Mechanistic study of the biomimetic synthesis of flavonolignan diastereoisomers in milk thistle

Article abstract of DOI:10.1021/jo4011377

The mechanism for the biomimetic synthesis of flavonolignan diastereoisomers in milk thistle is proposed to proceed by single-electron oxidation of coniferyl alcohol, subsequent reaction with one of the oxygen atoms of taxifolin's catechol moiety, and finally, further oxidation to form four of the major components of silymarin: silybin A, silybin B, isosilybin A, and isosilybin B. This mechanism is significantly different from a previously proposed process that involves the coupling of two independently formed radicals.

Full text of DOI:10.1021/jo4011377

Article
pubs.acs.org/joc
Mechanistic Study of the Biomimetic Synthesis of Flavonolignan
Diastereoisomers in Milk Thistle
Hanan S. Althagafy, Maria Elena Meza-Avina, Nicholas H. Oberlies,* and Mitchell P. Croatt*
̃
Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, 435 Sullivan Science Building, P.O. Box
26170, Greensboro, North Carolina 27402, United States
S
* Supporting Information
ABSTRACT: The mechanism for the biomimetic synthesis of flavonolignan diastereoisomers in milk thistle is proposed to
proceed by single-electron oxidation of coniferyl alcohol, subsequent reaction with one of the oxygen atoms of taxifolin’s catechol
moiety, and finally, further oxidation to form four of the major components of silymarin: silybin A, silybin B, isosilybin A, and
isosilybin B. This mechanism is significantly different from a previously proposed process that involves the coupling of two
independently formed radicals.
development of tools to discern and quantify flavonolignans by
1H NMR spectroscopy, despite near-identical spectra.36
INTRODUCTION
■
Milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)] has
been used as a medicinal herb since antiquity. As outlined in
several reviews, modern pharmacological studies typically focus
on the hepatoprotective properties1−3 (as milk thistle is the top
herbal supplement for hepatitis C patients2), the prostate cancer
chemopreventive properties4−6 (where promising results have
been observed, especially for isosilybin B7−10), or both. The two
most studied formulations are either silymarin, an extract of the
seeds that contains at least seven major flavonolignans, or
silibinin, a roughly equimolar mixture of silybin A and silybin B
(Figure 1); a recent review delineates the somewhat confusing
nomenclature surrounding the various permutations of milk
thistle.11
Structurally, flavonolignans are characterized by the amalga-
mation of a flavonoid moiety (taxifolin) and a phenylpropane
unit (coniferyl alcohol) (Scheme 1).1,37,38 Silibinin (silybin A and
silybin B) and isosilibinin (isosilybin A and isosilybin B) each
exist as a pair of trans diasteroisomers with respect to the relative
configuration at positions C7″ and C8″ in the 1,4-benzodioxane
ring.26,27,35,36 Silychristin (5) and isosilychristin (6) have
coumaran ring systems, or dihydrobenzofuran bicycles, but the
position of this bicycle differs by being formed at either the C4′
and C5′ positions in silychristin or the C2′ and C3′ positions in
isosilychristin. Silydianin (7) is the most structurally complex of
the flavonolignans in silymarin because it contains a
bicyclo[2.2.2]octenone with a transannular hemiketal.19,22,24
The first biomimetic synthesis of milk thistle flavonolignans
was reported in 1977 by Schrall and Becker,39 who used
horseradish peroxidase and a cell-free extract of S. marianum
suspension cultures to produce silibinin from taxifolin (8) and
coniferyl alcohol (9) (Scheme 1). Two years later, Merlini and
co-workers17 reported an enzyme-free oxidative coupling of
taxifolin and coniferyl alcohol using Ag2O to yield a mixture of
silibinin and isosilibinin. On the basis of those results, multiple
researchers have reported biomimetic syntheses of flavonoli-
gnans and related analogues, typically using a silver
The chemistry of milk thistle extract has been investigated
since the 1960s, and impressive strides were made in the isolation
and structure determination of the individual flavonolignans
throughout the 60s, 70s, and 80s, particularly by the competing
groups of Hansel and colleagues12−19 and Wagner and
̈
colleagues.20−25 However, likely because of improvements in
chromatographic technology, the individual diastereoisomers
were not isolated and characterized completely until 2003.26,27
Subsequently, gram-scale purifications of the individual
flavonolignans were developed.28−30 Those materials likely
facilitated several chemistry-driven investigations, including the
generation of analogues,31−34 an X-ray crystallographic study to
verify the structures of the four main isomers,35 and the
Received: May 28, 2013
© XXXX American Chemical Society
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Figure 1. Flavonolignans from milk thistle; silibinin is a 1:1 mixture of 1 and 2, while isosilibinin is a 1:1 mixture of 3 and 4.11
Scheme 1. Mechanistic Options for the Biomimetic Synthesis of Flavonolignans
B
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Scheme 2. Biomimetic Synthesis of Silibinin, Isosilibinin, and Other Byproducts
Figure 2. UPLC chromatogram of biomimetic reaction using conditions in Scheme 2. UPLC was conducted using a CH3OH/H2O (0.1% formic acid)
gradient that was initiated at 5:95, increased to 50:50 over 10 min, and then held at that ratio for 2 min at a flow rate of 0.6 mL/min (50 °C) using an
HSST3 column monitored at 288 nm.
oxidant.18,31,40−42 The mechanism presented for these, termed
Freudenberg’s hypothesis, is based on the synthesis of lignin
from coniferyl alcohol43 and has been a topic of controversy in
recent years.44,45
that alternative mechanisms could be taking place, and as such, a
definitive mechanism cannot be absolutely determined.
As mentioned earlier, the mechanism proposed in the
literature involves simultaneous single-electron oxidation of
both coniferyl alcohol and taxifolin, followed by a combination of
the resultant radicals to form an ether (14) which undergoes
rapid addition of the phenol to the electrophilic p-quinone
methide to yield silibinin (Option 1; Scheme 1). The same
mechanism is possible using the other phenoxy radical of taxifolin
to produce isosilibinin (omitted from Scheme 1 for clarity).
Although this mechanism has been proposed based on
Freudenberg’s hypothesis for lignin biosynthesis,43 it was difficult
to support due to the low concentration of each radical.
Moreover, taxifolin and coniferyl alcohol would need to oxidize
at nearly identical rates, otherwise dimerization would be the
major pathway.
A second mechanistic option proceeds via two sequential
oxidations of taxifolin to yield an o-quinone (12). This pathway
seemed more plausible, given the precedence for o-quinones to
react with alkenes in the Diels−Alder reaction.48 Additionally,
the stereospecific nature of the Diels−Alder reaction would
conserve the relative configuration of the dienophile (i.e., the
trans configuration of the alkene in coniferyl alcohol would
deliver the trans configuration at C7″ and C8″ as observed in
both silibinin and isosilibinin).35
The mechanism that has been proposed for the biomimetic
synthesis of flavonolignans 1−4 involves single electron
oxidation of both coniferyl alcohol and taxifolin individually,
followed by a combination of these two radicals to produce
silibinin and isosilibinin (Scheme 1, Option 1). While there is a
possibility that this type of pathway could occur within or near
the active site of an enzyme,46,47 the probability of two radicals
being formed independently in solution from Ag2O and then
combining in a productive manner18 is unlikely based on first
principles of reaction kinetics. Because the concentration of both
radicals will be extremely low, the reaction rate will be essentially
zero. Given the absence of an alternative mechanism for the
biomimetic synthesis of flavonolignans in the current literature,
we pursued a more thorough exploration of this process.
RESULTS AND DISCUSSION
■
The conversion of coniferyl alcohol and taxifolin to silibinin and
isosilibinin necessarily requires an oxidation. This oxidation is
presumably enzyme-catalyzed in nature,46,47 but it has been
shown that silver salts can also efficiently effect this
conversion.18,31,40−42 Because silver oxidations typically occur
through a series of single electron transfers, various mechanistic
options were explored and resolved (Scheme 1). Specifically, the
reaction involves three steps: two single electron oxidations and
the coupling of taxifolin to coniferyl alcohol (or an oxidized
variant of either partner). The five options discussed below cover
logical combinations of these three processes, although it is noted
Options 3 and 4 are similar to one another because they both
begin with initial oxidation of either taxifolin (Option 3) or
coniferyl alcohol (Option 4) followed by coupling to either
coniferyl alcohol or taxifolin, respectively. The resultant radical
(13) or (15) would be further oxidized to yield silibinin. Unlike
Option 1, Options 3 and 4 both seemed plausible, because both
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Scheme 3. Mechanistic Investigation for the o-Quinone Diels−Alder Option
Scheme 4. Individual Oxidations of Taxifolin and Coniferyl Alcohol
coniferyl alcohol and taxifolin are electron-rich and are therefore
susceptible to oxidation or reaction with an electrophile.
The final option considered had two sequential oxidations of
coniferyl alcohol and subsequent coupling to taxifolin (Option
5). It has been shown that coniferyl alcohol undergoes oxidation
to yield coniferyl aldehyde (16);43 however, an unlikely redox
reaction of this aldehyde and taxifolin would be required to yield
silibinin. For the sake of this study, all five of these options were
considered while the mechanism was investigated. However,
Option 1 seemed unlikely because it required a sufficient
concentration of both reactive radical intermediates (10 and 11),
whereas Options 2−5 were only dependent on the concentration
of either of the singly oxidized radicals.
The biomimetic synthesis was performed with natural
taxifolin, isolated from milk thistle extract in >90% purity (data
not shown), and commercially available coniferyl alcohol. The
reactions were explored initially on a small scale (∼1 mg
taxifolin) and monitored by HPLC; they were later scaled up to
>100 mg of taxifolin. The reaction conditions were based upon
the procedure described by Merlini and co-workers,17 with
moderate optimizations. Several solvents, oxidizing agents, and
temperatures were tested, and the best results were obtained
when 1 equiv of taxifolin was reacted with 2 equiv of coniferyl
alcohol in ethyl acetate containing 4 equiv of Ag2O at 75 °C for
96 h (Scheme 2). These conditions afforded a mixture of silybin
A (1), silybin B (2), isosilybin A (3), and isosilybin B (4)
(Scheme 2) in a combined 52% yield with nearly equimolar
amounts of each flavonolignan (Figure 2 and Supporting
Information).
isolation and NMR analysis. It had been determined previously
that silymarin consists of silybin A (16.0%), silybin B (23.8%),
isosilybin A (6.4%), isosilybin B (4.4%), silychristin (11.6%),
isosilychristin (2.2%), silydianin (16.7%), and taxifolin (1.6%;
see Supporting Information).10 The biomimetic conditions
described herein increased the yield of isosilybin B relative to
other flavonolignans. This was noteworthy given studies that
demonstrate its potential in prostate cancer chemopreven-
tion7−10 and also the challenges it presents when isolating it on a
multigram scale.30 Flavonolignans 1−4 were isolated by HPLC
in greater than 99% purity, and their structures were confirmed
by NMR spectroscopy (see Supporting Information).
To explore the mechanistic possibilities involved in this
biomimetic process, the oxidative coupling of cis-coniferyl
alcohol (19) and taxifolin using Ag2O was examined (Scheme
3). If Option 2 (Scheme 1) occurred, the product would maintain
a cis relationship at C7″ and C8″, because the Diels−Alder
reaction of an o-quinone has been shown to be stereospecific
with respect to the relative configuration of the dienophile.48
With all of the other options, the stereochemical information of
coniferyl alcohol would be lost when either radical 11 or 13 was
formed, and a trans relationship at C7″ and C8″ would be
produced as the major product because this is thermodynami-
cally more favorable. By running the oxidative coupling of cis-
coniferyl alcohol with taxifolin, it was determined that the
identical products (1−4) as trans-coniferyl alcohol were
generated and that the cis related products (20−23) were not
observed. Although it was determined that cis-coniferyl alcohol
isomerized to trans-coniferyl alcohol under the reaction
conditions, it occurred much more slowly than the rate of
formation of silibinin and isosilibinin. Unless either the Diels−
Alder reaction is not concerted, which is inconsistent with prior
results,48 or cis related products 20−23 rapidly isomerize to trans
related products 1−4, Option 2 is not viable. On the basis of the
In addition to flavonolignans 1−4, coniferyl aldehyde 16 and
lignan 18 were produced in moderate amounts in the biomimetic
reaction (Figure 2). Trace amounts of flavonolignans 5−7 also
formed during some of the reactions, as determined by UPLC−
MS, but the quantities were never sufficient to confirm by
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Scheme 5. Attempted Coupling of Coniferyl Aldehyde to Taxifolin
Scheme 6. Mechanistically Supported Biomimetic Synthesis of Flavonolignans
results of the reaction of cis-coniferyl alcohol (Scheme 3), Option
2 was considered unlikely, but as it could not be definitively
excluded, additional reactions were performed.
considered was Option 4, where coniferyl alcohol was oxidized;
radical 11 reacted with taxifolin, and finally, the compound
oxidized further to yield silibinin and isosilibinin (Scheme 6).
This pathway accounts for the formation of flavonolignans 1−4,
coniferyl aldehyde 16, and lignan 18. Although not specifically
tested, it is plausible that the silver salts are involved in the
process. Beyond acting as the single-electron oxidants, they may
coordinate the phenols to position and stabilize the various
charges and radicals.
The next reactions that were examined were the oxidation of
either coniferyl alcohol or taxifolin individually with Ag2O in the
absence of the other compound (Scheme 4). Interestingly,
coniferyl alcohol was rapidly oxidized by Ag2O, whereas taxifolin
was practically inert to those conditions (Scheme 4 and
Supporting Information). The lack of reactivity toward oxidation
of taxifolin implies that Options 1−3 in Scheme 1 were all not
viable mechanisms and simplifies the possibilities to only
Options 4 or 5. Importantly, further scrutiny of the oxidation
of coniferyl alcohol in the absence of taxifolin revealed the
production of two major products, coniferyl aldehyde and lignan
18. This verified that two oxidations of coniferyl alcohol yielded
coniferyl aldehyde, as expected, and that the initial radical from
single-electron oxidation was prone to react with an electron-rich
phenol of a different molecule of coniferyl alcohol. Hypotheti-
cally, if taxifolin was in solution with the oxidized radical of
coniferyl alcohol, the nucleophilic catechol moiety could
similarly react to give silibinin and isosilibinin (Option 4).
As mentioned earlier, it was not anticipated that coniferyl
aldehyde 16 would react with taxifolin to undergo a redox
reaction and yield silibinin and isosilibinin. To test this, the
reaction of coniferyl aldehyde with taxifolin was examined in
both the presence and absence of Ag2O (Scheme 5). As
anticipated, silibinin and isosilibinin were not observed with this
reaction. Thus, the only remaining viable mechanism of those
CONCLUSIONS
■
In summary, a biomimetic synthesis of four major flavonolignans
present in silymarin is reported, and the analyses of related
reactions were used to support or refute possible mechanisms.
From this analysis it is proposed that the mechanism for the
biomimetic synthesis of flavonolignans proceeds by single
electron oxidation of coniferyl alcohol, addition of taxifolin,
and finally oxidation to yield silibinin and isosilibinin. This is
contrary to the mechanism proposed previously for this
process,18 which involved the coupling of two independently
formed radicals. While the study presented herein has exclusively
examined oxidative couplings using Ag2O instead of enzymes to
form flavonolignans, it is proposed that similar reactivity should
be considered for the biosynthesis of related compounds such as
lignans.
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(dd, J = 8.1, 1.7 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 51.9, 56.1,
85.5, 86.1, 114.1, 114.2, 114.7, 125.8, 146.3, 146.6. HRMS (APCI) (m/
z): 179.0698 [M + H]+ calcd for C10H11O3; found, 179.0703).
(Z)-4-(3-Hydroxyprop-1-en-1-yl)-2-methoxyphenol or cis-
Coniferyl Alcohol 19. To a stirred solution of 4-(3-hydroxyprop-1-
yn-1-yl)-2-methoxyphenol (72 mg, 0.40 mmol) and Lindlar’s catalyst
(0.016 g, 37 mol %) in 10 mL of methanol was added an atmosphere of
hydrogen gas at ambient temperature. The reaction mixture was stirred
for 1 h, filtered through Celite, concentrated under reduced pressure,
and purified by silica gel column chromatography (80:20 hexanes/ethyl
acetate) to yield 37 mg of cis-coniferyl alcohol (51% yield) as a white
solid. The 1H NMR spectrum was consistent with previously reported
data (Supporting Information).50
EXPERIMENTAL SECTION
■
General Information. All reactions were carried out under a N2
atmosphere with anhydrous conditions. All reagents and solvents were
purchased and used without further purification. NMR experiments
were conducted using a spectrometer operating at 500 MHz for 1H and
125 MHz for 13C. Accurate mass measurements were acquired using an
Orbitrap mass analyzer and an electrospray ionization (ESI) source for
compounds 1−4 in negative ionization mode via a liquid chromato-
graphic/autosampler system that consisted of a UPLC system. Accurate
mass measurements of 4-(3-hydroxyprop-1-yn-1-yl)-2-methoxyphenol
were accomplished using an atmospheric pressure chemical ionization
(APCI) source under direct infusion flow conditions in positive mode
ionization. HPLC and UPLC samples were analyzed using a photodiode
array (PDA) detector. For preparative HPLC, a YMC ODS-A (5 μm,
250 × 20 mm) column was used at a 7 mL/min flow rate, and a
pentafluorophenyl propyl (PFP; 5 μm, 250 × 21 mm) column was used
at a 21.2 mL/min flow rate. For analytical HPLC, a YMC ODS-A (5 μm,
150 × 4.6 mm) column and a PFP (5 μm, 150 × 4.6 mm) column were
used, both at a 1 mL/min flow rate. For UPLC, an HSST3 (1.8 μm, 2.1 ×
100 mm) column was used at 50 °C at a 0.6 mL/min flow rate and
monitored at 288 nm.
ASSOCIATED CONTENT
■
S
* Supporting Information
UPLC chromatograms, 1H and 13C NMR spectra, and tabulated
comparisons between isolated and synthesized compounds. This
material is available free of charge via the Internet at http://pubs.
acs.org.
Procedure of Biomimetic Synthesis. Taxifolin was isolated in
>90% purity from milk thistle extract (silymarin) via two successive
reverse phase HPLC methods. The first method utilized a gradient of
15:85 to 50:50 MeOH/H2O over 60 min using the YMC ODS-A (5 μm,
250 × 20 mm) column and detected at 288 nm. The second method
utilized a gradient of 5:90 to 70:30 CH3CN/H2O (0.1% formic acid)
over 30 min using a PFP (5 μm, 250 × 21 mm) column.
AUTHOR INFORMATION
Corresponding Author
*E-mail: mpcroatt@uncg.edu (M.P.C.) and nicholas_oberlies@
uncg.edu (N.H.O.).
Notes
■
The authors declare no competing financial interest.
To a 100 mL round-bottom flask with a stirred solution of taxifolin
(106 mg, 0.348 mmol) and trans-coniferyl alcohol (125 mg, 0.696
mmol) in ethyl acetate (30 mL, 0.01 M) under a nitrogen atmosphere at
ambient temperature was added Ag2O (323 mg, 1.39 mmol). The flask
was covered with foil and equipped with a reflux condenser. The
solution was stirred and heated to 75 °C for 96 h under an atmosphere of
nitrogen gas. The reaction mixture was cooled to room temperature,
filtered through Celite, and washed with ethyl acetate. A yellow filtrate
was concentrated under reduced pressure, dissolved in ethyl acetate (2
mL), and centrifuged through a polypropylene Eppendorf tube filter
(0.22 μm) to remove any residual silver salts. The crude product (225
mg) was purified by reverse-phase HPLC as described below to afford
flavonolignans with a total yield of 82.6 mg, 52% (21.4 mg, 21.1 mg, 20.7
mg, and 19.5 mg for 1, 2, 3, and 4, respectively). In addition to the four
major compounds, coniferyl aldehyde 16 (2.3 mg) and lignan 18 (13.6
mg) were isolated. In addition to having 1H and 13C NMR spectra that
were identical to prior reports,36 coinjection of coniferyl aldehyde or the
individual natural flavonolignans by UPLC was used to confirm their
identity (see Supporting Information). 1H and 13C NMR data were used
to confirm the structure of known lignan 18.49
To purify the reaction mixtures, two different reverse-phase columns
were utilized, ODS-A (5 μm, 250 × 20 mm) and PFP (5 μm, 250 × 21
mm). The reaction mixture was first purified using a gradient of 20:80 to
50:50 CH3OH/H2O over 90 min and then held for 20 min. Partially
purified fractions were chromatographed using a similar procedure.
Then, for the final purification, the PFP column was used with a gradient
of 20:80 to 40:60 CH3CN/H2O (0.1% formic acid) over 30 min. Each
synthetic flavonolignan was purified until >99% pure, as measured by
analytical UPLC (see Supporting Information).
4-(3-Hydroxyprop-1-yn-1-yl)-2-methoxyphenol. To a stirred
solution of 4-bromo-2-methoxyphenol (1.00 g, 4.93 mmol), CuI (282
mg, 0.148 mmol, 3 mol %), and bis(triphenylphosphine)palladium(II)
dichloride (104 mg, 0.148 mmol, 3 mol %) in triethylamine (10 mL)
under a nitrogen atmosphere at ambient temperature was added
propargyl alcohol (440 mg, 7.9 mmol). The reaction was heated to 95
°C for 4 h, cooled to room temperature, filtered through Celite, and
concentrated under reduced pressure. The crude extract was purified by
silica gel column chromatography (85:15 hexanes/ethyl acetate) to yield
80 mg of 4-(3-hydroxyprop-1-yn-1-yl)-2-methoxyphenol (9% yield) as a
white solid. 1H NMR (500 MHz, CDCl3) δ = 3.89 (s, 3H), 4.48 (s, 2H),
5.74 (s, 1H), 6.86 (d, J = 8.1 Hz, 1H), 6.95 (d, J = 1.5 Hz, 1H), 7.0 ppm
ACKNOWLEDGMENTS
■
This research was supported in part by the National Institutes of
Health/National Center for Complementary and Alternative
Medicine via Grant R01 AT006842. H.S.A. was supported by the
King Abdullah Scholarship Program from the Ministry of Higher
Education of Saudi Arabia. The authors thank Mr. T. N. Graf
(UNCG) for helpful discussions regarding milk thistle and Dr. I.
F. D. Hyatt (UNCG) for assistance with this mechanistic study.
The authors thank Dr. Brandie M. Ehrmann (UNCG) for
assistance with the high resolution mass spectrometry data at the
Triad Mass Spectrometry Laboratory at the University of North
Carolina at Greensboro.
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