Nonenzymatic Biomimetic Synthesis of Black Tea Pigment Theaflavins

Article abstract of DOI:10.1055/s-0036-1588529

Theaflavins are reddish-orange black tea pigments with a benzotropolone chromophore, and their various biological activities have been reported. Theaflavins are produced by oxidative coupling between catechol-type and pyrogallol-type catechins via bicyclo[3.2.1]octane-type intermediates. In this study, a new method for nonenzymatic biomimetic synthesis of theaflavins was developed using the DPPH radical as an oxidizing agent.

Full text of DOI:10.1055/s-0036-1588529

SYNLETT
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1
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9
6
© Georg Thieme Verlag Stuttgart · New York
2017, 28, A–D
letter
A
en
Y. Matsuo et al.
Letter
Synlett
Nonenzymatic Biomimetic Synthesis of Black Tea Pigment
Theaflavins
Yosuke Matsuo*
0
0
0
0
0-
0
0
2
6-
4
6
2
9-
7
2
7
OH
OH
Ryosuke Oowatashi
Yoshinori Saito
OH
OH
O
OH
O
OH
epigallocatechin
HO
+ H2O
O
O
Takashi Tanaka*
0
0
0
0
0-
0
0
1
7-
7
6
2
7-
4
3
2
OH
H
OH
OH
r.t.,
15 min
HO
O
r.t.,
15 min
Graduate School of Biomedical Sciences, Nagasaki University,
1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
y-matsuo@nagasaki-u.ac.jp
OH
OH
OH
OH
OH
O
DPPH
t-tanaka@nagasaki-u.ac.jp
OH
acetone
r.t., 1 h
theaflavin
O
epicatechin
o-quinone
Received: 07.06.2017
Accepted after revision: 04.07.2017
Published online: 11.08.2017
tane-type intermediate produced by coupling of a pyrogal-
lol-type B-ring with an o-quinone form of catechol-type B-
ring.9 The bicyclo[3.2.1]octane-type intermediate is stable
in aprotic solvents; however, in aqueous solution, the car-
bonyl group of the intermediate readily reacts with a water
molecule and successive oxidation and decarboxylation
afford the benzotropolone structure (Scheme 1).9
DOI: 10.1055/s-0036-1588529; Art ID: st-2017-u0442-l
Abstract Theaflavins are reddish-orange black tea pigments with a
benzotropolone chromophore, and their various biological activities
have been reported. Theaflavins are produced by oxidative coupling be-
tween catechol-type and pyrogallol-type catechins via bicyclo[3.2.1]oc-
tane-type intermediates. In this study, a new method for nonenzymatic
biomimetic synthesis of theaflavins was developed using the DPPH radi-
cal as an oxidizing agent.
OH
OH
OH
OR1
B
OH
HO
O
A
O
HO
O
Key words theaflavins, quinones, black tea, benzotropolones, bicy-
clo[3.2.1]octane, biomimetic synthesis
R
OH
OH
OH
HO
O
OH
OG
OH
5: R =
6: R =
9: R =
OR2
Plant polyphenols have attracted great attention due to
their various health benefits,1 and black tea is one of the
most important beverages with abundant polyphenols.
Black tea is produced from the leaves of Camellia sinensis,
which contain tea catechins as major polyphenols.2 In the
process of black tea production, tea catechins are oxidized
enzymatically to afford various catechin dimers, such as
theaflavins, theasinensins, and theacitrins, along with high-
molecular-weight products known as thearubigins.3
Theaflavins are reddish-orange pigments with a benzotro-
polone chromophore and are mainly composed of four
compounds, theaflavin (1), theaflavin-3-O-gallate (2),
theaflavin-3′-O-gallate (3), and theaflavin-3,3′-di-O-gallate
(4) (Figure 1).4 Many studies on the biological activities of
theaflavins, such as α-amylase inhibition,5 α-glucosidase
inhibition,6 lipase inhibition,7 and anti-inflammatory activ-
ity,8 have been reported. Theaflavins are produced by oxi-
dative condensation between catechol-type catechins (epi-
catechin (5) and epicatechin-3-O-gallate (6)) and pyrogallol-
type catechins (epigallocatechin (7) and epigallocatechin-3-
O-gallate (8)). Yanase et al. reported that the benzotropo-
lone moiety of theaflavins is formed via a bicyclo[3.2.1]oc-
OH
OH
OH
OH
1: R1 = R2 = H
2: R1 = G, R2 = H
3: R1 = H, R2 = G
4: R1 = R2 = G
HO
O
OR
O
OH
OH
7: R = H
8: R = G
G =
OH
OH
Figure 1 Structures of theaflavins 1–4 and tea catechins 5–9
Up to now, several methods of synthesizing theaflavins
by enzymatic or nonenzymatic oxidation have been report-
ed. Enzymatic methods were performed with polyphenol
oxidase (tyrosinase and laccase),10 peroxidase,11 a plant ho-
mogenate with high polyphenol oxidase activity,12 or C. sin-
ensis cell culture.13 Polyphenol oxidase contained in many
plants has substrate specificity for catechol rather than py-
rogallol,14 which gives a good yield of theaflavins. However,
these enzymatic methods also afford other products, such
as dimers of pyrogallol-type catechins. On the other hand,
there have been a few studies on nonenzymatic synthesis of
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–D

B
Y. Matsuo et al.
Letter
Synlett
OH
OH
H+
O
H+
OH
O
OH
O
O
OH
OH
OH
OH
O
OH
− CO2
oxidation
OH
pyrogallol
OH
OH
HO
H
O
H2O
OH
OH
O
O
OH
oxidation
O
O
OH
bicyclo[3.2.1]octane-type intermediate
OH
benzotropolone
o-quinone
catechol
Scheme 1 Proposed production mechanism of benzotropolone moiety in theaflavins
theaflavins. In the 1960–1970s, the synthesis of theaflavins
was performed by using K3[Fe(CN)6] in water (yield: 5–
19%).4a,d However, pyrogallol-type catechin dimers are also
generated under these conditions, as is the case in the en-
zymatic oxidation.15 Recently, several groups have reported
the biomimetic synthesis of benzotropolone derivatives in-
cluding the formation of the bicyclo[3.2.1]octane-type
structure as a key intermediate in aprotic solvent. Yanase et
al. reported the synthesis of simple benzotropolone deriva-
tives by using the Fetizon reagent (Ag2CO3/Celite) as an oxi-
dant.9,16 Kan et al. reported the synthesis of theaflavins, al-
though their method needs protection and deprotection of
hydroxy groups at the catechin A-ring (total yield of 1 from
7: 5.7%).17 The protection of hydroxy groups at the catechin
A-ring was unavoidable due to an oxidation process using
Pb(OAc)4.
If a catechol-type B-ring could be oxidized to the corre-
sponding o-quinone selectively without protection of A-
ring hydroxy groups, the synthesis of theaflavins will be ac-
complished by a much simpler procedure. In this study, we
developed a new method for nonenzymatic biomimetic
synthesis of theaflavins without a protecting group.
Previously, it was reported that the oxidation of epicate-
chin (5) and catechin (9) with the 2,2-diphenyl-1-picrylhy-
drazyl (DPPH) radical in acetone affords the o-quinones 5a
and 9a, respectively.18,19 Production of the quinones was
confirmed by derivatization with o-phenylenediamine as
well as 13C NMR spectra of the reaction mixture.18 Based on
this, we proposed the following synthetic route for theafla-
vins: Firstly, a catechol-type catechin is oxidized with the
DPPH radical in acetone to prepare its o-quinone without
protecting A-ring hydroxy groups. Secondly, a pyrogallol-
type catechin is added to form the bicyclo[3.2.1]octane in-
termediate. Finally, addition of water to the reaction mix-
ture causes ring cleavage followed by spontaneous oxida-
tion and decarboxylation to afford theaflavins.
The optimized reaction conditions for the synthesis of
theaflavin (1) are as follows: four equivalents of DPPH were
added to an acetone solution of epicatechin (5, 2 equiv) and
stirred for one hour at room temperature to generate the o-
quinone 5a. Then, epigallocatechin (7, 1 equiv) was added
and stirred for 15 min to form a bicyclo[3.2.1]octane-type
intermediate.20 Finally, water was added and stirred for 15
1
min to afford 1 (47% from 7, Scheme 2).21 The H NMR and
13C NMR data of 1 were completely consistent with the lit-
erature data.4c,d,11,22 A small amount of theanaphthoqui-
none (10, 1.6% from 7), an oxidation product of 1, was also
afforded as a byproduct (Figure 2);23 however, dimers of 7
were not obtained. A large excess of the DPPH radical of
over four equivalents increased the production of 10 from
1. Direct addition of the DPPH radical to the mixture of 5
and 7 in acetone did not give 1 and only oxidation of 7 was
OH
OH
O
OH
OH
HO
O
OH
G =
OH
OR
OR1
OH
O
OH
OH
O
O
(1 equiv)
HO
O
O2N
H
OH + H2O
OH
OH
7: R = H
8: R = G
r.t.,
15 min
OH
r.t.,
N
N
NO2
HO
O
15 min
OH
OH
OH
O
O
O2N
OR2
HO
O
bicyclo[3.2.1]octane-type
intermediates
HO
O
DPPH (4 equiv)
acetone, r.t., 1 h
OH
1: R1 = H; R2
2: R1 = G; R2
=
R
OH (47% from 7)
OH (30% from 8)
OH (20% from 7)
=
R
OH
11: R1 = H; R2
=
(2 equiv)
OH
OH
OH
5: R =
9: R =
OH
OH
5a: R =
9a: R =
Scheme 2 Nonenzymatic biomimetic method for the synthesis of theaflavins
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–D

C
Y. Matsuo et al.
Letter
Synlett
observed. This is because the redox potential of pyrogallol-
type catechins is much lower than that of catechol-type cat-
echins.24 Using the same method, theaflavin-3-O-gallate (2)
was synthesized from 5 and epigallocatechin-3-O-gallate
(8) (30% from 8). However, theaflavin-3′-O-gallate (3) and
theaflavin-3,3′-di-O-gallate (4) could not be synthesized
from epicatechin-3-O-gallate (6) with 7 or 8. These results
suggest that the presence of the galloyl group in pyrogallol-
type catechins does not affect the production of benzotro-
polone. On the other hand, the presence of the galloyl group
in catechol-type catechins prevents the generation of the o-
quinone of the catechol ring. It is considered that the galloyl
group is oxidized by the DPPH radical instead of the cate-
chol ring. Neotheaflavin (11), a minor pigment contained in
black tea,4d was also able to be synthesized from (+)-cate-
chin (9) and epigallocatechin (7) in the same way (20% from
7). By using this method, other benzotropolone derivatives
could be synthesized from catechol and pyrogallol deriva-
tives. From epicatechin (5) and myricitrin (12), a non-natu-
ral benzotropolone derivative 13 was synthesized by the
same method (12% from 12) (Figure 2).
Funding Information
This work was supported by JSPS KAKENHI Grant Numbers
JP16K07741 and JP17K08338
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Acknowledgment
The authors are grateful to Mr. K. Inada and Mr. N. Tsuda (Center for
Industry, University and Government Cooperation, Nagasaki Univer-
sity) for collecting NMR and MS data. The authors would like to thank
Enago (www.enago.jp) for the English language review.
Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/s-0036-1588529.
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References and Notes
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OH
OH
OH
OH
OH
HO
O
O
HO
O
OH
O
O
O
HO
O
OH
HO
O
OH
HO
OH
OH
10
12
nol Research;5Vo.
l
Yoshida, K.; Cheynier, V.; Quideau, S., Eds.; Wiley-
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13
Figure 2 Structures of theanaphthoquinone (10), myricitrin (12), and 13
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In summary, we have developed a new method for non-
enzymatic biomimetic synthesis of black tea pigment
theaflavins without a protecting group. Benzotropolone de-
rivatives are rare in nature,25 and several compounds have
been reported, such as purpurogallin glycosides,26 fomenta-
riol,27 aurantricholone,28 crocipodin,29 and goupiolone A.30
Our method is considered to be applicable to the synthesis
of these benzotropolone derivatives. In addition, there are
many catechol and pyrogallol derivatives as natural prod-
ucts;1 therefore, various non-natural benzotropolone deriv-
atives could be synthesized by applying this method.
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–D

D
Y. Matsuo et al.
Letter
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Catal. B: Enzym. 2017, DOI: 10.1016/j.molcatb.2017.03.009.
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ated Beverages, Health Benefits, Physiological Effects, and Chemis-
column of MCI-gel CHP20P (3.0 × 24 cm; 0–80% aq MeOH, 10%
stepwise, each 200 mL) to afford a crude theaflavin (1) fraction
along with 5 (154 mg, 77%). The crude fraction of 1 was purified
with Sephadex LH-20 (3.0 × 20 cm; 40–100% aq MeOH, 10%
stepwise, each 200 mL) to afford 14c,d,11,22 (91.3 mg, 0.16 mmol,
47% from 7) and theanaphthoquinone (10)23 (2.9 mg, 0.0054
mmol, 1.6% from 7).
Analytical Data for Theaflavin (1)
Reddish-orange amorphous powder, [α]D –234 (c 0.105,
25
MeOH). FAB-MS: m/z = 565 [M + H]+, 587 [M + Na]+. HRMS–FAB:
m/z calcd for C29H25O12: 565.1346; found: 565.1340 [M + H]+. IR:
3406, 2935, 1627, 1604, 1518, 1470, 1419, 1310, 1227 cm–1. UV
(MeOH): λmax (log ε) = 462 (3.49), 378 (3.90), 267 (4.27). 1H NMR
(400 MHz, acetone-d6 + D2O, 95:5): δ = 8.00 (s, H-g), 7.93 (s, H-
e), 7.53 (s, H-c), 6.04 (d, J = 2.2 Hz, H-6′), 6.02 (d, J = 2.4 Hz, H-6),
5.99 (d, J = 2.2 Hz, H-8′), 5.95 (d, J = 2.4 Hz, H-8), 5.69 (s, H-2′),
4.98 (s, H-2), 4.45 (m, H-3′), 4.36 (m, H-3), 2.95 (dd, J = 16.8, 4.4
Hz, H-4′a), 2.89 (dd, J = 16.8, 4.4 Hz, H-4a), 2.80 (dd, J = 16.8, 2.3
Hz, H-4b), 2.77 (br d, J = 16.8 Hz, H-4′b). 13C NMR (100 MHz,
acetone-d6 + D2O, 95:5): δ = 185.1 (C-a), 157.8, 157.7, 157.6,
157.5 (C-5, 7, 5′, 7′), 157.0 (C-8′a), 156.5 (C-8a), 154.6 (C-b),
150.4 (C-i), 146.1 (C-h), 135.0 (C-d), 131.7 (C-f), 128.6 (C-k),
126.9 (C-e), 123.9 (C-g), 121.7 (C-j), 118.9 (C-c), 99.8 (C-4′a),
99.3 (C-4a), 96.5 (2 C, C-6, 6′), 95.6 (C-8′), 95.3 (C-8), 81.2 (C-2),
76.6 (C-2′), 66.2 (C-3), 65.0 (C-3′), 29.5 (C-4′), 29.2 (C-4).
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7
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l
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clo[3.2.1]octane intermediate by converting it into a stable
derivative. The details will be reported in the near future.
(21) Experimental Procedure for Synthesis of Theaflavin (1)
DPPH (543 mg, 1.4 mmol) was added to an acetone solution
(100 mL) of epicatechin (5, 200 mg, 0.69 mmol). After stirring
for 1 h at r.t., an acetone solution (50 mL) of epigallocatechin (7,
106 mg, 0.35 mmol) was added to the reaction mixture and
stirred for 15 min at r.t. Then, water (150 mL) was added to the
reaction mixture and stirred for 15 min. The reaction mixture
was concentrated in vacuo, and the residue was applied to a
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