) isomerization in EtOAc showed the formation of the 23-O-
acetyl-2,3-cis-10,11-trans-isomer 11 within the first 3 hours,
The choice of solvent was limited by the low solubility of then it slowly disappeared, and finally 23-O-acetyl-2,3-trans-
silybin in most organic solvents or by their incompatibility with 10,11-cis-silybin B (12) was formed (Scheme 2). In EtOAc
the Lewis acid BF3·OEt2. The following solvents were tested: (48 h, 80 °C), 1a isomerized into the 23-O-acetyl-2,3-cis-10,11-
EtOAc, DMF, CH3CN, DMSO, CHCl3 at 0, 25, and 50 °C. In trans-isomer 12 and a minor 23-O-acetyl-2,3-trans-10,11-cis-
Figure 3: Structures of cis-derivatives obtained by the isomerization of 1 using BF3·OEt2 in EtOAc.
Table 1: Screening of suitable Lewis and protic acids for silybin isomerization.
Lewis acid
conversion (%)a
note
BF3·OEt2
15–20
SnCl4
12–15
TiCl4
–
strong complexation, quantitative oxidation to 2,3-dehydrosilybin
strong complexation
FeCl3
no reaction
no reaction
–
ZnCl2
BBr3
decomposition
toluene-4-sulfonic acid
no reaction
aReaction conditions: silybin, Lewis or protic acid (>10 equiv), DMF, 50 °C, 1–3 h.
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Beilstein J. Org. Chem. 2014, 10, 1047–1063.
Scheme 1: Silybin A and silybin B isomerizations into their 2,3-cis-isomers (DMF).
Scheme 2: Silybin B isomerization in EtOAc.
isomer 13 (Scheme 3). Surprisingly, the absolute configuration Separation and purification of 2,3-cis-silybins
at C-2, C-3 of compound 13 was the opposite of 1a (2S,3S vs The separation of 2,3-cis-silybin 7 from unreacted 2,3-trans-
2R,3R), which was confirmed by a comparison of their elec- silybin 1a (or 10 from 1b) on silica gel was not feasible, analo-
tronic circular dichroism (ECD) data. Formation of the 10,11- gous to the preparative separation of silybin stereoisomers 1a
cis-isomer with the 2R,3R configuration was not observed and 1b (Figure 1), which was a challenge for several decades. It
during/after the isomerization of 1a in EtOAc. All the reactions was accomplished as the separation of the respective silybin
in EtOAc were accompanied by C-23 O-acetylation.
glycosides for the first time [13,14], later by HPLC [3], and at
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Beilstein J. Org. Chem. 2014, 10, 1047–1063.
Scheme 3: Isomerization of silybin A in EtOAc.
the preparatory scale by lipase-catalyzed discrimination [4,5]. obtain suitable crystals using various crystallization conditions
However, chromatographic separation of 2,3-cis- and 2,3-trans- were unsuccessful. Pure silybins and their congeners are known
silybins is feasible after their C-23 O-acetylation, which can be for their poor crystallization or the formation of small crystals,
accomplished with Novozym 435 in an acetone/vinyl acetate which are not suitable for X-ray analysis. The absolute con-
mixture, giving a nearly quantitative yield of the respective figuration of silybins A (1a) and B (1b) was determined indi-
acetates (without the risk of further isomerization) (Scheme 4, rectly on the basis of a comparison of their physicochemical
part I). As the 2,3-trans-isomer was more abundant than the data (NMR, ECD, OR). Later, these data were matched to those
2,3-cis-isomer its excess was removed before enzymatic acety- of their 10,11-regioisomer, isosilybin A (5) [11]. The same ap-
lation by crystallizing the crude mixture from MeOH/H2O 9:1, proach was used to determine the absolute configuration of
which increased the ratio of the cis/trans-isomer in the mixture isosilybin B (6). The only analogue that yielded suitable crys-
from 1:4 to 1:2.
tals, and hence an unequivocal determination of its absolute
configuration, was 7-(4-bromobenzoyl)isosilybin A [15].
Deacetylation of cis-23-O-acetylsilybins
All pure cis-silybins were obtained as their C-23 acetates. The In this study the determination of the absolute configuration
deacetylation proved to be rather difficult, as both acidic and was based on NMR and ECD spectroscopy. Their relative
basic hydrolysis as well as mild transesterification (NaOMe, configurations, i.e., the determination of trans/cis isomers at
MeOH) failed and/or resulted in decomposition or reversed C-2, C-3 and C-10, C-11, are set by the 1H NMR coupling
isomerization to the original trans-isomers. We eventually had constants J2,3 and J10,11, respectively. The trans-isomers ex-
to use enzymatic alcoholysis with a large excess of Novozym hibit J2,3 of ca. 11 Hz and J10,11 of ca. 8 Hz, while the cis-con-
435 and a longer reaction time (5:1, wt. enzyme/substrate, figuration is indicated by a value of J2,3 or J10,11 of ca. 2–3 Hz
3–5 d, 45 °C) compared to the alcoholysis of the respective [11,12].
acetates of the trans-silybins. This method finally yielded pure
2,3-cis-silybins A (9) and B (10) as well as 10,11-cis-silybin B For the assignment of the absolute configuration, experimental
(14) (Scheme 4, parts II and III).
ECD spectra of compounds 9, 10, 13, and 14 were compared to
those of related compounds with known absolute configura-
tions [2]. The assignment of Cotton effects (CE) to particular
regions in this molecule is based on the rough assumption that
Determination of absolute configuration of
cis-silybins
The determination of the absolute configuration of the new cis- silybin and its analogues are considered to be composed of two
silybins by X-ray analysis is not possible, since all attempts to separate π-conjugated subsystems, the 3-hydroxyflavonone
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Beilstein J. Org. Chem. 2014, 10, 1047–1063.
Scheme 4: Schematic flowchart of the procedures for the preparation and the isolation of cis-silybin isomers (cis-isomers are underlined).
moiety (with the stereogenic centers C-2 and C-3), and the 1,4- ranges were utilized, which were clearly rationalized in terms of
benzodioxane moiety (C-10 and C-11) (see Figure 4). In this the corresponding electronic transitions [22,23]. A pair of posi-
simplification we cannot avoid the adverse effects caused by tive–negative CEs at both spectral ranges is characteristic of the
neglecting the effects of local perturbations on specific stereo- (2R,3R) configuration of the 3-hydroxyflavonone moiety.
genic centers. Thus, the interpretation of our experimental ECD Nevertheless, it should be kept in mind that there are also other
spectra is mainly based on empirical comparison and correla- absorption bands at shorter wavelengths giving rise to observ-
tion to those of i) the dihydroflavonols taxifolin (15) and epitax- able CEs near 230 nm, which are relatively constant in pattern
ifolin (16) isolated from Thujopsis dolobrata [16] and ii) [24-28].
various benzodioxanes, e.g., the pair of benzodioxanes 17 and
18 synthesized from (+)-ephedrine; the natural benzodioxanes The ECD spectrum of compound 10: (θ [deg·cm2·dmol−1], CEs:
eusiderin (19) and eusiderin C (20) [17]; 21 and 22, isolated 339 nm, θ = +24971; 294 nm, θ = −50028; 237 nm θ = +16914;
from Juniperus chinensis [18]; and benzodioxane 23 from 224 nm, θ = −8082; 213 nm, θ = −44628; 203 nm, θ = +162457;
Licaria chrysophylla [19] (Figure 5), for both the C-2, C-3 and 193 nm, θ = −71457) (Figure S1, Supporting Information
C-10, C-11 pairs, respectively [20].
File 1) displayed the coupling constant J2,3 = 2.5 Hz corres-
ponding to a cis-configuration and, therefore, compound 10 was
The ECD of the 3-hydroxyflavonone moiety was discussed with assigned to the 2R,3S-isomer. The ECD spectrum of compound
the aim to determine absolute configuration [21]. UV absorp- 9: (θ [deg·cm2·dmol−1], CEs: 338 nm, θ = +26771; 292 nm,
tion bands in the respective 270–290 nm and 330–320 nm θ = −60650; 233 nm, θ = −58000; 214 nm, θ = −9828; 207 nm,
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Beilstein J. Org. Chem. 2014, 10, 1047–1063.
Figure 4: ECD spectrum of silybin B (1b) and its separation (in a crude approximation) into two π-conjugated moieties (3-hydroxyflavonone with
stereogenic centers C-2, C-3 (in blue) and 1,4-benzodioxane with C-10,C-11) (in red) according to [2].
gous to (+)-taxifolin (2R,3R), according to its J2,3 value of
11.4 Hz and its ECD spectrum (14: θ [deg·cm2·dmol−1], CEs:
327 nm, θ = +9568; 293 nm, θ = −46824; 240 nm,
Δε = +36917 deg·cm2·dmol−1; 230 nm, θ = −8890; 211 nm,
θ = −63590) (Figure S3, Supporting Information File 1).
Accordingly, its absolute configuration at C-2, C-3 should be
2R,3R. Interestingly, compound 13 seems to be 2,3-trans-10,11-
cis-silybin according to its NMR spectra (J2,3 = 11.3 Hz,
J10,11 = 2.9 Hz). However, its ECD spectrum (13:
θ [deg·cm2·dmol−1], CEs: 328 nm, θ = −12870; 294 nm,
θ = +44453; 253 nm, θ = −7535; 242 nm, θ = +7451; 229 nm,
θ = −71888; 205 nm, θ = −1116600; 194 nm, θ = +62990)
(Figure S4, Supporting Information File 1) corresponds to (−)-
taxifolin. According to these results, the absolute configuration
at C-2, C-3 is the opposite of the starting compound 1a (2R,3R).
Therefore, the absolute configuration of 13 is 2S,3S.
To assign the absolute configuration at C-10 and C-11 of silybin
we combined the corresponding coupling constants J10,11 with
Figure 5: Synthetic and natural (benzodioxane-type) compounds
related to silybin stereoisomers with known absolute configurations.
the CEs in 200–280 nm spectral range. It was shown that a sign
of the CE at ca. 236 nm could be used to determine the stereo-
chemistry at C-3 (i.e. C-10 of silybin) (negative CE corre-
θ = +83000; 197 nm, θ = +77685) (Figure S2, Supporting Infor- sponds to the R configuration) [2,17,20]. Thus, the negative CE
mation File 1) together with the J2,3 coupling constant of 2.4 Hz around 230 nm for 13, corroborated by the vicinal 1H–1H
(cis-configuration) are similar to (+)-epitaxifolin [16]. There- coupling constants (J10,11 = 2.9 Hz, e.g., cis-configuration)
fore, the absolute configuration of 9 at C-2, C-3 is 2R,3S. The (Figure S4, Supporting Information File 1) indicates an absolute
absolute configuration of compound 14 at C-2, C-3 is analo- configuration of 2S,3S,10R,11S. In contrast to the ECD spec-
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Beilstein J. Org. Chem. 2014, 10, 1047–1063.
trum of silybin B (1b) (Figure S3, Supporting Information IIa is more stable than the complex at O-1 by ca. 6 kcal·mol−1.
File 1), the J10,11 coupling constant (2.8 Hz, cis-configuration) In the benzodioxane moiety, complexation at O-12 is favored
of compound 14 and its ECD spectrum (positive/negative CE (compared to O-9) by 4 kcal·mol−1. The coordination with
around 240 nm) implies the absolute configuration at C-10, boron initiates isomerization at C-2, C-3 and C-10, C-11 by
C-11 to be 10S,11R, so that the absolute configuration of 14 is charge rearrangements (e.g., proton release and formation of
2R,3R,10S,11R.
IIb in Scheme 5). The mechanisms related to both sites can be
considered separately, as both moieties are electronically inde-
The absolute configuration at C-10, C-11 of the 2,3-cis-10,11- pendent.
trans-silybins 9 and 10, as inferred from the coupling constants
(J10,11 ca 8 Hz, trans-configuration) and the ECD spectra
Table 2: Chemical hardness (η, eV) of BF3 and silybin A (1a), in three
(Figure S1 and Figure S2, Supporting Information File 1), is
2R,3S,10R,11R (9) and 2R,3S,10S,11S (10). Here we would like
to stress that the absolute configuration at C-10, C-11 of 9 and
10 remains the same as in the starting compounds, namely 1a
(10R,11R) and 1b (10S,11S), respectively.
different solvents (obtained at the IEFPCM B3P86/6-31+G(d,p) level of
theory).
η
solvent
BF3
silybin A
benzene
EtOAc
DMF
7.4
7.3
7.0
4.7
4.8
4.8
Mechanism of silybin isomerization
Based on the absolute configuration of the new cis-silybin
isomers, we propose mechanisms for the stereospecific isomer-
ization.
Table 3: Electronic energies (ΔE, kcal·mol−1) of BF3 complexation with
silybin A (1a) at the different O-atoms.
The isomerization process is initiated by BF3 complexation.
Boron trifluoride may complex silybin through a coordinate
bond, in which the two electrons originate from the oxygen
atoms of silybin. Silybin and BF3 are hard bases (η = 4.8 eV in
EtOAc) and acids (η = 7.3 eV in EtOAc), respectively
(Table 2), favoring such a coordinate bond. The hardness of
BF3 and silybin A is not significantly modified when the
solvent polarity is increased (Table 2). The hardness of silybin
B was not calculated, as the stereochemistry is not expected to
EtOAc
DMF
O-1
−6.3
−6.8
C-4=O
O-9
−12.4
−6.9
−12.8
−7.7
O-12
−10.9
−11.3
significantly modify this parameter. The hardness calculation is The isomerization of flavanonols at C-2, C-3 is not without
based on the HOMO (highest occupied molecular orbital) and precedent, as it was described for taxillusin – the flavanonol
LUMO (lowest unoccupied molecular orbital) energies, which glycoside from Taxillus kaempferi (Japanese mistletoe)
are the same for silybin A and B. The quantum calculations containing the 3-hydroxy-2,3-dihydro-2-phenylchromen-4-one
show that the coordination complex at C-4=O (IIa in Scheme 5) moiety [29]. Taxillusin (24, (2R,3R)-taxifolin 3-β-D-glucopy-
is the most stable (Table 3). The corresponding complex IIa ranoside 6''-gallate, Figure 6) was subjected to both, basic and
exhibits a strong O–B bond of 1.55 Å, lower than, e.g., 1.73 Å acidic hydrolysis during its structure elucidation. However,
in the complex at O-1 (Table 4). In the benzopyranone moiety, under these conditions four stereoisomers of the original
Table 4: Bond distance and atomic charge of interest in BF3-silybin A at C-4=O (complex IIa) and O-1, in a) EtOAc and b) DMF.
a)
complex at O-1
complex at C-4=O (IIa)
d(O–B)
1.734
1.485
0.023
1.557
1.441
0.030
d(O1–C2)
atomic charge at C3
b)
complex at O-1
complex at C-4=O (IIa)
d(O–B)
1.718
1.486
0.035
1.553
1.442
0.010
d(O1–C2)
atomic charge at C3
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Beilstein J. Org. Chem. 2014, 10, 1047–1063.
Scheme 5: Proposed mechanisms of Lewis acid catalyzed isomerization at the benzopyranone ring of 23-O-acetylsilybin A (2a) in EtOAc.
aglycon (2R,3R)-taxifolin were formed. Base-catalyzed isomer- In principle, the isomerization can be initiated by the formation
ization of another taxifolin glycoside, (2R,3R)-2,3-dihydro- of coordination complexes at either C-4=O or O-1 (see
quercetin-3-α-L-rhamnopyranoside (pyridine; aqueous Scheme 5). Nevertheless, based on the quantum chemical calcu-
solution), was reported earlier [30-33]. The thermal or enzy- lations (Table 3 and Table 4) together with the representation of
matic rearrangement of taxifolin to alphitonin yielded four taxi- cis-isomers in the isomerization mixture, the most stable coordi-
folin isomers [34]. With flavanonols (3-O-glycosides or the nation complex IIa at C-4=O is stabilized by a strong O–B
respective aglycons), isomerization can be explained by the bond of 1.55 Å, having an atomic charge at C-3 close to zero
benzopyranone ring enolization, which can be initiated by either (Table 4). This favors a proton release from this position and
acids or bases. A resonance effect can further cause reversible the formation of the negatively charged species IIb. The release
C-ring opening, thus allowing isomerization at C-2.
of BF3 probably occurs from IIb by two different routes. The
major pathway (route I) proceeds without ring opening and after
re-protonation it leads to the major cis-isomer 12. The minor
route (route II), accompanied by a ring opening of the benzopy-
ranone, proceeds via intermediate IIc, which after ring closure
and protonation yielded a mixture of 2a and 13. This reversible
benzopyranone ring opening/closure process is probably re-
sponsible for the inversion of the absolute configuration at C-2,
C-3 of 13 compared to 2a (Scheme 5).
The limited degree of isomerization at C-2 of silybin requiring
the ring opening of the benzopyranone is in agreement with a
related study of taxifolin isomerization [34]. A deuterium
incorporation NMR study supported by quantum chemical
calculations showed that the intermediate of taxifolin with an
Figure 6: Taxillusin, (2R,3R)-taxifolin 3-β-D-glucopyranoside 6''-gallate
(24).
The proposed mechanism for the isomerization of silybin at open benzopyranone ring (α-hydroxychalcone related to IIc in
C-2, C-3 was inspired by these previous studies, i.e., involving our study) is very short-lived and the energy barrier for its for-
an enolization stage (route II in Scheme 5) [29-34]. In EtOAc, mation is relatively high [35]. It must be noted that IIc contains
the isomerization can occur from 1a or from its acetylated coun- additional substitution at the ortho-dihydroxyphenol moiety,
terpart (2a), since the 23-O-acetylation proceeds in parallel with which probably further decreases the possibilities for resonance
the isomerization and is substantially faster than the isomeriza- stabilization compared to unsubstituted α-hydroxychalcone
tion reaction.
(quinone methide formation is disabled).
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Interestingly, the course of 1a and 1b isomerization was slightly The isomerization of 1 at the benzodioxane moiety is also initi-
different in DMF than in EtOAc, since it only yielded the cis- ated by BF3 complexation, followed by a multistep mechanism
isomers 9 and 10. The isomer with an inverted absolute con- including the ring opening of benzodioxane. As the change in
figuration at C-2, C-3 (the non-acetylated form of 13) was not configuration takes place exclusively at C-11, it is probably
observed at all. This is easily explained by the charge distribu- initiated by the acid-catalyzed dioxane ring opening at O-12
tion of complex IIa. In DMF, the atomic charge at C-3 is closer (Scheme 6). This site is highly nucleophilic in EtOAc
to zero than in EtOAc (Table 4). This clearly indicates that the (Figure 7a), with lone electron pairs, which is in favor of BF3
formation of the open intermediates (IIc) is not supported in complexation as confirmed by the complexation energies
DMF.
(Table 3). Following complexation, the D-ring opening
Figure 7: Spatial distribution of nucleophilic f−(r) (blue) and electrophilic f+(r) (blue) Fukui functions of silybin A (1a) in both a) EtOAc and b) DMF.
Scheme 6: Proposed mechanism of Lewis acid catalyzed isomerization of benzodioxane part of 23-O-acetylsilybin B (2b) in EtOAc.
1056

Beilstein J. Org. Chem. 2014, 10, 1047–1063.
proceeds (Scheme 6), allowing molecular rearrangements and HPLC pump, Shimadzu SIL-20AC cooling autosampler,
isomerization after ring closure.
Shimadzu CTO-10AS column oven, and Shimadzu SPD-20MA
diode array detector. The monolithic column was a Chromolith
The observation of the isomerization of 1 (or 2) at C-11 that Performance, RP-18e, 100 × 3 mm equipped with a guard
occurs in EtOAc but not in DMF can be explained either by a column (5 × 4.6 mm) with an isocratic mobile phase CH3CN/
solvent effect or by the participation of the acetyl group. MeOH/H2O/HCO2H 2:37:61:0.1, flow rate 1.5 mL/min at
However, the second possibility is less probable, since the parti- 25 °C, detection at 285 nm; tr (silybin A, 1a) = 3.1 min,
cipation of the acetyl group should lead to the formation of an tr (silybin B, 1b) = 3.7 min, tr (23-O-acetylsilybin A) =
isomer with a retained configuration at C-11 (two subsequent 12.7 min, tr (23-O-acetylsilybin B, 2b) = 14.1 min. For the
nucleophilic substitutions with two Walden inversions). The semipreparatory HPLC, a chromolith SemiPrep RP-18e mono-
possibility of acetyl group participation was refuted by the reac- lithic column, 100 × 10 mm was used with an isocratic mobile
tion of 2 in DMF, so that no isomerization took place at C-11. phase CH3OH/H2O 50:50. The flow rate was set to 5 mL/min at
Moreover, in DMF the f(r) Fukui function is totally displaced 25 °C, the UV detection to 285 nm; tr (2a) = 6.6 min, tr (13) =
into the E-ring (Figure 7b), so that the O-12 atom loses its 7.8 min.
nucleophilic character, which partially rationalizes the solvent
effect observed here.
Materials
Silybin (a mixture of diastereoisomers A (1a) and B (1b), ca.
Conclusion
1:1) was kindly provided by Dr. L. Cvak (division TAPI, Teva
We report here the first syntheses of hitherto undescribed cis- Czech Industries s.r.o., IVAX Pharmaceuticals, CZ). Silybin
isomers of silybin in optically pure form starting from silybin A diastereoisomers were separated by lipase-catalyzed discrimin-
(1a) and B (1b). The determination of their absolute configur- ation [5]. Candida antarctica lipase B (Novozym 435) was
ation was based on an analysis of NMR coupling constants and from Novozymes (DK). All other reagents were of analytical
a comparison of their ECD spectra with model compounds with grade and used without further purification.
well-defined absolute configurations. Moreover, the absolute
configuration of these novel 2,3-cis- and 10,11-cis-isomers of (2R,3S,10R,11R)-23-O-Acetylsilybin (12): BF3·OEt2 (2 mL,
silybin enabled us to propose mechanisms for the cis–trans 15.926 mmol, 50% solution in OEt2) was added to a stirred
isomerization of silybin. Although analogous isomerizations of solution of 1a (2 g, 4.146 mmol, >99% optical purity) in DMF
similar compounds at respective C-2, C-3 centers have been (15 mL) and further stirred for 1 h at 50 °C. The reaction was
described, we present here parallel isomerizations on both chiral quenched by the addition of an ice-cold solution of saturated
centers under kinetic control. cis-Silybins were obtained by the NaHCO3 (100 mL), extracted with EtOAc (3 × 50 mL), the
chemoenzymatic separation methods mostly due to the sensi- combined organic layers were dried (Na2SO4) and evaporated.
tivity of new cis-derivatives to the reversed isomerization.
The residue was dissolved in MeOH (10 mL) and a few drops
of water were added. After several hours, the crystals formed
were filtered off (ca. 1.1 g, virtually pure trans-isomer) and the
mother liquor was evaporated (0.84 g, ca. 30% of cis-isomer,
Experimental
General experimental procedures
NMR spectra were recorded in DMSO-d6 (30 °C) by using a according to HPLC). The evaporated residue was then dissolved
Bruker AVANCE 600 NMR spectrometer (600 MHz for 1H, in a mixture of acetone/vinyl acetate (100 mL, 9:1, v/v),
151 MHz for 13C) with the residual solvent peak as the internal Novozym 435 (0.84 g, ≥10000 U/g, 100% w/w) was added, and
standard. Mass spectra were measured in an APEX-Ultra FTMS the mixture was shaken at 45 °C and 650 rpm for 48 h. After
with ESI ionization. The high-resolution ESI–MS spectra were enzyme removal by filtration, the solution was evaporated and
measured by using a GCT Premier benchtop orthogonal acceler- the crude mixture was purified by column chromatography
ation time-of-flight mass spectrometer.
(CHCl3/acetone/HCO2H 95:5:1, twice) yielding 2a (0.6 g, 30%
yield, >98% optical purity) and 12 (0.2 g, 10% yield, >97%
Optical rotations were measured with a Rudolph Autopol optical purity). 1H and 13C NMR data see Table 5. HRMS
polarimeter in acetone at 25 °C, and ECD spectra were recorded (ESI–TOF) m/z: [M − H]− calcd for C27H23O11, 523.1246;
in a Jasco-815 spectrometer in MeOH from 200 to 400 nm with found, 523.1245.
a scanning speed 20 nm/min, time response 8 s with a 2 mm
quartz cell and a sample concentration of ca. 1 mmol/L.
(2R,3S,10S,11S)-23-O-Acetylsilybin (11) – Method A: This
compound was prepared analogously to 12 starting from pure
HPLC analyses were carried out in a Shimadzu Prominence LC 1b (2 g, 4.146 mmol, >99% optical purity) to yield 343 mg of
analytical system consisting of a Shimadzu LC-20AD binary 11 (17% yield, >97% optical purity). 1H and 13C NMR data, see
1057

Beilstein J. Org. Chem. 2014, 10, 1047–1063.
Table 5. HRMS (ESI–TOF) m/z: [M − H]− calcd for >98% optical purity). 1H and 13C NMR data, see Table 5.
C27H23O11, 523.1246; found, 523.1247.
HRMS (ESI–TOF) m/z: [M − H]− calcd. for C27H23O11,
523.1246; found, 523.1246.
(2R,3S,10S,11S)-23-O-Acetylsilybin (11) – Method B:
BF3∙OEt2 (2.3 mL, 18.3 mmol, 50% solution in OEt2) was (2R,3S,10R,11R)-Silybin (9): Novozym 435 (1 g, ≥10000 U/g)
added to a stirred solution of 1b (3 g, 6.2 mmol, >99% optical was added to a solution of 12 (200 mg, 0.381 mmol) in a mix-
purity) in EtOAc (100 mL) and the mixture was stirred for 3 h ture of MTBE/n-butanol (30 mL, 9:1, v/v), and the mixture was
at 80 °C. The reaction mixture was quenched by the addition of shaken at 45 °C and 650 rpm for 100 h. The enzyme was
an ice-cold solution of saturated NaHCO3 (100 mL), and after removed by filtration, the solution was evaporated, and the
stirring for 10 min both phases were separated. The aqueous crude mixture purified by column chromatography (CHCl3/
phase was extracted with EtOAc (3 × 50 mL), the combined acetone/HCO2H from 95:5:1 to 70:30:1) yielding title com-
organic layers were dried (Na2SO4) and evaporated. The pound 9 (140 mg, 70% yield, >97% purity). 1H and 13C NMR
residue after flash chromatography (CHCl3/acetone/HCO2H, data, see Table 6. [α]D22 −51.6 (c 0.091, acetone); ECD spec-
97:3:1, twice) yielded title compound 11 (50 mg, 2% yield, trum, see Supporting Information File 1, Figure S2; HRMS
Table 5: NMR spectroscopic data (600 MHz, DMSO-d6, 30 °C) of compounds 12 and 11.
(2R,3S,10R,11R)-23-O-acetylsilybin (12)
(2R,3S,10S,11S)-23-O-acetylsilybin (11)
position
δC
ma
δH (m, J in Hz)
δC
ma
δH (m, J in Hz)
2
80.50
d
d
s
s
s
d
s
d
s
d
d
s
d
s
d
d
s
s
d
s
s
d
d
t
5.460 (d, 2.4)
80.52
d
d
s
s
s
d
s
d
s
d
d
s
d
s
d
d
s
s
d
s
s
d
d
m
d
d
s
s
s
d
s
m
5.461 (d, 2.4)
3
70.78
4.109 (br.d, 2.4, 6.4)
70.79
4.113 (dd, 2.4, 6.4)
4
195.29
100.27
164.00
96.04
–
195.31
100.29
164.01
96.05
–
4a
5
–
–
–
–
6
5.908 (d, 2.1)
5.912 (d, 2.1)
7
166.86
95.05
–
166.85
95.05
–
8
5.936 (d, 2.1)
5.940 (d, 2.1)
8a
10
11
12a
13
14
15
16
16a
17
18
19
20
21
22
23
162.49
75.01
–
162.50
75.02
–
4.491 (ddd, 8.0, 5.2, 2.7)
4.492 (ddd, 7.9, 5.2, 2.7)
75.85
4.913 (d, 8.0)
75.86
4.915 (d, 7.9)
143.06
116.26
129.51
120.70
116.30
142.59
126.67
111.74
147.80
147.34
115.50
120.60
62.65
–
143.07
116.27
129.52
120.70
116.31
142.60
126.68
111.74
147.81
147.34
115.51
120.61
62.66
–
7.080 (d, 2.0)
7.083 (d, 2.0)
–
–
7.020 (dd, 8.4, 2.0)
6.976 (d, 8.4)
–
7.022 (dd, 8.3, 2.0)
6.978 (d, 8.3)
–
–
–
7.013 (d, 1.9)
–
7.014 (d, 2.0)
–
–
–
6.809 (d, 8.0)
6.861 (dd, 8.0, 1.9)
4.081 (dd, 2.7, 12.4)
3.926 (dd, 5.2, 12.4)
6.223 (d, 6.4)
11.861 (s)
10.805 (br s)
3.773 (s)
6.812 (d, 8.0)
6.863 (dd, 2.0, 8.0)
4.084 (dd, 2.7, 12.4)
3.928 (dd, 5.2 , 12.4)
6.227 (d, 6.4)
11.863 (s)
10.813 (br s)
3.775 (s)
3-OH
–
–
–
–
q
–
s
q
–
5-OH
–
–
7-OH
–
–
19-OMe
20-OH
23-C=O
23-Ac
55.75
–
55.75
–
9.164 (s)
9.167 (s)
170.06
20.46
–
170.07
20.47
–
2.021 (s)
2.022 (s)
amultiplicity of 13C signals.
1058

Beilstein J. Org. Chem. 2014, 10, 1047–1063.
Table 6: NMR spectroscopic data (600 MHz, DMSO-d6, 30 °C) of compounds 9 and 10.
(2R,3S,10R,11R)-silybin (9)
(2R,3S,10S,11S)-silybin (10)
position
δC
ma
δH (m, J in Hz)
δC
ma
δH (m, J in Hz)
2
80.60
d
d
s
s
s
d
s
d
s
d
d
s
d
s
d
d
s
s
d
s
s
d
d
t
5.441 (ddd, 2.4, 0.7, 0.6)
80.60
d
d
s
s
s
d
s
d
s
d
d
s
d
s
d
d
s
s
d
s
s
d
d
t
5.443 (ddd, 2.5, 0.6, 0.4)
3
70.81
4.092 (br.d, 2.4)
70.82
4.087 (m, -)
4
195.21
100.17
164.03
96.08
–
195.25
100.22
164.04
96.07
–
4a
5
–
–
–
–
6
5.897 (d, 2.1)
5.903 (d, 2.1)
7
167.15
95.09
–
167.04
95.08
–
8
5.925 (d, 2.1)
5.934 (d, 2.1)
8a
10
11
12a
13
14
15
16
16a
17
18
19
20
21
22
23
162.54
78.14
–
162.57
78.15
–
4.151 (ddd, 7.9, 4.7, 2.5)
4.144 (ddd, 7.8, 4.7, 2.6)
75.86
4.889 (d, 7.9)
75.90
4.891 (d, 7.8)
143.11
116.14
129.09
120.46
116.18
143.19
127.56
111.76
147.67
147.06
115.37
120.54
60.23
–
143.21
116.15
129.07
120.47
116.19
143.11
127.57
111.79
147.68
147.07
115.38
120.55
60.24
–
7.055 (dd, 2.0, 0.7)
7.052 (dd, 2.0, 0.4)
–
–
6.998 (ddd, 8.3, 2.0, 0.6)
7.001 (ddd, 8.3, 2.0, 0.6)
6.945 (d, 8.3)
6.947 (d, 8.3)
–
–
–
–
7.002 (d, 2.0)
–
7.003 (d, 1.9)
–
–
–
6.802 (d, 8.1)
6.858 (dd, 8.1, 2.0)
3.534 (dd, 2.5, 12.2)
3.341 (dd, 4.7, 12.2)
6.216 (br.s)
11.884 (br.s)
n.d.
6.803 (d, 8.1)
6.860 (dd, 1.9, 8.1)
3.532 (m)
3.340 (m)
6.212 (br.d, 5.5)
11.881 (s)
n.d.
3-OH
–
–
–
–
q
–
–
–
–
–
q
–
d
5-OH
–
7-OH
–
19-OMe
20-OH
23-OH
55.75
3.777 (s)
55.76
3.780 (s)
9.118 (br.s)
–
–
9.151 (br.s)
amultiplicity of 13C signals.
(ESI–TOF) m/z: [M + H]+ calcd for C25H23O10, 483.1291; (100 mL) and the mixture was kept for 48 h at 80 °C. The reac-
found, 483.1296.
tion mixture was quenched by the addition of an ice-cold solu-
tion of saturated NaHCO3 (100 mL), and after stirring for
(2R,3S,10S,11S)-Silybin (10): This compound was prepared 10 min both phases were separated. The aqueous phase was
analogously to 9 starting from pure 11 (343 mg, 0.654 mmol) to extracted with EtOAc (3 × 50 mL). The combined organic
yield 8 (220 mg, 64% yield, >96% purity). 1H and 13C NMR layers were dried (Na2SO4) and evaporated to dryness. The
data, see Table 6. [α]D22 −40.4 (c 0.39, acetone); ECD spec- crude mixture was purified by column chromatography (CHCl3/
trum, see Supporting Information File 1, Figure S1; HRMS acetone/toluene/HCO2H, 95:5:5:1, twice) yielding a mixture of
(ESI–TOF) m/z: [M + H]+ calcd. for C25H23O10, 483.1291; 2b and 8, which was then dissolved in a mixture of MTBE/n-
found, 483.1294.
butanol (150 mL, 9:1 v/v), Novozym 435 (0.25 g, ≥10000 U/g,
100% w/w) was added, and the mixture was shaken at 45 °C
(2R,3R,10S,11R)-23-O-Acetylsilybin (8): BF3∙OEt2 (1.5 mL, and 650 rpm for 37 h until the ratio of 8/2b was 96:4 (HPLC).
12.2 mmol, 50% solution in OEt2) was added to a stirred solu- After enzyme removal by filtration, the solution was evapo-
tion of 1b (2 g, 4.1 mmol, >99% optical purity) in EtOAc rated, and the crude mixture purified by column chromatog-
1059

Beilstein J. Org. Chem. 2014, 10, 1047–1063.
raphy (CHCl3/acetone/HCO2H 90:10:1) yielding 1b (0.898 g, were dried (Na2SO4) and evaporated. The crude mixture was
45% yield, >99% purity) and 12 (0.06 g, 3% yield, >98% purified by column chromatography (CHCl3/acetone/HCO2H
purity). 1H and 13C NMR data, see Table 7. HRMS (ESI–TOF) 95:5:1, twice), yielding 12 (0.32 g; 12%, >96% purity) and a
m/z: [M − H]− calcd for C27H23O11, 523.1246; found, mixture of 2a and 13. After evaporation of the solvent, the
523.1244.
residue containing compounds 2a and 13 (1 g) was then
dissolved in a mixture of MTBE/n-butanol (30 mL, 9:1, v/v),
(2S,3S,10R,11S)-23-O-Acetylsilybin (13): BF3∙OEt2 (2.7 mL, Novozym 435 (0.3 g, ≥10000 U/g, 30% w/w) was added and
21.9 mmol, 50% solution in OEt2) was added to a stirred solu- the mixture was shaken at 45 °C and 650 rpm for 72 h to give a
tion of 1a (2.7 g, 5.6 mmol, >99% optical purity) in EtOAc 13/2a ratio of ca. 2:3 (HPLC). After enzyme removal by filtra-
(30 mL) and the mixture was kept at 80 °C for 48 h. The reac- tion, the solution was evaporated, and the crude mixture was
tion mixture was quenched by the addition of an ice-cold solu- purified by column chromatography (CHCl3/acetone/HCO2H
tion of saturated NaHCO3 (100 mL), and after stirring for 90:10:1) to remove 1a. Subsequent preparative HPLC sep-
10 min both phases were separated. The aqueous phase was aration (A Chromolith SemiPrep RP-18e monolithic column,
extracted with EtOAc (3 × 50 mL), the combined organic layers 100 × 10 mm, Merck) of the mixture of 2a and 13 was carried
Table 7: NMR spectroscopic data (600 MHz, DMSO-d6, 30 °C) of compounds 8 and 13.
(2R,3R,10S,11R)-23-O-acetylsilybin (8)
(2S,3S,10R,11S)-23-O-acetylsilybin (13)
position
δC
ma
δH (m, J in Hz)
δC
ma
δH (m, J in Hz)
2
82.48
d
d
s
s
s
d
s
d
s
d
d
s
d
s
d
d
s
s
d
s
s
d
d
t
5.101 (d, 11.4)
82.44
d
d
s
s
s
d
s
d
s
d
d
s
d
s
d
d
s
s
d
s
s
d
d
t
5.085 (dd, 0.4, 11.3)
3
71.46
4.618 (dd, 6.1, 11.4)
71.46
4.597 (dd, 6.3, 11.3)
4
197.71
100.48
163.32
96.13
–
197.32
100.21
163.36
96.31
–
4a
5
–
–
–
–
6
5.923 (d, 2.1)
5.879 (br.s, –)
7
166.86
95.07
–
167.00
95.31
–
8
5.883 (d, 2.1)
5.848 (br.s, –)
8a
10
11
12a
13
14
15
16
16a
17
18
19
20
21
22
23
162.48
73.83
–
162.44
73.87
–
4.801 (ddd, 2.8, 3.4, 8.2)
4.793 (ddd, 2.9, 3.4, 8.3)
74.60
5.340 (dd, 0.6, 2.8)
74.60
5.337 (ddd, 0.4, 0.7, 2.9)
142.43
116.95
130.86
121.79
116.92
141.73
126.52
110.73
147.62
146.65
115.59
118.67
60.38
–
142.47
116.87
130.95
121.97
116.93
141.70
126.53
110.65
147.63
146.65
115.59
118.66
55.73
–
7.167 (d, 2.0)
7.171 (d, 2.0)
–
–
7.066 (dd, 2.0, 8.3)
7.049 (ddd, 0.4, 2.0, 8.3)
7.005 (d, 8.3)
–
7.013 (d, 8.3)
–
–
–
7.023 (d, 2.0)
–
7.026 (dd, 0.4, 2.1)
–
–
–
6.811 (d, 8.2)
6.860 (ddd, 0.6, 2.0, 8.2)
4.004 (dd, 3.4, 12.2)
3.954 (dd, 8.2, 12.2)
5.783 (d, 6.1)
11.875 (s)
6.807 (d, 8.1)
6.859 (ddd, 0.7, 2.1, 8.1)
3.998 (dd, 3.4, 12.2)
3.995 (dd, 8.3, 12.2)
5.769 (d, 6.3)
11.902 (s, –)
10.860 (br.s, –)
3.757 (s, –)
9.119 (s, –)
–
3-OH
–
–
–
–
–
q
–
s
q
5-OH
–
–
7-OH
–
–
10.788 (br s)
3.757 (s)
19-OMe
20-OH
23-CO
23-Ac
55.73
–
55.73
–
q
9.093 (s)
170.08
20.46
–
170.13
20.51
s
1.941 (s)
q
1.940 (s, –)
amultiplicity of 13C signals.
1060

Beilstein J. Org. Chem. 2014, 10, 1047–1063.
out with an isocratic mobile phase CH3OH/H2O 50:50, a flow
rate of 5 mL/min at 25 °C, and UV detection at 285 nm; tr (2a)
= 6.6 min, tr (13) = 7.8 min.) yielding 2a (20 mg, 0.7% yield,
>98% purity) and 13 (7 mg, 0.3% yield, >96% purity). [α]D22
+69.7 (c 0.11, acetone); ECD spectrum, see Supporting Infor-
mation File 1, Figure S4; 1H and 13C NMR data, see Table 7;
HRMS (ESI–TOF) m/z: [M + Na]+ calcd for C27H24O11Na,
547.1211; found, 547.1209.
Table 8: NMR spectroscopic data (600 MHz, DMSO-d6, 30 °C) of
compound 14.
(2R,3R,10S,11R)-silybin (14)
position
δC [ppm]
ma
δH (m, J in Hz)
2
82.56
d
d
s
s
s
d
s
d
s
d
d
s
d
s
d
d
s
s
d
s
s
d
d
t
5.082 (dd, 0.4, 11.4)
3
71.44
4.608 (dd, 5.8, 11.4)
4
197.69
100.43
163.30
96.12
–
4a
5
–
(2R,3R,10S,11R)-Silybin (14): Novozym 435 (0.350 g, 500%
w/w, ≥10000 U/g) was added to 12 (60 mg, 0.11 mmol)
dissolved in a mixture of MTBE/n-butanol (15 mL, 9:1, v/v),
and the mixture was shaken at 45 °C and 650 rpm for 120 h.
The enzyme was then filtered off, the solution evaporated, and
the crude mixture purified by column chromatography (CHCl3/
acetone/HCO2H from 97:3:1 to 70:30:1) yielding title com-
pound 14 (40 mg, 67% yield, >98% purity). [α]D22 −59.2 (c
0.13, acetone); ECD spectrum, see Supporting Information
File 1, Figure S3; 1H and 13C NMR data, see Table 8; HRMS
(ESI–TOF) m/z: [M + H]+ calcd for C25H23O10, 483.1291;
found, 483.1289
–
6
5.913 (d, 2.1)
7
166.94
95.08
–
8
5.873 (d, 2.1)
8a
10
11
12a
13
14
15
16
16a
17
18
19
20
21
22
23
162.49
77.36
–
4.464 (ddd, 2.8, 4.0, 7.9)
75.13
5.272 (ddd, 0.5, 0.5, 2.8)
142.69
116.74
130.53
121.36
116.85
142.47
127.23
111.21
147.44
146.49
115.44
119.05
58.20
–
7.106 (d, 2.0)
–
7.037 (ddd, 0.4, 2.0, 8.3)
6.986 (d, 8.3)
–
–
Calculation methods
6.966 (dd, 0.5, 1.8)
–
All geometries and energies, including the zero-point correc-
tion (V), enthalpies (H) and Gibbs energies (G) at 298 K of the
reactants, intermediates and products were determined at the
(U)B3P86/6-31+G(d,p) level, well-adapted for polyphenol re-
activity. Solvent effects were implicitly taken into account by
using a PCM (polarizable continuum model) method; the
IEFPCM (integral equation formalism PCM) method coupled to
UA0 radii was used. The mechanisms were studied with both
EtOAc and DMF solvents with a dielectric constant ε of 5.99
and 37.21, respectively.
–
6.770 (d, 8.2)
6.791 (ddd, 0.5, 1.8, 8.2)
3.409 (dd, 7.9, 11.6)
3.338 (dd, 4.0, 11.6)
5.766 (d, 5.8)
11.876 (s, –)
10.490 (br.s)
3.732 (s, –)
9.032 (s, –)
4.833 (br.s)
3-OH
–
–
–
–
q
–
–
5-OH
–
7-OH
–
19-OMe
20-OH
23-OH
55.72
–
–
The hardness η is related to the hard–soft-acid-basis (HSAB)
principle. According to this theory, hard acids react with hard
bases whereas soft acids react with soft bases. The hardness is
amultiplicity of 13C signals.
given by
where I and A are the adiabatic ionization
past few years. The Fukui function fk(r) has become one of
potential and the adiabatic electron affinity, respectively. The
hardness of silybin B was not calculated, as the stereochemistry
is not expected to significantly modify this parameter. The hard-
ness calculation is based on the HOMO (highest occupied mole-
cular orbiral) and LUMO (lowest occupied molecular orbital)
energies, which are the identical for silybin A and B. The hard-
ness was calculated in benzene, EtOAc, and DMF to study the
impact of the solvent polarity on the HSAB principle. The hard-
ness of BF3 and silybin A is not significantly modified when the
solvent polarity is increased (Table 2).
these powerful tools, providing an atomic picture of hardness.
For a given atom k, it is given by
where
fk+(r) and fk−(r) are the electrophilic and nucleophilic contribu-
tions of the Fukui function calculated as follows:
and
, where qk(N), qk(N−1) and
qk(N + 1) are the electronic population of atom k in its neutral,
radical-cation and radical-anion forms, respectively. In this
New methods to rationalize chemical reactivity have been study, the Fukui function is used to partially rationalize BF3
developed in the field of quantum mechanical methods over the complexation. In this case, the nucleophilic contribution is the
1061

Beilstein J. Org. Chem. 2014, 10, 1047–1063.
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Supporting Information File 1
1H and 13C NMR spectra of new compounds, ECD spectra
of new compounds, HPLC chromatograms of new
compounds, table of retention times and purity of the new
compounds, XYZ coordinates of optimized silybin A and B
and absolute energies.
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This work was supported by the grant P207/10/0288 (R.G.)
from the Czech Science Foundation, by projects LH13097 and
LD13041 from the Ministry of Education of the Czech Republic
(V.K.), by the ESF COST Chemistry project CM0804 (P.T. and
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F.D.M. wish to thank “Conseil Régional du Limousin” for
financial support and CALI (CAlcul en LImousin) for their
computing facilities. P.T. gratefully acknowledges the support
of the Operational Program Research and Development for
Innovation-European Regional Development Fund (project
CZ.1.05/2.1.00/03.0058 of the Ministry of Education, Youth
and Sports of the Czech Republic). The authors would like to
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