Synthesis of oolongtheanins and their inhibitory activity on micellar cholesterol solubility in vitro

Article abstract of DOI:10.1016/j.bmcl.2015.01.002

The synthesis of oolongtheanins (1a-d) was accomplished from EGC and/or EGCg in three steps. Oolongtheanin-3′-O-gallate (1b) showed more potent inhibitory activity on micellar cholesterol solubility than did EGCg.

Full text of DOI:10.1016/j.bmcl.2015.01.002

Bioorganic & Medicinal Chemistry Letters 25 (2015) 749–752
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of oolongtheanins and their inhibitory activity on micellar
cholesterol solubility in vitro
⇑
Kazuki Ogawa, Sayumi Hirose, Hitomi Yamamoto, Masaya Shimada, Satoshi Nagaoka, Emiko Yanase
Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of oolongtheanins (1a–d) was accomplished from EGC and/or EGCg in three steps. Oolong-
theanin-30-O-gallate (1b) showed more potent inhibitory activity on micellar cholesterol solubility than
did EGCg.
Received 11 November 2014
Revised 25 December 2014
Accepted 5 January 2015
Available online 9 January 2015
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Catechin
Oolongtheanins
Micelle
Cholesterol
Tea (Camellia sinensis) is a globally popular beverage. It is clas-
sified as green tea, oolong tea and black tea according to the fer-
mentation conditions of the tea leaves. Oolong tea has attracted
attention because of its health benefits, such as its anti-obesity
effects.1 Obesity and hyperlipidemia increases the risk of various
diseases, such as heart disease and cerebral arterial disease.2
Oolong tea is traditionally reported to be effective for the preven-
tion of obesity and improvement of lipid metabolism. Recently,
anti-hypocholesterolemic activity has been reported in the green
tea catechins and the polymerized polyphenols of black tea and
oolong tea.3–9 Oolongtheanins (1a–1d; Fig. 1)10 are one of the char-
acteristic polyphenols found in oolong tea and are also predicted to
exert various bioactive effects.11,12 Recently their chemical struc-
tures were revised by Tanaka et al.2 However, detailed studies on
their activity have thus far been limited because the numerous
components found in the extract leads to a complicated mixture
and thus, preparative scale isolation is difficult. A few synthetic
studies towards 1a and 1b have been reported using enzymatic
oxidation.13 The generation of oolongtheanin-30-O-gallate (1b)
was reported as a byproduct during the synthesis of theasinensin
A (2) using CuCl2 as an oxidizing agent.14,15 However, an efficient
synthetic method for oolongtheanins (1a–d) has not been reported.
In a previous study, we proved the mechanism for the formation of
1a and 1b from EGC (3) or EGCg (4).16 In this study, we investigated
effective synthetic methods towards 1a and 1b, which are
homodimers of 3 and 4, and the syntheses of 1c and 1d, which
are heterodimers of 3 and 4.
We also examined the ability of synthetic oolongtheanins to
decrease the micellar solubility of cholesterol using a model
micelle system in vitro.17
First, the synthesis of 1a and 1b, which are homodimers of 3
and 4, was investigated (Scheme 1). Previously, we achieved the
transformation of 4 to 1b in three steps via two intermediates.
Therefore, we now moved on to optimizing each step of the
reaction to inhibit formation of byproducts and to increase the
yields of oolongtheanins as follows: for the first step, a solution
of 4 was treated with CuCl2ꢀ2H2O in 30% aq MeOH under optimal
conditions. The reaction was monitored by HPLC and three new
peaks were observed (data not shown). The chemical structures
corresponding to these peaks were determined by LC/MS and
NMR to be 2, 5b, and the MeOH adduct of 5 (5MeOH), respectively.
The transformation of 5MeOH to 5b was not observed. Hence, the
same reaction was carried out in 30% aq 1,4-dioxane, which would
not form an adduct, and the yield of 5b was improved. The result-
ing solution was subjected to HP20SS column chromatography,
and was eluted with CH3CN after washing with water to remove
CuCl2ꢀ2H2O, providing crude product 5b, which was used directly
in the next reaction. The transformation of 3 to 5a was performed
in the same manner.
Abbreviations: EC, (ꢁ)-epicatechin; EGC, (ꢁ)-epigallocatechin; ECg, (ꢁ)-epicat-
echin-3-O-gallate; EGCg, (ꢁ)-epigallocatechin-3-O-gallate.
Crude 5b was transformed to 6b by a dehydration and rear-
rangement reaction. The reaction was performed under anhydrous
⇑
Corresponding author. Tel./fax: +81 08 58 293 2914.
E-mail address: e-yanase@gifu-u.ac.jp (E. Yanase).
http://dx.doi.org/10.1016/j.bmcl.2015.01.002
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

750
K. Ogawa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 749–752
OH
GO
OH
R1O
2"'
3"
O
OH
OH
HO
HO
HO
OH
OH
O
OH
2"
6"'
O
O
OH
O
H
OH
OH
HO
HO
O
OH
2
O
OH
6'
OG
G =
OR2
OH
3
OH
OH
1
: R , R2 = H, Desgaloyl oolongtheanin
2 : theasinensin A
1a
1b : R1, R2 = G, Oolongtheanin-3'- O-gallate
1c : R1 = H, R2 = G, Oolongtheanin
1d : R1 = G, R2 = H
Figure 1. Structure of tea polyphenols.
OH
OR1
O
OH
O
R3O
OH
OH
O
HO
OH
OH
CuCl2
O
HO
H
30% Dioxane/H2O
Dioxane
1
OH
OH
OR
HO
O
Δ
OH
OR2
1
3
R = H: ( )
OH
R1 = G: (4)
R1 , R2 , R3= H: Dehydrotheaninensin C (5a)
R1 , R2= G, R3= H: Dehydrotheaninensin A (5b)
2
3
R1 , R = G, R = Me: (
)
5
MeOH
R1=H, R2=G, R3= H: (1c)
R1=G, R2=H, R3= H: (1d)
OH
OH
R1O
1
O
OR
OH
HO
O
O
CO2
O
OH
HO
O
O
O
H
H
H2O
HO
OH
HO
O
O
OH
OH
OR2
OH
OR2
OH
OH
R1 , R2 = H: (1a)
R1 , R2 = H: Desgalloyl pro-oolongtheanin(6a)
2
O
6b
R1 , R = G: (
)
2
R1 , R = G: Pro-oolongtheanin-3'- -gallate(
)
1b
R1=H, R2=G: (1c)
R1=G, R2=H: (1d)
R1=H, R2=G: (6c)
R1=G, R2=H: (6d)
Scheme 1. Synthesis of oolongtheanins (1a–d).
conditions for efficient conversion. A solution of 5b in anhydrous
1,4-dioxane was heated at 60 °C to give crude 6b. The same proce-
dure could be used for the conversion of 5a to 6a.
formation mechanism of oolongtheanin involves a nucleophilic
attack of either 3 or 4 on o-quinone, which is formed from another
molecule of 3 or 4.16 Hence, the upper unit of oolongtheanin is
derived from the oxidant of 3 or 4, while the lower unit is derived
from nucleophilic attack of 3 or 4. For the preparation of each
homodimer, the reaction rate from 3 was slightly faster than that
from 4. This result suggests that 3 is more susceptible to oxidation
or is a better nucleophile than 4. Therefore, the selective synthesis
of either one of the heterotype dimers could be possible. We opti-
mized the reaction conditions for the transformation of 3 and 4 to
5a–d. The results are shown in Table 1. The product ratio of the
reaction mixture under each set of conditions were calculated on
the basis of an integrated value of peaks in the 1H NMR spectrum.
The results indicate that the ratio for generation of the analogues of
5 was influenced by the nature of the solvent. When the mixture of
The transformation of 6b to 1b was carried out using water as
the solvent, for hydrolysis of the lactone moiety. The synthesis of
1b was completed in 39% yield from 4. Although the transforma-
tion of 6a to 1a was performed in the same manner, the yield of
1a was significantly lower than 1b due to the generation of an
unidentified byproduct. No byproduct was observed when using
50% aq CH3CN instead of water and the synthesis of 1a was com-
pleted in 40% yield from 3.
Next, the synthesis of 1c and 1d, which are heterodimers of 3
and 4, were investigated. When oxidation of the mixture of 3 and
4 was performed in the same manner, 1a–d were prepared in
almost equal amounts. It is assumed that the initial step for the

K. Ogawa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 749–752
751
Table 1
Oxidative coupling reaction of 3 and 4
OH
OH
HO
O
OH
OH
OR1
O
OH
OH
O
OH
O
HO
HO
3
OH
OH
CuCl2
H
OH
OH
HO
O
HO
O
OH
OH
OR2
O
OH
5a
OH
OH
OH
: R1 = R 2= H
O
5b: R1 = R2 = gallate
5c: R1 = H, R2 = gallate
5d: R1 = gallate, R2 = H
4
OH
Entry
Solvent
Product ratio
5a
5b
5c
5d
1
2
3
4
30% aq dioxane
50% aq dioxane
10% aq dioxane
H2O
1
1
1
2
1
1
1
1
1
1
1
2
1
1
1
1
The product ratio of the reaction mixture was calculated on the basis of the integrals of the peaks in the 1H NMR spectrum.
Table 2
1H NMR (500 MHz) data for oolongtheanins in d6-acetone (ppm)
Position
1a
1b
1c
1d
2
5.16
4.00
4.35
4.38
6.90
6.61
4.58
5.97
5.96
5.83
5.75
5.44
5.44
4.68
5.75
6.88
6.56
4.67
6.06
6.01
5.99
5.89
7.09
7.06
5.42
5.40
4.38
4.42
6.84
6.65
4.67
6.05
5.99
5.98
5.78
7.08
5.19
4.02
4.67
5.77
6.93
6.54
4.55
6.02
6.00
5.88
5.87
7.08
3
200
300
6’
2000
6000
A ring
Galloyl 2,6
Figure 2. Inhibitory activity on micellar cholesterol solubility. Values are expressed
as means SEM (n = 4). Means with different superscript letters are significantly
different (P <0.05) by Tukey’s test.
3 and 4 was treated with CuCl2ꢀ2H2O in ꢂ10–50% aq 1,4-dioxane,
compounds 5a–d were generated in almost equal amounts (entries
1–3). On the other hand, when this treatment was carried out in
water, generation of compounds 5b and 5d was suppressed and
5a and 5c were generated as the major products (entry 4).
good agreement with previous reports which had shown that cat-
echins with a galloyl moiety have a higher inhibitory ability than
free catechins.3,9,12 Intriguingly, although 1a does not contain a
galloyl moiety, a relatively strong inhibitory activity was shown
(26.39 4.10%). These data suggested that not only the presence
of the galloyl moiety, but also the presence of the 5,5,6 ring system
within 1a and 1b contributed to the enhanced inhibitory ability.
In conclusion, we have succeeded in the effective synthesis of
oolongtheanins 1a–d based on their formation via oxidative cou-
pling. Moreover, 1b showed strong inhibitory ability, comparable
to cholestyramine, for the micellar solubility of cholesterol in vitro.
The mixture of 5a–d was treated in the same manner as previ-
ously to afford a mixture of 1a–d (Scheme 1). Compounds 1a–d
were separated by preparative HPLC and their chemical structures
were determined through 1H NMR spectroscopy (Table 2). Com-
pound 5d, which was difficult to obtain from this method, was suc-
cessfully generated from 5b by selective hydrolysis of the gallate
group using the enzymatic reaction of tannase (data not shown).
The micellar solubility of cholesterol with 1a, 1b, 3, 4, and cho-
lestyramine, which is currently used as a medicine, was measured
in vitro by a previously described method (Fig. 2).17 As a result, 1b
showed a strong inhibitory ability on the micellar solubility of cho-
lesterol (1.40 0.14%), which was comparable to the positive con-
trol, cholestyramine (1.53 0.08%). In addition, catechins and
oolongtheanins with a galloyl moiety showed stronger inhibitory
effects than those without the galloyl moiety. This result was in
Acknowledgment
This work was supported by a Grant-in-Aid for Young Scientists
(B) No. 24780123 from Japan Society for the Promotion of Science.

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K. Ogawa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 749–752
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We would like to thank Editage (www.editage.jp) for English lan-
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Supplementary data
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Supplementary data associated with this article can be found, in
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