Resolution of diastereomeric flavonoid (1S)-(-)-camphanic acid esters via reversed-phase HPLC

Article abstract of DOI:10.1016/j.phytochem.2007.01.022

Prenylflavonoids are an unique class of phytochemicals found in the inflorescences of the hop plant (Humulus lupulus). These flavonoids have demonstrated a wide range of biological activities, which may be influenced by their stereochemical configuration.

Full text of DOI:10.1016/j.phytochem.2007.01.022

PHYTOCHEMISTRY
Phytochemistry 68 (2007) 1206–1211
www.elsevier.com/locate/phytochem
Resolution of diastereomeric flavonoid (1S)-(ꢀ)-camphanic acid
esters via reversed-phase HPLC
*
Casey S. Philbin, Steven J. Schwartz
Department of Food Science and Technology, The Ohio State University, 2015 Fyffe Road, Columbus, OH 43210, USA
Received 18 October 2006; received in revised form 21 December 2006
Available online 23 March 2007
Abstract
Prenylflavonoids are an unique class of phytochemicals found in the inflorescences of the hop plant (Humulus lupulus). These flavo-
noids have demonstrated a wide range of biological activities, which may be influenced by their stereochemical configuration. Addition-
ally, recent studies suggest that hop prenylflavonoids are subject to biotransformations which could alter or enrich their stereochemistry.
In order to facilitate studies of the stereoisomers of flavanones, a facile method was developed for resolving the diastereomeric esters of
flavanones via reversed-phase HPLC. Herein, a method for forming the tri-(1S)-(ꢀ)-camphanic acid esters of the 40,5,7-trihydroxy flav-
anones naringenin, 8-prenylnaringenin and 6-prenylnaringenin, is described. The respective diastereomers were separated using analy-
tical reversed-phase HPLC. Diastereomeric esters were isolated by preparative HPLC to >98% d.e. based on HPLC, with their
absolute configurations established by application of CD spectrometry.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Humulus lupulus; Cannabaceae; Hops; High performance liquid chromatography; Chiral resolution; Naringenin; 8-Prenylnaringenin;
6-Prenylnaringenin; Prenylflavonoids; Camphanic acid chloride
1. Introduction
had comparable binding affinities for the two isoforms of
human recombinant estrogen receptors ERa and ERb,
although (S)-3 showed slightly higher affinity for ERb than
(R)-3. In contrast, Schaefer et al. (2003) found racemic 3 to
be selective for human recombinant ERb, and (S)-3 to
show moderately higher affinity for both receptors in a
competitive binding assay.
Although 3 consumed directly from hops or beer is
likely to be found as a racemic mixture (Stevens and Page,
2004), recent research suggests that other compounds in
hops and beer may act as precursors to 3 in vivo. The major
prenylflavonoid in beer, 1, may be O-demethylated to 3 by
endogenous microflora in the gut. This conversion has been
observed in vitro using fecal cultures (Possemiers et al.,
2005), and in vivo as demonstrated by the presence of 3
in human urine samples after oral administration of 1 (Pos-
semiers et al., 2006). In addition, in vitro digestion studies
using liver microsomes have shown that 1 may undergo
O-demethylation (Nikolic et al., 2005), catalyzed by
CYP1A2 enzymes (Guo et al., 2006).
Prenylflavonoids represent a unique class of phyto-
chemicals which can be found in the vine Humulus lupulus,
more commonly known as hops, and have demonstrated a
wide range of biological activities (Gerhauser, 2005; Ste-
vens and Page, 2004). Hops are traditionally used in beer
brewing, and hop prenylflavonoids are generally found in
beer as flavanones (Fig. 1), formed by isomerization of
their corresponding chalcones during boiling: the most
abundant prenylflavanone in beer is isoxanthohumol (1),
followed by 6-prenylnaringenin (2) and 8-prenylnaringenin
(3) (Stevens et al., 1999).
Of the most abundant flavanones found in beer, 3 has
recently received a great deal of interest as a potent phyto-
estrogen (Milligan et al., 1999, 2000, 2002; Schaefer et al.,
2003). Milligan et al. (2002) found that both enantiomers
*
Corresponding author. Tel.: +1 614 292 2934; fax: +1 614 292 4233.
E-mail address: schwartz.177@osu.edu (S.J. Schwartz).
0031-9422/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.01.022

C.S. Philbin, S.J. Schwartz / Phytochemistry 68 (2007) 1206–1211
1207
3, and subsequently separating them using reversed-phase
high performance liquid chromatography (HPLC). Opti-
cally pure diastereomers were isolated using preparative
chromatography, and the diastereomeric purity and abso-
lute configuration were confirmed by HPLC and CD
spectrometry.
2. Results and discussion
Racemic 3 was prepared from 4 using previously pub-
lished methods (Gester et al., 2001). However, in our
hands, published procedures for the synthetic preparation
of 2 were unsatisfactory. Direct C-prenylation of 4 under
basic conditions (Mizobuchi and Sato, 1984) and using
Lewis acid catalysts (Jain et al., 1978) resulted in poor
yields. A total synthesis of 2 has recently been described,
but, final yields were still low (Bu et al., 1997). One draw-
back of direct nuclear prenylation is poor regioselectivity,
yielding mixtures of various C- and O-alkylated products.
It has been suggested that strongly hydrogen bonding sol-
vents favor C-alkylation of phenoxide ions (Kornblum
et al., 1963) due to solvation interactions interfering with
the nucleophilic activity of the phenolate anion. By carry-
ing out this reaction under aqueous conditions (Scheme 1),
C-alkylation was favored, and at low temperatures, 2 was
formed as the major product. Although over-alkylation
was still apparent under these conditions, yield was
satisfactory compared with previously described C-prenyl-
ation methods (Jain et al., 1978; Mizobuchi and Sato,
1984).
Fig. 1. Structures of the hop flavanones found in hops and beer.
The optical purity of 3 from hops has previously been
examined using chiral chromatography, whereby the
authors found that approximately equal levels of each
enantiomer were present (Milligan et al., 2002). Although
chiral chromatography may be used to determine the
enantiomeric purity of compounds such as 3 (Kitaoka
et al., 1998; Milligan et al., 2002), chiral columns can
be costly. In order to overcome this shortcoming, we
have developed a facile method for producing the diaste-
reomeric (1S)-(ꢀ)-camphanic acid esters of racemic
naringenin (4), a flavanone derived from citrus, 2 and
Naringenin (4) was used as a model flavonoid in order to
optimize the esterification and chromatographic separation
of 40,5,7-trihydroxyflavanones due to its commercial avail-
Scheme 1.
Scheme 2.

1208
C.S. Philbin, S.J. Schwartz / Phytochemistry 68 (2007) 1206–1211
ability and established stereochemical properties (Gaffield,
1970). In order to produce diastereomeric esters that could
be separated by column chromatography, (1S)-(ꢀ)-cam-
phanoyl chloride was chosen as a resolving agent. (1S)-
(ꢀ)-camphanic acid esters have been previously employed
in resolving chroman derivatives via crystallization (Este-
rhuysen et al., 2005; Zehnter and Gerlach, 1995), as well
as several different classes of compounds including carote-
noids (Vecchi and Muller, 1979), binaphthols (Hua et al.,
2001), and helicenols (Thongpanchang et al., 2000) either
through crystallization or chromatographic methods. In
order to avoid complex mixtures of mono-, di- and tri-
esters, 4 was treated with a large excess of (1S)-(ꢀ)-cam-
phanoyl chloride (Scheme 2), resulting in quantitative
conversion to the tri-camphanoate (5) based on LCMS
analysis and a characteristic hypsochromic shift in the
UV spectrum.
Using a tertiary solvent system including aqueous
MeOH and MeCN, near-baseline separation of 5a and 5b
was achieved (Fig. 2a). These methods were applied to
the hop prenylflavonoids 2 and 3, and quantitative conver-
sion to the tri-camphanoyl esters 6 and 7 was observed for
all compounds based on HPLC. Due to the non-polar nat-
ure of the prenylflavonoids 6 and 7 relative to 5, the mobile
phase was adjusted accordingly to reduce retention time
with minimal loss in resolution, and near-baseline separa-
tion (Fig. 2b and c).
In order to isolate each diastereomer for further analy-
sis, several iterations of preparative HPLC were employed
using chromatographic conditions analogous to analytical
methods, eventually yielding diastereomers of >98% d.e.,
diastereomeric excess (Fig. 2). The isolated diastereomers
of 5, 6 and 7 were analyzed using CD spectrometry, and
the observed spectra were similar to those previously
observed for 1 (Gaffield, 1970; Prescott et al., 2002) and
3 (Kitaoka et al., 1998). It is known that 2S flavanones
exhibit a positive Cotton effect in the n ! p* region, and
a negative Cotton effect in the p ! p* region of the CD
spectrum (Gaffield, 1970). Based on the signs of the Cotton
effects in these regions, absolute configurations were
assigned to each diastereomer.
Fig. 2. Chromatographic separations of the (1S)-(ꢀ)-camphanic acid
esters of (a) 5, (b) 6, and (c) 7.
evaluate the effects of various treatments such as extrac-
tions or hydrolysis on the enantiomeric purity of those
compounds when access to chiral chromatography is
limited.
2.1. Concluding remarks
Herein, a facile method is thus described for the for-
mation and chromatographic resolution of the diastereo-
meric (1S)-(ꢀ)-camphanic acid esters of prenylated and
non-prenylated flavanones using RP-HPLC. Despite the
advantages in circumventing chiral chromatography via
a simple and inexpensive esterification, recovering enanti-
omerically enriched flavanones using this method would
require alkaline hydrolysis of isolated diastereomers. Fur-
ther investigation is necessary to determine the extent of
isomerization and racemization, which could occur via a
chalcone intermediate, under various alkaline hydrolysis
conditions. However, this method may be used to analyze
the enantiomeric purity of various flavanones, and to
3. Experimental
3.1. General
All reagents were purchased from Sigma–Aldrich (St.
Louis, MO) and HPLC-grade solvents were purchased
from Fisher Scientific (Fairlawn, NJ). UV/Vis spectra were
obtained on an HP8453 diode-array spectrophotometer
1
(Agilent, Palo Alto, CA). H NMR spectra were obtained
in CDCl3 using a Bruker 600 MHz spectrometer (Bruker

C.S. Philbin, S.J. Schwartz / Phytochemistry 68 (2007) 1206–1211
1209
Biospin, Rheinstetten, Germany) at ambient temperatures.
3.3.2. 8-Prenylnaringenin (3)
rac-8-Prenylnaringenin was produced synthetically from
4 (58 mg, 28% overall yield) as previously described by
CD spectra were recorded using an AVIV 202 Circular
Dichroism Spectrometer (AVIV Biomedical, Lakewood,
NJ). ESIMS were recorded on a Micromass Q-TOF
(Micromass, Manchester, UK) measured at 30 eV, coupled
to an HP1100 HPLC gradient system (Agilent, Palo Alto,
CA) equipped with DAD and applying the chromato-
graphic methods described below.
Gester et al. (2001). Rt: 7.5 min (Method A). UV kMeOH
:
max
1
293, 334 sh. H NMR spectroscopic data were in agree-
ment with previously published spectra (Mizobuchi and
Sato, 1984). ESIMS (30 eV) m/z (rel. int.): 341.2 [MH]+
(100), 285.2 [MHꢀC4H8]+ (9); ESI-CAD-MS m/z (rel.
int.): 285.2 [MHꢀC4H8]+ (12), 165.1 [MHꢀC11H12O2]+
(100).
3.2. HPLC methods
Analytical separations were carried out on a Waters
2695 System coupled to a 2996 PDA detector, using a
4 lm Waters (Milford, MA) Nova-Pakꢁ C18 column
(3.9 · 150 mm) equipped with a Nova-Pakꢁ guard car-
tridge (4 lm, 3.9 · 20 mm). The following solvent systems
were employed at a flow rate of 1 mL/min: (A) 40–80%
MeCN in aq. AcOH (1%) at a flow rate of 1 mL/min over
40 min; (B) 40% MeOH, 24% MeCN, 36% aq. AcOH (1%)
isocratic; (C) 50% MeOH, 20% MeCN, 30% aq. AcOH
(1%) isocratic.
Preparative separations were carried out on an HP1050
quaternary gradient system coupled to a Waters 2996 PDA
detector, using a 5 lm Waters Sunfireꢂ Prep C18 column
(19 · 150 mm) equipped with a Sunfireꢂ Prep guard car-
tridge (5 lm, 19 · 10 mm). The following isocratic solvent
systems were employed at a flow rate of 10 mL/min: (D)
40% MeCN in aq. AcOH (1%); (E) 45% MeOH, 22%
MeCN, 33% 1% aq. AcOH; (F) 55% MeOH, 18% MeCN,
27% aq. AcOH (1%).
3.4. Typical (1S)-(ꢀ)-camphanoylation of flavanones
To a solution of 3 (0.23 mmol) in pyridine (1.2 mL) was
added (1S)-(ꢀ)-camphanoyl chloride (2.06 mmol, 9 equiv.).
The resulting solution almost immediately became hetero-
geneous, and was stirred for 2 h. Quantitative conversion
was observed by an HPLC analysis (Method B), and excess
pyridine was removed under N2. The resulting paste was
dissolved in CH2Cl2 (5 mL) methylene chloride, washed
10% aq. NaHCO3 (3 · 5 mL), dried (MgSO4) and filtered.
Solvent was removed under N2, providing crude 7a+b
(250 mg). The crude diastereomeric mixture was purified
by preparative reversed-phase HPLC (Method F). Diaste-
reomerically enriched fractions were combined and re-puri-
fied several times to yield each diastereomer >98% d.e. by
HPLC (Method B). Fractions were stored at ꢀ20 ꢃC to
prevent hydrolysis of camphanic acid esters. Solvent was
removed under nitrogen flow and lyophilization yielding
12 mg (35% yield) of each diastereomer.
3.3. Prenylflavonoids
3.4.1. (R)-40,5,7-tris-O-[(1S)-(ꢀ)-camphanoyl]-8-
prenylnaringenin (7a)
3.3.1. 6-Prenylnaringenin (2)
Rt: 34.1 min (Method C). CD: [h]341 ꢀ15000; [h]309
To a suspension of 4 (0.5 mmol) in H2O (0.5 mL), stir-
red in an ice bath, was added dropwise 5 N aq. NaOH
(2.0 mmol). The suspension was removed from the ice bath
until the suspension became homogeneous, returned to the
ice bath and allowed to cool to 0 ꢃC before adding neat 3,3-
dimethylallyl bromide (1.0 mmol). This emulsion became
homogeneous, then heterogeneous as an insoluble tar
began to form over 15 min. The heterogeneous mixture
was washed with hexanes (3 · 2 mL) before acidifying with
5% aq. HCl. The aqueous suspension was extracted with
EtOAc (3 · 4 mL), with organics combined and washed
with 10% NaHCO3 (4 · 5 mL), dried (Na2SO4), filtered
and the solvent removed under nitrogen flow. Crude prod-
uct was subjected to preparative HPLC (Method D) and
the major product was collected and recovered by lyophili-
zation yielding 27 mg (16%) of desired product as an off-
+17000; [h]238 +300; [h]258 +7900; [h]275 +1800 (MeCN).
MeCN
max
UV k
(loge): 258 (3.95), 319. ESIMS (30 eV) m/z
(rel. int.): 881.6 [MH]+ (100); ESI-CAD-MS m/z (rel.
int.): 881.7 [MH]+ (30), 825.6 [MHꢀC4H8]+ (16), 727.5
[MHꢀcamphonolactone]+ (29), 645.5 [MHꢀCpꢀC4H8]+
(100), 581.5 [MHꢀCpꢀC8H8O]+ (63).
3.4.2. (S)-40,5,7-tris-O-[(1S)-(ꢀ)-camphanoyl]-8-
prenylnaringenin (7b)
Rt: 35.7 min (Method C). CD: [h]341 +15000; [h]309
ꢀ17000; [h]238 +300; [h]254 ꢀ6500; [h]275 ꢀ800 (MeCN).
MeCN
max
UV k
(loge): 258, (3.95), 319.0. ESIMS (30 eV) m/z
(rel. int.): 881.6 [MH]+ (100); ESI-CAD-MS m/z (rel.
int.): 881.7 [MH]+ (33), 825.6 [MHꢀC4H8]+ (16), 727.5
[MHꢀcamphonolactone]+ (26), 645.5 [MHꢀCpꢀC4H8]+
(100), 581.5 [MHꢀCpꢀC8H8O]+ (71).
white powder. Rt: 11.5 min (Method A). UV kMeOH: 293,
max
1
334 sh. H NMR spectroscopic data were in agreement
3.5. (1S)-(ꢀ)-camphanoylation of prenylflavanones
with previously published spectra (Haensel and Schulz,
1988; Tabata et al., 1997). ESIMS (30 eV) m/z (rel. int.):
341.2 [MH]+ (32), 285.2 [MHꢀC4H8]+ (100); ESI-CAD-
MS m/z (rel. int.): 285.2 [MHꢀC4H8]+ (42), 165.1
[MHꢀC11H12O2]+ (100).
This method was used to separate the diastereomeric
tris-(1S)-(ꢀ)-camphanate esters 6a and b providing 7 mg
(15% yield) of each diastereomer, and the diastereomeric
naringenin tris-(1S)-(ꢀ)-camphanate esters 5a and b were

1210
C.S. Philbin, S.J. Schwartz / Phytochemistry 68 (2007) 1206–1211
Guo, J., Nikolic, D., Chadwick, L.R., Pauli, G.F., van Breemen, R.B.,
2006. Identification of human hepatic cytochrome P450 enzymes
involved in the metabolism of 8-prenylnaringenin and isoxanthohu-
mol from hops (Humulus lupulus L.). Drug Metab. Dispos. 34,
1152–1159.
separated using preparative chromatography (Method E),
providing 10 mg (30% yield) of each diastereomer. The
configuration of each respective diastereomer was charac-
terized by CD spectrometry.
Haensel, R., Schulz, J., 1988. Demethylxanthohumol: isolation from hops
and cyclization to flavanones. Arch. Pharm. 321, 37–40.
Hua, D.H., Nguyen, T.X.C., McGill, J.W., Chen, Y., Robinson, P.D.,
2001. Resolution of 80-alkoxy- and hydroxy-1,10-binaphthalen-8-ols.
Tetrahedron: Asymmetr. 12, 1999–2004.
3.5.1. (R)-40,5,7-tris-O-[(1S)-(ꢀ)-camphanoyl]-
naringenin (5a)
Rt: 35.0 min (Method B). CD: [h]336 ꢀ9600; [h]307
+13000; [h]274 300; [h]256 +3300 (MeCN). UV kMeCN (loge):
max
Jain, A.C., Gupta, R.C., Sarpal, P.D., 1978. Synthesis of ( )-lupinifolin,
di-O-methylxanthohumol and isoxanthohumol and related com-
pounds. Tetrahedron 34, 3563–3567.
255 (3.90), 311. ESIMS (30 eV) m/z (rel. int.): 813.5 [MH]+
(100); ESI-CAD-MS m/z (rel. int.): 813.6 [MH]+ (40), 767.6
[MHꢀCH3O2]+ (23), 659.4 [MHꢀcamphonolactone]+
(100), 633.4 [MHꢀCp]+ (22).
Kitaoka, M., Kadokawa, H., Sugano, M., Ichikawa, K., Taki, M.,
Takaishi, S., Iijima, Y., Tsutsumi, S., Boriboon, M., Akiyama,
T., 1998. Prenylflavonoids: a new class of non-steroidal phyto-
estrogen (Part 1). Isolation of 8-isopentenylnaringenin and an
initial study on its structure–activity relationship. Planta Med. 64,
511–515.
3.5.2. (S)-40,5,7-tris-O-[(1S)-(ꢀ)-camphanoyl]-
naringenin (5b)
Rt: 37.3 min (Method B). CD: [h]336 +11000; [h]307
Kornblum, N., Berrigan, P.J., Noble, W.J.L., 1963. Solvation as a
factor in the alkylation of ambident anions: the importance of the
hydrogen bonding capacity of the solvent. J. Am. Chem. Soc. 85,
1141–1147.
MeCN
max
ꢀ14000; [h]272 +600; [h]256 ꢀ4720 (MeCN). UV k
(loge): 255 (3.90), 311. ESIMS (30 eV) m/z (rel. int.):
813.5 [MH]+ (100); ESI-CAD-MS m/z (rel. int.): 813.6
[MH]+ (50), 767.6 [MHꢀCH3O2]+ (20), 659.4 [MHꢀcam-
phonolactone]+ (100), 633.4 [MHꢀCp]+ (23).
Milligan, S.R., Kalita, J.C., Heyerick, A., Rong, H., De Cooman, L., De
Keukeleire, D., 1999. Identification of a potent phytoestrogen in hops
(Humulus lupulus L.) and beer. J. Clin. Endocrinol. Metab. 84, 2249–
2252.
Milligan, S.R., Kalita, J.C., Pocock, V., Van De Kauter, V., Stevens,
J.F., Deinzer, M.L., Rong, H., De Keukeleire, D., 2000. The
endocrine activities of 8-prenylnaringenin and related hop (Humu-
lus lupulus L.) flavonoids. J. Clin. Endocrinol. Metab. 85, 4912–
4915.
3.5.3. (R)-40,5,7-tris-O-[(1S)-(ꢀ)-camphanoyl]-6-
prenylnaringenin (6a)
Rt: 32.7 min (Method C). CD: [h]345 ꢀ15000; [h]313
MeCN
max
+19000; [h]269 0; [h]254 +3000 (MeCN). UV k
(loge):
Milligan, S., Kalita, J., Pocock, V., Heyerick, A., De Cooman, L.,
Rong, H., De Keukeleire, D., 2002. Oestrogenic activity of the
hop phyto-oestrogen, 8-prenylnaringenin. Reproduction 123, 235–
242.
256 (3.94), 322. ESIMS (30 eV) m/z (rel. int.): 881.6
[MH]+ (100); ESI-CAD-MS m/z (rel. int.): 881.7 [MH]+
(25), 825.6 [MHꢀC4H8]+ (75), 727.5 [MHꢀcamphonolac-
tone]+ (15), 645.5 [MHꢀCpꢀC4H8]+ (100).
Mizobuchi, S., Sato, Y., 1984. A new flavanone with antifungal activity
isolated from hops. Agric. Biol. Chem. 48, 2771–2775.
Nikolic, D., Li, Y., Chadwick, L.R., Pauli, G.F., van Breemen, R.B., 2005.
Metabolism of xanthohumol and isoxanthohumol, prenylated flavo-
noids from hops (Humulus lupulus L.), by human liver microsomes. J.
Mass Spectrom. 40, 289–299.
3.5.4. (S)-40,5,7-tris-O-[(1S)-(ꢀ)-camphanoyl]-6-
prenylnaringenin (6b)
Rt: 34.5 min (Method C). CD: [h]345 +15000; [h]313
MeCN
max
Possemiers, S., Heyerick, A., Robbens, V., De Keukeleire, D., Verstraete,
W., 2005. Activation of proestrogens from hops (Humulus lupulus L.)
by intestinal microbiota; conversion of isoxanthohumol into 8-pren-
ylnaringenin. J. Agric. Food Chem. 53, 6281–6288.
ꢀ17000; [h]269 0; [h]254 ꢀ6000 (MeCN). UV k
(loge):
256 (3.94), 322. ESIMS (30 eV) m/z (rel. int.): 881.6
[MH]+ (100); ESI-CAD-MS m/z (rel. int.): 881.7 [MH]+
(30), 825.6 [MHꢀC4H8]+ (76), 727.5 [MHꢀcamphonolac-
tone]+ (15), 645.5 [MHꢀCpꢀC4H8]+ (100).
Possemiers, S., Bolca, S., Grootaert, C., Heyerick, A., Decroos, K.,
Dhooge, W., De Keukeleire, D., Rabot, S., Verstraete, W., Van de
Wiele, T., 2006. The prenylflavonoid isoxanthohumol from hops
(Humulus lupulus L.) is activated into the potent phytoestrogen 8-
prenylnaringenin in vitro and in the human intestine. J. Nutr. 136,
1862–1867.
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