Chemo-enzymatic transformation of naturally abundant naringin to luteolin, a flavonoid with various biological effects

Article abstract of DOI:10.1016/j.molcatb.2013.03.002

Luteolin [3′,4′,5,7-tetrahydroxyflavone], having multiple biological effects such as anti-inflammation, anti-allergy and anti-cancer, was prepared by chemo-enzymatic synthesis from naringin, a naturally abundant flavonoid glycoside. On the occasion of Candida antarctica lipase B (Novozym 435)-catalyzed transesterification on peracetylated form of naringin, an acetate on C-4′ was exclusively deprotected to give the key intermediate. The oxidation with 2-iodoxybenzoic acid (IBX) followed by the reductive workup provided regioselectively C-3′and C-4′ catechol functionality. After protection of the above-mentioned diol with methoxymethyl (MOM) groups and subsequent hydrolysis of all acetyl groups, a dehydrogenative introduction of double bond between C-2 and C-3 was done by the treatment with I₂. Acid-catalyzed simultaneous removal of MOM groups and glycoside provided luteolin in total 8 steps and 36% overall yield from the starting material. Throughout the synthesis, diglycoside side chain effectively worked as the protective group on C-7 hydroxy group.

Full text of DOI:10.1016/j.molcatb.2013.03.002

Journal of Molecular Catalysis B: Enzymatic 92 (2013) 14–18
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Chemo-enzymatic transformation of naturally abundant naringin to luteolin, a
flavonoid with various biological effects
Ryohei Kobayashi, Takasi Itou, Kengo Hanaya, Mitsuru Shoji, Noriyasu Hada, Takeshi Sugai∗
Department of Pharmaceutical Science, Keio University, 1-5-30, Shibakoen, Minato-ku, Tokyo 105-8512, Japan
a r t i c l e i n f o
a b s t r a c t
Luteolin [3ꢀ,4ꢀ,5,7-tetrahydroxyflavone], having multiple biological effects such as anti-inflammation,
anti-allergy and anti-cancer, was prepared by chemo-enzymatic synthesis from naringin, a naturally
abundant flavonoid glycoside. On the occasion of Candida antarctica lipase B (Novozym 435)-catalyzed
transesterification on peracetylated form of naringin, an acetate on C-4ꢀ was exclusively deprotected to
give the key intermediate. The oxidation with 2-iodoxybenzoic acid (IBX) followed by the reductive
workup provided regioselectively C-3ꢀand C-4ꢀ catechol functionality. After protection of the above-
mentioned diol with methoxymethyl (MOM) groups and subsequent hydrolysis of all acetyl groups, a
dehydrogenative introduction of double bond between C-2 and C-3 was done by the treatment with I2.
Acid-catalyzed simultaneous removal of MOM groups and glycoside provided luteolin in total 8 steps and
36% overall yield from the starting material. Throughout the synthesis, diglycoside side chain effectively
worked as the protective group on C-7 hydroxy group.
Article history:
Received 1 February 2013
Received in revised form 2 March 2013
Accepted 3 March 2013
Available online 15 March 2013
Keywords:
Lipase
Transesterification
Regioselective reaction
Flavonoids
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
arylalkyne was demonstrated for the synthesis of luteolin as its
permethylated form [8].
Luteolin, 3ꢀ,4ꢀ,5,7-tetrahydroxyflavone (1a, Fig. 1), is a com-
mon flavone, which is one of the categories of flavonoids and
exists in various plants including fruits, vegetables, and medici-
nal herbs. It has been shown to possess potent antioxidant [1] and
anti-inflammatory/anti-allergic activities [2], and the recent review
gives an account of the pharmacological data and many references
for luteolin concerning its biological effect, comparing it to other
flavonoids [3]. Moreover, it displays the anti-cancer action, and
research papers have been reporting cancer preventive effect and
anti-cancer effects [4]. Inhibitory activity for osteoclast differenti-
ation has also been reported [5].
We turned our attention to naringin (2a, Fig. 1), which is daily
produced in large quantity as the by-product in industries of citrus
beverages and foods and envisaged a supply of 1a as sole aglycone
flavonoid, starting from 2a. By the combination of regioselective
introduction of one hydroxy group on C-3ꢀ, dehydrogenation to
flavanone to flavone, and the subsequent hydrolytic cleavage of
glycosidic bond, the desired molecule would be produced in an
expeditious manner. Herein we describe the successful chemo-
enzymatic approach, which was inspired by the Candida antarctica
lipase B-catalyzed regioselective transesterification and hydrolysis
on aromatic hydroxy groups and acetates [9,10].
However, the amount of luteolin is generally low compared
to some of the major flavonols like quercetin. While it is only
a minor flavonoid component in food and traditional medicine,
high amounts can be isolated from peanut hulls and Reseda lute-
ola L. that has been used as a dyeing plant due to its high
luteolin content since ancient times. The yield is still ca. 0.4%
[6]. Luteolin has been synthesized by the traditional Claisen
condensation [7] between 2,4,6-trihydroxyacetophenone and bis-
t-butyldimethylsilylated form of methyl 3,4-dihydroxybenzoate,
but the latter component is quite expensive. Recently, palladium-
catalyzed carbonylative coupling between an aryl iodide and an
2. Experimental
IR spectra were measured as ATR on a Jeol FT-IR SPX60 spec-
trometer. 1H NMR spectra were measured at 400 MHz on an Agilent
400-MR or at 500 MHz on an Agilent INOVA-500 or at 600 MHz on a
JEOL FT-NMR ECP-600 spectrometers. 13C NMR spectra were mea-
sured at 100 MHz on an Agilent 400-MR or at 125 MHz on an Agilent
INOVA-500 spectrometer. Optical rotation values were recorded
on a Jasco P-1010 polarimeter. High resolution mass spectra were
recorded on a Jeol JMS-700 MStation spectrometer at 70 eV. Silica
gel 60 N (spherical and neutral, 100–210 ␮m, 37560-79) of Kanto
Chemical Co. was used for column chromatography.
∗
Corresponding author. Tel.: +81 3 5400 2665; fax: +81 3 5400 2665.
E-mail address: sugai-tk@pha.keio.ac.jp (T. Sugai).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.03.002

R. Kobayashi et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 14–18
15
2.2. 5-Acetoxy-7-[hexa-O-acetyl-(2-O-˛-l-rhamnopyranoxyl-ˇ-
d-glucopyranosyl)oxy]-4ꢀ-hydroxyflavan-4-one
(2d)
To a solution of 2c (9.16 g, 10.0 mmol) in cyclopentanol (180 mL)
and cyclopentyl methyl ether (180 mL) were added an immobi-
lized form of C. antarctica lipase B (Novozymes, Novozym 435,
9.16 g). The mixture was stirred for 12 h at room temperature.
After removal of insoluble materials by filtration with a pad of
Celite, the filtrate was concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (200 g). Elution with
hexane/AcOEt = 1:1 afforded 2d (8.44 g, 96%) as slightly yellow
amorphous solid. [˛]D −39 (c 1.00, EtOH); 1H NMR (400 MHz,
26
CDCl3): ı 1.18 (d, J = 6.3 Hz, 2.7H, rhamnose-6-CH3), 1.19 (d,
J = 6.3 Hz, 0.3H, rhamnose-6-CH3), 1.95, 1.96, 1.97, 2.01, 2.08, 2.12
(each s, total 18H, sugar-OAc), 2.36 (s, 3H, 5-OAc), 2.68 (dd, J = 2.7,
16.8 Hz, 0.7H, H-3a), 2.69 (dd, J = 2.7, 16.8 Hz, 0.3H, H-3a), 3.00 (dd,
J = 13.5, 16.8 Hz, 0.7H, H-3b), 3.02 (dd, J = 13.5, 16.8 Hz, 0.3H, H-
3b), 3.81–4.21 (total 5H), 4.97–5.09 (total 5H), 5.12 (d, J = 2.9 Hz,
0.67H, rhamnose-H-1), 5.15 (d, J = 2.9 Hz, 0.33H, rhamnose-H-1),
5.29 (d, J = 9.4 Hz, 0.13H, glucose-H-1), 5.30 (d, J = 9.4 Hz, 0.37H,
glucose-H-1), 5.309 (d, J = 9.4 Hz, 0.13H, glucose-H-1), 5.313 (d,
J = 9.4 Hz, 0.37H, glucose-H-1), 5.37 (dd, J = 2.7, 13.5 Hz, 0.25H, H-
2), 5.38 (dd, J = 2.7, 13.5 Hz, 0.75H, H-2), 6.30 (d, J = 2.4 Hz, 1H, H-8),
6.505 (d, J = 2.4 Hz, 0.3H, H-6), 6.510 (d, J = 2.4 Hz, 0.7H, H-6), 6.84
(d, J = 8.4 Hz, 1.2H, H-2ꢀ, H-6ꢀ), 6.85 (d, J = 8.4 Hz, 0.8H, H-2ꢀ, H-6ꢀ),
7.28 (d, J = 8.4 Hz, 2H, H-3ꢀ, H-5ꢀ); 13C NMR (100 MHz, CDCl3): ı 17.4,
20.37, 20.40, 20.44, 20.53, 20.55, 20.71, 20.93, 44.6, 61.7, 66.8, 68.1,
68.3, 69.9, 70.7, 72.0, 74.0, 76.4, 79.5, 97.9, 98.0, 102.1, 105.7, 109.3,
115.5, 127.9, 129.3, 151.7, 157.0, 161.7, 164.1, 169.3, 169.6, 169.8,
170.01, 170.05, 170.1, 170.6, 189.4; the signals 115.5 and 127.9
included two carbons. IR: 1743, 1686, 1616, 1520, 1439, 1365, 1211,
Fig. 1. Luteolin (1a), naringin (2a), and its aglycone, naringenin (2b).
2.1. 4ꢀ,5-Diacetoxy-7-[hexa-O-acetyl-(2-O-˛-l-
rhamnopyranoxyl-ˇ-d-glucopyranosyl)oxy]flavan-4-one
(2c)
1138, 1038, 895, 837 cm−1
.
To a solution of naringin dihydrate (2a, 5.80 g, 10.0 mmol)
in pyridine (50 mL) were added Ac2O (50 mL) and 4-N,N-
dimethylaminopyridine (DMAP, 122 mg, 1.00 mmol, 0.1 equiv.)
under argon atmosphere. The mixture was stirred for 2 h at room
temperature, then the reaction was quenched by adding water.
The organic materials were extracted with AcOEt, and the com-
bined extracts were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (150 g). Elution with hexane/AcOEt = 1:1
2.3. 5-Acetoxy-7-[hexa-O-acetyl-(2-O-˛-l-rhamnopyranoxyl-ˇ-
d-glucopyranosyl)oxy]-3ꢀ,4ꢀ-dihydroxyflavan-4-one
(3a)
To a solution of 2d (150 mg, 0.172 mmol) in CHCl3 (1.4 mL) and
MeOH (0.35 mL) were added 2-iodoxybenzoic acid (IBX, 62.6 mg,
0.224 mmol, 1.3 equiv.). The mixture was stirred for 1 h at room
temperature, then the reaction was quenched by adding aque-
ous Na2S2O4 solution. The organic materials were extracted twice
with CHCl3, and the combined extracts were washed with satu-
rated aqueous NaHCO3 solution and brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (4.50 g). Elution with CHCl3/MeOH = 100:1
25
afforded 2c (9.08 g, 99%) as colorless amorphous solid. [˛]D −32
(c 1.00, EtOH); 1H NMR (400 MHz, CDCl3): ı 1.17 (d, J = 6.3 Hz, 3H,
rhamnose-6-CH3), 1.93, 1.94, 1.95, 1.99, 2.06, 2.09 (each s, total 18H,
sugar-OAc), 2.27 (s, 3H, 4ꢀ-OAc), 2.33 (s, 3H, 5-OAc), 2.70 (dd, J = 2.7,
16.8 Hz, 0.7H, H-3a), 2.71 (dd, J = 2.7, 16.8 Hz, 0.3H, H-3a), 2.98 (dd,
J = 13.5, 16.8 Hz, 0.7H, H-3b), 3.00 (dd, J = 13.5, 16.8 Hz, 0.3H, H-3b),
3.80–4.20 (total 5H), 4.94–5.09 (total 5H), 5.10 (d, J = 2.9 Hz, 0.5H,
rhamnose-H-1), 5.13 (d, J = 2.9 Hz, 0.5H, rhamnose-H-1), 5.27 (d,
J = 9.6 Hz, 0.13H, glucose-H-1), 5.28 (d, J = 9.6 Hz, 0.37H, glucose-
H-1), 5.295 (d, J = 9.6 Hz, 0.13H, glucose-H-1), 5.299 (d, J = 9.6 Hz,
0.37H, glucose-H-1), 5.42 (dd, J = 2.7, 13.5 Hz, 1H, H-2), 6.30 (d,
J = 2.4 Hz, 1H, H-8), 6.51 (d, J = 2.4 Hz, 1H, H-6), 7.107 (d, J = 8.6 Hz,
0.6H, H-2ꢀ, H-6ꢀ), 7.112 (d, J = 8.6 Hz, 1.4H, H-2ꢀ, H-6ꢀ), 7.41 (d,
afforded 3a (111 mg, 73%) as slightly yellow amorphous solid.
25
[˛]D
−40 (c 0.67, MeOH); 1H NMR (400 MHz, CDCl3): ı 1.17
(d, J = 6.3 Hz, 2.4H, rhamnose-6-CH3), 1.20 (d, J = 6.3 Hz, 0.6H,
rhamnose-6-CH3), 1.966, 1.970, 1.98, 2.02, 2.09, 2.12 (each s, total
18H, sugar-OAc), 2.36 (s, 3H, 5-OAc), 2.71 (dd, J = 2.9, 16.6 Hz,
0.8H, H-3a), 2.82 (dd, J = 3.5, 16.6 Hz, 0.2H, H-3a), 2.96 (dd,
J = 13.0, 16.6 Hz, 0.8H, H-3b), 3.01 (dd, J = 10.5, 16.6 Hz, 0.2H, H-
3b), 3.81–4.23 (total 5H), 4.97–5.09 (total 5H), 5.14 (d, J = 3.2 Hz,
0.37H, rhamnose-H-1), 5.17 (d, J = 3.2 Hz, 0.37H, rhamnose-H-1),
5.19 (d, J = 3.2 Hz, 0.13H, rhamnose-H-1), 5.22 (d, J = 3.2 Hz, 0.13H,
rhamnose-H-1),5.28 (d, J = 9.3 Hz, 0.13H, glucose-H-1), 5.30 (d,
J = 9.3 Hz, 0.37H, glucose-H-1), 5.31 (d, J = 9.3 Hz, 0.13H, glucose-H-
1), 5.32 (d, J = 9.3 Hz, 0.37H, glucose-H-1), 5.33 (dd, J = 2.9, 13.0 Hz,
0.8H, H-2), 5.41 (dd, J = 3.5, 10.5 Hz, 0.2H, H-2), 5.47 (s, 1H, OH),
5.79 (s, 0.8H, OH), 6.19 (s, 0.2H, OH), 6.31 (d, J = 2.4 Hz, 1H, H-8),
6.52 (d, J = 2.4 Hz, 0.8H, H-6), 6.55 (d, J = 2.4 Hz, 0.2H, H-6), 6.77
(dd, J = 1.9, 8.2 Hz, 0.2H, H-6ꢀ), 6.81(d, J = 1.9, 8.2 Hz, 0.8H, H-6ꢀ),
6.83 (d, J = 8.2 Hz, 0.2H, H-5ꢀ), 6.87 (d, J = 8.2 Hz, 0.8H, H-5ꢀ), 6.92
(d, J = 1.9 Hz, 0.2H, H-2ꢀ), 6.96 (d, J = 1.9 Hz, 0.8H, H-2ꢀ); 13C NMR
J = 8.6 Hz, 0.6H, H-3ꢀ, H-5ꢀ), 7.42 (d, J = 8.6 Hz, 1.4H, H-3ꢀ, H-5ꢀ); 13
C
NMR (100 MHz, CDCl3): ␦ 17.4, 20.38, 20.46, 20.48, 20.57, 20.59,
20.77, 20.95, 20.99, 44.9, 61.8, 66.8, 68.15, 68.24, 69.9, 70.7, 72.2,
74.0, 76.4, 79.1, 98.0, 98.1, 102.2, 105.9, 109.5, 122.0, 127.3, 135.5,
150.9, 151.8, 161.7, 163.8, 169.03, 169.17, 169.51, 169.62, 169.88,
169.92, 169.97, 170.31, 188.5; the signals 122.0 and 127.3 included
two carbons. The 1H and 13C NMR spectra were very complex, as
the stereochemistry at C-2 is prone to epimerize upon acetylation of
phenolic hydroxy groups. In the following sections from 2.2 to 2.5,
compounds 2d, 3a, 3b and 3c were also diastereomeric mixtures.
IR: 1743, 1686, 1616, 1439, 1365, 1219, 1037, 910, 748 cm−1
.

16
R. Kobayashi et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 14–18
(125 MHz, CDCl3): ı 17.5, 20.5, 20.57, 20.62, 20.70, 20.73, 20.9, 21.1,
44.7, 61.8, 66.9, 68.2, 68.5, 70.0, 70.8, 72.2, 74.1, 76.4, 79.4, 98.0,
98.1, 102.1, 106.0, 109.5, 113.5, 115.4, 119.0, 130.6, 144.2, 144.6,
151.7, 161.8, 164.1, 169.6, 169.7, 170.08, 170.11, 170.2, 170.3, 170.7,
189.3. IR: 3435, 1741, 1686, 1616, 1570, 1520, 1439, 1369, 1217,
then the reaction was quenched by saturated aqueous Na2S2O3
solution. The organic materials were extracted with CHCl3 three
times and the combined extracts were washed with brine, dried
over Na2SO4, and concentrated in vacuo to afford 1b (26.0 mg)
as yellow solid. 1H NMR (600 MHz, CD3OD): ı 1.32 (d, J = 6.1 Hz,
3H, rhamnose-6-CH3), 3.38–3.95 (total 16H), 5.20 (d, J = 7.6 Hz, 1H,
glucose-H-1), 5.28 (d, J = 1.6 Hz, 1H, rhamnose-H-1), 5.29 (s, 2H,
1034 cm−1
.
2.4. 5-Acetoxy-7-[hexa-O-acetyl-(2-O-˛-l-rhamnopyranoxyl-ˇ-
d-glucopyranosyl)oxy]-3ꢀ,4ꢀ-bis(methoxymethoxy)flavan-4-one
(3b)
O CH2 O), 5.30 (s, 2H, O CH2 O), 6.47 (d, J = 2.1 Hz, 1H, H-8), 6.70
(s, 1H, H-3), 6.80 (d, J = 2.1 Hz, 1H, H-6), 7.29 (d, J = 8.7 Hz, 1H, H-5ꢀ),
7.67 (dd, J = 2.1, 8.7 Hz, 1H, H-6ꢀ), 7.73 (d, J = 2.1 Hz, 1H, H-2ꢀ). This
was employed for the next step without further purification.
To a solution of 3a (70.0 mg, 0.0786 mmol) in CH2Cl2 (0.8 mL)
were added methoxymethyl (MOM) chloride (18 ␮L, 0.236 mmol,
3 equiv.) and diisopropylethylamine (DIPEA, 55 ␮L, 0.314 mmol,
4 equiv.). The mixture was stirred for 1 h at room temperature, then
the reaction was quenched by saturated aqueous NH4Cl solution.
The organic materials were extracted with AcOEt twice and the
combined extracts were washed with brine, dried over Na2SO4,
and concentrated in vacuo to afford 3b (77.0 mg) as yellow amor-
phous solid. 1H NMR (400 MHz, CDCl3): ı 1.19 (d, J = 6.2 Hz, 3H,
rhamnose-6-CH3), 1.95, 1.976, 1.978, 2.02, 2.08, 2.12 (each s, total
18H, sugar-OAc), 2.35 (s, 3H, 5-OAc), 2.68 (dd, J = 2.7, 16.8 Hz,
0.7H, H-3a), 2.69 (dd, J = 2.7, 16.8 Hz, 0.3H, H-3a), 3.03 (dd, J = 13.6,
16.8 Hz, 0.7H, H-3b), 3.04 (dd, J = 13.6, 16.8 Hz, 0.3H, H-3b), 3.50 (s,
3H, OCH3), 3.51 (s, 3H, OCH3), 3.83–4.24 (total 5H), 4.97–5.11 (total
5H), 5.127 (d, J = 3.0 Hz, 0.37H, rhamnose-H-1), 5.131 (d, J = 3.0 Hz,
0.13H, rhamnose-H-1), 5.15 (d, J = 3.0 Hz, 0.37H, rhamnose-H-1),
5.16 (d, J = 3.0 Hz, 0.13H, rhamnose-H-1), 5.21–5.25 (total 4H,
2.7. 3ꢀ,4ꢀ,5,7-Tetrahydroxyflavone (luteolin, 1a)
To a solution of 1b (26.0 mg) in MeOH (0.4 mL) and THF (0.4 mL)
were added conc. HCl (0.2 mL). The mixture was stirred for 14 h
at 60 ◦C, then the reaction was quenched by saturated aqueous
NaHCO3 solution. The organic materials were extracted with AcOEt
twice and the combined extracts were washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was purified
by silica gel column chromatography (2.00 g). Elution with CHCl3
afforded 1a (11.6 mg, 52% over 4 steps from 3a). Mp > 300 ◦C (uncor-
rected); 1H NMR (500 MHz, acetone-d6): ı 6.24 (d, J = 1.5 Hz, 1H,
H-8), 6.51 (d, J = 1.5 Hz, 1H, H-6), 6.57 (s, 1H, H-3), 6.99 (d, J = 8.3 Hz,
1H, H-5ꢀ), 7.47 (dd, J = 2.2, 8.3 Hz, 1H, H-6ꢀ), 7.49 (d, J = 2.2 Hz,
1H, H-2ꢀ); 13C NMR (125 MHz, acetone-d6): ı 94.6, 99.6, 104.2,
105.3, 114.1, 116.6, 120.1, 123.8, 146.4, 150.0, 158.8, 163.4, 164.8,
165.1, 183.0. IR: 3421, 1651, 1598, 1498, 1443, 1362, 1265, 1161,
1120, 1030 cm−1. HRMS (FAB+): m/z 287.0579 (M+H)+; calc. for
O
CH2 O), 5.29 (d, J = 9.4 Hz, 0.13H, glucose-H-1), 5.30 (d, J = 9.4 Hz,
0.37H, glucose-H-1), 5.31 (d, J = 9.4 Hz, 0.13H, glucose-H-1), 5.32 (d,
J = 9.4 Hz, 0.37H, glucose-H-1), 5.37 (dd, J = 2.7, 13.6 Hz, 0.7H, H-2),
5.38 (dd, J = 2.7, 13.6 Hz, 0.3H, H-2), 6.307 (d, J = 2.3 Hz, 0.7H, H-8),
6.311 (d, J = 2.3 Hz, 0.3H, H-8), 6.52 (d, J = 2.3 Hz, 1H, H-6), 7.01 (dd,
J = 2.1, 8.4 Hz, 0.7H, H-6ꢀ), 7.02 (dd, J = 2.1, 8.4 Hz, 0.3H, H-6ꢀ), 7.18 (d,
J = 8.4 Hz, 1H, H-5ꢀ), 7.23 (d, J = 2.1 Hz, 1H, H-2ꢀ). This was employed
for the next step without further purification.
C
15H11O6: 287.0556. Elemental analysis suggests the hemihydrate
form. Anal. Calcd for C15H10O6 + 0.5H2O: C 61.02, H 3.76; found: C
61.04, H 3.86. Its NMR spectral data were identical with those of an
authentic sample (Tokyo Chemical Industry Co., Ltd., Catalog No.
T2682).
2.8. 4ꢀ,5,7-Triacetoxyflavan-4-one (2e)
2.5. 5-Hydroxy-7-[(2-O-˛-l-rhamnopyranosyl-ˇ-d-
glucopyranosyl)oxy]-3ꢀ,4ꢀ-bis(methoxymethoxy)flavan-4-one
(3c)
In a similar manner as described for the acetylation of 2a, treat-
ment of 2b (100 mg, 0.37 mmol) with Ac2O (1 mL) and pyridine
(1 mL) gave 2e (129 mg, 88%) as solid. 1H NMR (400 MHz, CDCl3):
ı 2.28 (s, 3H, 4ꢀ-OAc), 2.30 (s, 3H, 7-OAc), 2.36 (s, 3H, 5-OAc), 2.76
(dd, J = 2.8, 16.8 Hz, 1H, H-3a), 3.01 (dd, J = 13.6, 16.8 Hz, 1H, H-3b),
5.47, (dd, J = 2.8, 13.6 Hz, 1H, H-2), 6.52 (d, J = 2.4 Hz, 1H, H-8), 6.76
(d, J = 2.4 Hz, 1H, H-6), 7.13 (d, J = 8.8 Hz, 2H, H-2ꢀ, H-6ꢀ), 7.44 (d,
J = 8.8 Hz, 2H, H-3ꢀ, H-5ꢀ).
To a solution of 3b (77.0 mg) in MeOH (1.0 mL) were added
K2CO3 (33 mg, 0.236 mmol). The mixture was stirred for 19 h at
room temperature, then the mixture was acidified with Amberlite
IR-120B (H+ form) to pH 5 and then filtered. The filtrate was con-
centrated in vacuo to afford 3c (50.0 mg) as yellow solid. 1H NMR
(600 MHz, CD3OD): ı 1.28 (d, J = 6.1 Hz, 3H, rhamnose-6-CH3), 2.79
(dd, J = 3.0, 17.1 Hz, 0.5H, H-3a), 2.80 (dd, J = 3.0, 17.1 Hz, 0.5H, H-
3a), 3.13 (dd, J = 12.7, 17.1 Hz, 0.5H, H-3b), 3.14 (dd, J = 12.7, 17.1 Hz,
0.5H, H-3b), 3.34–3.93 (total 16H), 5.08 (d, J = 7.6 Hz, 0.5H, glucose-
H-1), 5.10 (d, J = 7.6 Hz, 0.5H, glucose-H-1), 5.197 (s, 2H, O CH2 O),
5.203 (s, 2H, O CH2 O), 5.24 (d, J = 2.0 Hz, 0.5H, rhamnose-H-1),
5.25 (d, J = 2.0 Hz, 0.5H, rhamnose-H-1), 5.39 (dd, J = 3.0, 12.7 Hz,
0.5H, H-2), 5.41 (dd, J = 3.0, 12.7 Hz, 0.5H, H-2), 6.15 (d, J = 2.2 Hz,
1H, H-8), 6.198 (d, J = 2.2 Hz, 0.5H, H-6), 6.202 (d, J = 2.2 Hz, 0.5H, H-
6), 7.08 (dd, J = 2.2, 8.2 Hz, 0.5H, H-6ꢀ), 7.09 (dd, J = 2.2, 8.2 Hz, 0.5H,
H-6ꢀ), 7.15 (d, J = 8.2 Hz, 0.5H, H-5ꢀ), 7.16 (d, J = 8.2 Hz, 0.5H, H-5ꢀ),
7.27 (d, J = 2.2 Hz, 1H, H-2ꢀ). This was employed for the next step
without further purification.
2.9. C. antarctica lipase B-catalyzed transesterification of 2e
In a similar manner as described for the transesterification of
2c, a solution of 2e (25.0 mg, 0.063 mmol) in cyclopentanol (2 mL)
and cyclopentyl methyl ether (2 mL) was added Novozym 435
(50.0 mg). The mixture was stirred for 24 h at room temperature.
After removal of insoluble materials by filtration with a pad of
Celite, the filtrate was concentrated in vacuo to give a mixture of 2f
and 2g.
2f: 1H NMR (400 MHz, CDCl3): ı 2.28 (s, 3H, 7-OAc), 2.37 (s, 3H,
5-OAc), 2.71 (dd, J = 2.8, 16.8 Hz, 1H, H-3a), 3.02 (dd, J = 13.6, 16.8 Hz,
1H, H-3b), 5.37, (dd, J = 2.8, 13.6 Hz, 1H, H-2), 6.50 (d, J = 2.4 Hz, 1H,
H-8), 6.74 (d, J = 2.4 Hz, 1H, H-6), 6.84 (d, J = 8.4 Hz, 2H, H-2ꢀ, H-6ꢀ),
7.28 (d, J = 8.8 Hz, 2H, H-3ꢀ, H-5ꢀ).
2.6. 5-Hydroxy-7-[(2-O-˛-l-rhamnopyranosyl-ˇ-d-
glucopyranosyl)oxy]-3ꢀ,4ꢀ-bis(methoxymethoxy)flavone
(1b)
2g (400 MHz, CDCl3): 1H NMR: ı 2.37 (s, 3H, 5-OAc), 2.67 (dd,
J = 2.8, 16.4 Hz, 1H, H-3a), 2.98 (dd, J = 13.2, 16.4 Hz, 1H, H-3b),
5.35, (dd, J = 2.8, 13.2 Hz, 1H, H-2), 6.17 (d, J = 2.4 Hz, 1H, H-8), 6.29
(d, J = 2.4 Hz, 1H, H-6), 6.84 (d, J = 8.4 Hz, 2H, H-2ꢀ, H-6ꢀ), 7.28 (d,
J = 8.4 Hz, 2H, H-3ꢀ, H-5ꢀ).
To a solution of 3c (50.0 mg) in pyridine (0.8 mL) were added I2
(22 mg, 0.0869 mmol). The mixture was stirred for 20 h at 100 ◦C,

R. Kobayashi et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 14–18
17
Scheme 1. Chemo-enzymatic transformation of naringin (2a) to luteolin (1a). Reagents and conditions: (a) Ac2O, DMAP, pyridine; (b) Candida antarctica lipase B (Novozymes,
Novozym 435), cyclopentanol, cyclopentyl methyl ether (CPME); (c) 2-iodoxybenzoic acid (IBX), CHCl3 MeOH (4:1); (d) Na2S2O4; (e) methoxymethyl (MOM) chloride,
diisopropylethylamine (DIPEA), CH2Cl2; (f) K2CO3, MeOH; (g) I2, pyridine and (h) conc. HCl, MeOH, THF.
3. Results and discussion
products, primary acetates work as good acyl donors to cause the
undesired reverse reaction catalyzed by the same lipase in the reac-
tion mixture.
As expected by so far reported examples [9,10], the reaction
was influenced by steric hindrance in the substrate, and proceeded
highly regioselectively to give 2d with only free hydroxy group at
the desired C-4ꢀ position (Scheme 1). The same result was obtained
by the action of Burkholderia cepacia lipase (Amano PS-IM). Interest-
ingly, no transesterification on primary acetate of glucose side chain
Toward this end, peracetylated form (2c) of naringin was treated
with C. antarctica lipase B (Novozym 435) under transesterfica-
tion conditions with cyclopentanol as nucleophile. The diminished
steric hindrance in cyclopentanol compared with conventional
secondary alcohol such as isopropyl alcohol is advantageous to gen-
erate higher reaction rate as nucleophile [11]. In contrast, primary
alcohols are poor nucleophiles [9], because the transesterification

18
R. Kobayashi et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 14–18
to a certain extent to give the mixture of 2f and 2g (Scheme 2).
In contrast, for naringin, the neohesperidosyl [–(1␤)Glc(2,1␣)Rha]
disaccharide side chain worked as the efficient protective group in
our approach.
4. Conclusion
Luteolin (1a) was efficiently prepared from abundantly avail-
able naringin (2a) in total 8 steps and 36% yield. “Protective group
technology” involving lipase-catalyzed regioselective opening as
well as the blocking with inherent disaccharide side chain on the
proper hydroxy groups, in a polyphenol, was demonstrated. This
approach would be applicable to the synthesis of flavanones, the
saturated forms such as eriodictyrol and pinocembrin, from an
abundant bioresource.
Scheme 2. Regioselectivity in lipase-catalyzed transesterification on peracetylated
form of naringenin (2e). Reagents and conditions: (a) C. antarctica lipase B, cyclopen-
tanol, CPME.
Acknowledgements
We thank Novozymes Japan for generous gift of Novozym 435,
and Amano Enzyme Inc. for lipase PS-IM. This work was supported
both by a Grant-in-Aid for Scientific Research (No. 23580152) and
MEXT-Supported Program for the Strategic Research Foundation at
Private Universities (Centers of Excellence for Research) in “molec-
ular nanotechnology for green innovation”, FY 2012–2016 and
acknowledged with thanks.
was observed, in contrast to the previous observation that primary
position showed certain reactivity [12]. The acetylated rhamnopy-
ranosyl group on the glucose was supposed to work as some kind
of “cap” to prevent the transesterification on the oligosaccharide
side chain.
For the introduction of 3ꢀ-OH, free phenol was oxidized with 2-
iodoxybenzoic acid (IBX) [13–15] followed by the reductive workup
with Na2S2O4 to give 3a. At this stage, the mixture of CHCl3 and
MeOH was superior to dimethyl sulfoxide (DMSO) as the solvent.
As the yield in the removal of other acetyl protective groups,
was unexpectedly low, then the resulting catechol moiety was
temporary protected. While the diol withstood the protection as
acetonide, the formation of bis(methoxymethyl) (MOM) ether pro-
ceeded smoothly to give 3b. Prior to the dehydrogenation by the
treatment with I2 [16], all of the acetyl protective groups in 3b
were hydrolyzed to free form 3c. The increase of hydrophilicity [16]
was really advantageous for dehydrogenation, as the attempts for
the dehydrogenation at the stage of acetate 3b only resulted in the
complex mixture. Finally, the remaining MOM protective groups
and glycosidic bonds in 1b were removed simultaneously under
acidic conditions to give 1a (Scheme 1).
References
[1] R. Hirano, W. Sasamoto, A. Matsumoto, H. Itakura, O. Igarashi, K. Kondo, J. Nutr.
Sci. Vitaminol. 47 (2001) 357.
[2] H. Ueda, C. Yamazaki, M. Yamazaki, Biol. Pharm. Bull. 25 (2002) 1197.
[3] G. Seelinger, I. Merfort, C.M. Schempp, Planta Med. 74 (2008) 1667.
[4] Y. Lin, R. Shi, X. Wang, H.-M. Shen, Curr. Cancer Drug Targets 8 (2008) 634.
[5] J.-W. Lee, J.-Y. Ahn, S. Hasegawa, B.-Y. Cha, T. Yonezawa, K. Nagai, H.-J. Seo,
W.-B. Jeon, J.-T. Woo, Cytotechnology 61 (2009) 125.
[6] D. Cristea, I. Bareau, G. Vilarem, Dyes Pigments 57 (2003) 267.
[7] D. Nagarathnam, M. Cushman, J. Org. Chem. 56 (1991) 4884.
[8] B.M. O’Keefe, N. Simmons, S.F. Martin, Tetrahedron 67 (2011) 4344.
[9] T. Miyazawa, M. Hamada, R. Morimoto, T. Murashima, T. Yamada, Tetrahedron
Lett. 48 (2007) 8334.
[10] T. Miyazawa, M. Hamada, R. Morimoto, T. Murashima, T. Yamada, Tetrahedron
Lett. 49 (2008) 175.
[11] K. Kitsuda, J. Calveras, Y. Nagai, T. Higashi, T. Sugai, J. Mol. Catal. B: Enz. 59
(2009) 197.
[12] I. Sultana, M. Shimamoto, R. Obata, S. Nishiyama, T. Sugai, Sci. Technol. Adv.
Mater. 7 (2006) 197.
[13] D. Magdziak, A.A. Rodriguez, R.W. Van De Water, T.R.R. Pettus, Org. Lett. 4 (2002)
285.
In addition to the low price, the choice of naringin, the glycosy-
lated form, as the starting material had another advantage, judging
from the control experiment shown below. Triacetylated form (2e)
of naringenin (2b), the aglycone, was treated under the same trans-
esterification conditions as for 2c. The cleavage of C-4ꢀ acetate was
predominantly occurred, but the cleavage of C-7 was accompanied
[14] A. Kuboki, T. Yamamoto, S. Ohira, Chem. Lett. 32 (2003) 420.
[15] M. Tada, T. Ohkanda, J. Kurabe, Chem. Pharm. Bull. 58 (2010) 27.
[16] J. Liu, L. Chen, S. Cai, Q. Wang, Carbohydr. Res. 357 (2012) 41.