Efficient Synthesis of Theaflavin 3-Gallate by a Tyrosinase-Catalyzed Reaction with (-)-Epicatechin and (-)-Epigallocatechin Gallate in a 1-Octanol/Buffer Biphasic System

Article abstract of DOI:10.1021/acs.jafc.8b05971

Theaflavins, the orange-red pigments contained in black tea, have attracted attention as a result of their health-promoting effects. However, their synthetic preparation, in which the enzymatic oxidation of catechol-type catechin is followed by the quinon

Full text of DOI:10.1021/acs.jafc.8b05971

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Biotechnology and Biological Transformations
Efficient synthesis of theaflavin-3-gallate by a tyrosinase-
catalyzed reaction with (-)-epicatechin and (-)-
epigallocatechin gallate in a 1-octanol/buffer biphasic system
Asako Narai-Kanayama, Yoshinori Uekusa, Fumiyuki Kiuchi, and Tsutomu Nakayama
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05971 • Publication Date (Web): 28 Nov 2018
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Journal of Agricultural and Food Chemistry
1 Efficient Synthesis of Theaflavin 3-gallate by a Tyrosinase-
2 Catalyzed Reaction with (-)-Epicatechin and (-)-Epigallocatechin
3 Gallate in a 1-Octanol/Buffer Biphasic System
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Asako Narai-Kanayama*,†, Yoshinori Uekusa‡, Fumiyuki Kiuchi‡, Tsutomu
Nakayama†,§
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† School of Veterinary Medicine and Life Science, Nippon Veterinary and Life Science
University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180-8602, Japan
‡ Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512,
Japan
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§ Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka,
Setagaya-ku, Tokyo 156-8502, Japan
*Corresponding author. Tel: +81-422-31-4151; Fax: +81-422-51-9984;
E-mail: a-narai@nvlu.ac.jp;
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Abstract
Theaflavins, the orange-red pigments contained in black tea, have attracted attention
due to their health-promoting effects. However, their synthetic preparation, in which the
enzymatic oxidation of catechol-type catechin is followed by the quinone-induced oxidative
dimerization of selectively combined catechol- and pyrogallol-type catechins, provides only a
low yield. In the present study, we found that a 1-octanol/buffer biphasic system improved the
yield of theaflavin 3-gallate in a tyrosinase-catalyzed synthetic reaction with (-)-epicatechin
and (-)-epigallocatechin gallate. When the enzymatic reaction proceeded in a buffer solution,
oxidized (-)-epigallocatechin gallate was preferentially used for self-dimerization. However,
self-dimerization was suppressed in the octanol phase, allowing oxidized (-)-epigallocatechin
gallate to participate in coupling with (-)-epicatechin quinone leading to effective production
of theaflavin 3-gallate. Furthermore, the preferential localization of theaflavin 3-gallate in the
octanol phase prevented (-)-epicatechin quinone-induced degradation.
Key words
theaflavin; tyrosinase; enzymatic synthesis; biphasic system; 1-octanol
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Introduction
Theaflavins, the characteristic orange or orange-red pigments in black tea leaves and their
exudates, are produced during the fermentation of Camellia sinensis leaves, in which
endogenous polyphenol oxidase (PPO) and peroxidase (POD) are involved.1, 2 Condensation
of different pairs of oxidized catechins, one with a dihydroxylated B-ring (catechol-type) and
the other with a trihydroxylated B-ring (pyrogallol-type), followed by decarboxylation affords
four kinds of theaflavins, theaflavin, 1, theaflavin 3-O-gallate, 2, theaflavin 3'-O-gallate, 3,
and theaflavin 3,3'-O-digallate, 4 (Figure 1).3, 4
Theaflavins contribute to the astringency of various brewed teas and have various
bioactivities, such as antioxidant activity,5, 6 anticancer activity,7, 8 antidiabetic effects,9 and
inhibitory activity against bone loss in models of osteoporosis.10 According to some in vitro
and in vivo studies, 2 specifically decreases the micellar solubility of cholesterol,11, 12
probably being responsible for the lowering of blood LDL cholesterol by the intake of black
tea.13, 14 In addition, a single oral administration of theaflavins elevates cremasteric blood flow
in rats, and the magnitude of this effect is in the order of 3 >> 2 >> 1 = 4.15 To elucidate their
structure-activity relationships and the mechanisms of their bioactivities, sufficient amounts
of theaflavins are required. However, the study and application of theaflavins have been
hampered by their fairly low amounts in black tea16 and their instability in aqueous solutions,
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especially under neutral or alkaline conditions.17-19
Researchers have investigated the mechanism of theaflavin synthesis using plant
homogenates,4 PPO purified from tea leaves,1, 20, 21 or tyrosinase (EC 1.14.18.1) from the
mushroom Agaricus bisporus, a copper-containing oxidase with both monophenol
monooxygenase and diphenol oxidase activities, as a commercially available PPO model.22
Through the prospective mechanism of PPO-catalyzed theaflavin synthesis (Figure 1), the
enzymatic and non-enzymatic reactions proceed concurrently: 1) catechol-type catechins are
enzymatically oxidized to the quinone form, 2) pyrogallol-type catechins are oxidized either
by enzymatic oxidation or a coupling redox reaction with catechol quinones, and 3)
theaflavins are produced by the condensation of a pair of catechol- and pyrogallol-type
quinones and subsequent decarboxylation. There is another explanation in which the
benzotropolone moiety in theaflavins is produced by direct coupling between catechin
quinone and pyrogallol.23, 24 This hypothesis concerning the first step is based on organic
reactions in aprotic media. The higher electron-donating property of the pyrogallol group25, 26
and our previous kinetic analyses of tyrosinase-catalyzed reactions with catechins in aqueous
media (i.e., catechol-type catechins decrease very slowly in spite of their higher constant
efficiency (kcat/Km) for tyrosinase until almost all of the combined pyrogallol-type ones are
consumed)22 strongly suggest that coupling redox reactions between catechol quinones and
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pyrogallol occur predominantly under aqueous conditions essential for enzymes. The sub-
products interfere with the synthesis of theaflavins; in particular, highly reactive quinones
attack theaflavins leading to oxidative degradation.4, 20, 22 Pyrogallol quinones are known to
cause self-dimerization,27-30 restricting the efficiency of the condensation critical for the
production of theaflavins as described above.22 In our previous study, we found that the
synthetic yields of galloylated theaflavins, such as 2, 3, and 4, by the tyrosinase-catalyzed
reaction are significantly lower compared with that of 1.22
Considering the differences in the partition coefficients of catechins,31 we conceived a 1-
octanol/buffer biphasic system that would allow for mild separation of hydrophobic (-)-
epigallocatechin gallate, 8, from the aqueous buffer phase, where tyrosinase preferentially
oxidizes (-)-epicatechin (EC), 5, a relatively hydrophilic catechol-type catechin (Figure 2).22
A coupling redox reaction involving EC quinone and 8 then rapidly occurs, and the
condensation of EC- and EGCg quinones synthesize 2. The partition of hydrophobic 2 into
the 1-octanol phase may prevent EC quinone-mediated oxidative degradation of 2. In this
study, we investigated the effects of a 1-octanol/buffer biphasic system on the tyrosinase-
catalyzed synthesis of 2 and found that this system effectively increased the synthetic yield of
2.
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Materials and methods
Materials
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(-)-Epicatechin (EC), 5, (-)-epicatechin gallate (ECg), 6, (-)-epigallocatechin (EGC), 7,
and (-)-epigallocatechin gallate (EGCg), 8, were kindly provided by Mitsui Norin Co., Ltd.
(Shizuoka, Japan). Tyrosinase from A. bisporus was obtained from Sigma-Aldrich Co. (St.
Louis, MO). 1-Octanol was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). o-
Phenylenediamine was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
All other reagents used were of analytical grade.
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Measurement of partition coefficients of catechins and theaflavins
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The respective four catechins were dissolved in pure water at 1 mM. An aliquot (200 L)
of aqueous catechin or theaflavin solution was mixed with an equal volume of 1-octanol.
After the mixture was vigorously shaken, it was centrifuged at approx. 2046  g for 3 min at
room temperature (25 C). Polyphenols in both the upper layer (1-octanol phase) and the
lower one (aqueous buffer phase) were analyzed by RP-HPLC. RP-HPLC was carried out as
described previously.22 Briefly, 30 L of sample were injected to a 150 mm × 4.6 mm i.d., 4
m, Synergi Polar-RP 80Å column (Phenomenex, Torrance, CA). Catechins and theaflavins
were eluted with aqueous 20% and 32% MeCN solutions containing 0.05% phosphoric acid at
flow rate of 1 mL/min, respectively, and were detected at 280 nm. Chromatogram analysis
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was performed with the data processing software Chromato-PRO (Run Time Corp., Tokyo,
Japan). Partition coefficients (log Pow values) were determined as follows:
log Pow = log10 [polyphenol in 1-octanol phase]/[polyphenol in aqueous phase]
Tyrosinase-catalyzed synthesis of 2 in the absence or presence of 1-octanol
A series of reaction mixtures containing 5, 8, and tyrosinase were constructed as shown
in Table 1. Final concentrations of respective catechins and tyrosinase were 2.5 mM and
0.025 mg/mL in 50 mM sodium phosphate buffer, pH 6.0. The enzymatic reaction was
conducted at 25 °C. At intervals of 15 min for up to 60 min, the reaction mixture was shaken
at 2800 rpm/min for 1 min with a vortex mixer, centrifuged at approximately 2046  g for 3
min, and then each aliquot (30 L) of the 1-octanol and buffer fractions was taken and added
into 180 L of the stop solution (a mixture of equal volumes of 25 mM citric acid and
ethanol). The stopped samples were analyzed by RP-HPLC using a 150 mm × 4.6 mm i.d., 4
m, Synergi Polar-RP 80Å column (Phenomenex, Torrance, CA). The mobile phase consisted
of 0.05% phosphoric acid in water (A) and 0.05% phosphoric acid in 80% MeCN (B).
Polyphenols were eluted by gradient elution according to the following program: 0-10-30
min/20-25-50% B at flow rate of 1 mL/min. They were detected at 280 nm to identify the
peaks of catechins and theaflavins.
Analysis of phenazine derivatives
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Each aliquot (30 L) of the 1-octanol and buffer fractions was taken and added into 180
L of 0.5 mg/mL o-phenylenediamine in 0.5% acetic acid/ethanol.29 Phenazine derivatives
were analyzed by RP-HPLC as described above. Some peak compounds were isolated by RP-
HPLC with similar eluting conditions, except that 0.2% acetic acid was substituted for 0.05%
phosphoric acid. They were concentrated by a centrifugal evaporator CC-105 (Tomy Seiko,
Tokyo, Japan) for mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses.
High resolution electrospray ionization time-of-flight (HR-ESI-TOF) MS spectra were
measured with a JMS-T100LP AccuTOF LC-plus (JEOL, Tokyo, Japan) operated in positive
ion mode. Monoisotopic masses were calculated by a Molecular Mass Calculator.32 MS
spectra of P1 and P2 showed [M+Na]+ peaks at m/z 1007.1744 and m/z 1007.1738,
respectively. Thus, both molecular formulae of P1 and P2 were identical, corresponding to
C50H36N2O20 (calcd for C50H36N2NaO20, 1007.1759). NMR spectra were recorded on an ECP-
600 FT-NMR (JEOL, Tokyo, Japan) spectrometer operating at 1H and 13C resonance
frequencies of 600.18 MHz and 150.93 MHz, respectively. A field-gradient system was used
for 2D-NMR measurements. Chemical shift values were referenced with respect to
tetramethylsilane (TMS) at 0 ppm.
Results and Discussion
Efficient synthesis of 2 by a tyrosinase-catalyzed reaction with 5 and 8 in the presence of
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1-octanol
The log Pow values of four catechins (Table 2) indicated that the order of their
hydrophobicity is 6>8>5>7. While 5 was partitioned almost evenly into both 1-octanol and
water fractions, the concentrations of 6 and 8 in 1-octanol were about 40- and 10-fold of those
in the water phase, respectively. The log Pow values of theaflavins (Table 2) also indicated that
galloylated theaflavins are more hydrophobic in the order of 4>3>2>1. As expected, 2 was
preferentially partitioned into the 1-octanol phase. After a small volume (10 L) of tyrosinase
solution (1 mg/mL in 50 mM sodium phosphate buffer, pH 6.0) was added into the 1-octanol
phase (400 L) containing 2 mM of 5 or 8, these two catechins hardly decreased (data not
shown), suggesting that the enzyme was denatured and inactivated by 1-octanol. Therefore,
we examined the tyrosinase-catalyzed reaction with 5 and 8 in the 1-octanol/buffer biphasic
system.
In a simple buffer solution, tyrosinase oxidized 5 (Figure 3A) and 8 individually (Figure
3B). As previously reported,22 the tyrosinase-catalyzed reaction with a mixture of 5 and 8
produced 2; however, the yield of 2 was very low: 0.20 mM of 2 at 60 min corresponding to
32% and 9% based on consumed 5 and 8, respectively (Figure 3C). On the other hand, in the
presence of 1-octanol, tyrosinase decreased 5 by itself in both 1-octanol and buffer phases,
keeping its distribution pattern between the two phases (Figure 4A). The decrease of 5
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accompanied an orange colorization of the buffer phase (data not shown). This is probably
due to oxidative polymerization of 5 and hydrophilicity of the resulting products. Although
the generated EC quinone is considered to be more hydrophobic than 5, it may be so highly
reactive as to induce the polymerization of 5 before its distribution into the 1-octanol phase.
In the case of the tyrosinase-catalyzed reaction with 8 by itself, a slightly pale-yellow
colorization in the buffer phase and a moderate decrease of 8 in the 1-octanol phase were
observed (Figure 4B). The latter might be due to compensation for the enzymatically
consumed 8 in the buffer phase, because the enzymatic oxidation of 8 did not occur in 1-
octanol as described above. The tyrosinase-catalyzed reaction with a mixture of 5 and 8 in the
1-octanol/buffer biphasic system showed efficient synthesis of 2 with a higher yield in the 1-
octanol fraction: 0.46 mM of 2 at 60 min corresponding to 91% and 34% based on consumed
5 and 8, respectively (Figure 4C). Preferential localization of 2 into the 1-octanol phase
(Figure 4C and D) was visually confirmed. Although it is still unclear in which of the two
phases the redox reaction between EC quinone and 8 occurs, the resulting product, EGCg
quinone, might be effectively utilized for synthesis of 2 through condensation with EC
quinone. The increase of 2 as well as the consumption of catechins reached a plateau after a
45-min reaction (Figure 4C and 4D) despite 5 remaining in both phases at higher
concentrations than with treatment of 5 alone (Figure 4A). A higher concentration of
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tyrosinase produced more 2: 0.70 mM of 2 at 60 min with 88% and 47% yields based on
consumed 5 and 8, respectively (Figure 5A), though the reaction rate also decreased after 45
min (Figure 5). These results imply that enzyme inactivation during the reaction restricts the
yield of 2. Thus, in order to improve such a biphasic reaction system, further examinations are
necessary into the adequate stirring conditions and technologies for enzyme immobilization
required to prevent the enzyme from being denatured by 1-octanol.
Suppression of self-dimerization of EGCg quinone in the 1-octanol/buffer biphasic
system
To investigate effects of the 1-octanol/buffer biphasic system on quinone production and
oxidative self-dimerization of 8,28, 29 we compared the formation of phenazine derivatives
between the reaction mixtures in the absence and presence of 1-octanol. In RP-HPLC
chromatograms of samples in the absence of 1-octanol, compared with the slight peaks
detected in the reaction of 5 alone with retention times of 9.4 min and 19.4 min (Figure 6A),
two more distinguishable peaks, P1 and P2, possibly corresponding to phenazine derivatives
were detected in both the individual 8 reaction (Figure 6B) and the mixture of 5 and 8 (Figure
6C). Although 2, P1, and P2 were also detected in the 1-octanol phase (Figure 7), the peak
area of 2 was remarkably larger than those of P1 and P2, the latter being smaller than those
from samples without 1-octanol (Figure 6C). The HR-ESI-TOF MS spectra of P1 and P2
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showed [M+Na]+ peaks at m/z 1007.1744 and m/z 1007.1738, respectively. These data
indicated that both molecular formulae of P1 and P2 were identical, corresponding to
C50H36N2O20. The 1H NMR spectrum of compound P2 showed signals for eight protons
derived from two C-rings (H 2.34–5.59), two pairs of meta coupled benzene protons (H 5.90
(d, J = 2.2 Hz), and H 5.95 (d, J = 2.2 Hz), H 6.08 (d, J = 2.2 Hz), and H 6.12 (d, J = 2.2
Hz)), two singlet benzene protons (H 7.12 and H 8.22, each 1H), two galloyl protons (H
6.98 and H 7.09, each 2H), and protons of the phenazine moiety (H 7.91–8.26) in the 1H
NMR spectrum (Table 3). In the 13C NMR spectrum, 45 signals including two signals for 2
carbons and one signal for 4 carbons were observed as shown in Table 3. These spectra were
analyzed by the aid of COSY, HMQC, and HMBC experiments, and the signals were
assigned. Based on the chemical and spectroscopic data, P2 was determined to be the
phenazine derivative as shown in Figure 8, which is identical to a compound prepared by
treatment of an ethanol extract of fermented tea leaves with o-phenylenediamine.27
Comparison of the 13C NMR data of P2 with those reported confirmed the identity.27
Similarly, the 1H NMR spectrum of P1 revealed two C-rings (H 1.93–3.02), two pairs of
doublets (H 5.88 (d, J = 2.2 Hz) and H 5.92 (d, J = 2.6 Hz), H 6.05 (d, J = 2.2 Hz), and H
6.06 (d, J = 2.2 Hz)), two galloyl moieties (H 7.11 and H 7.12, each 2H), and a phenazine
moiety (H 7.89–8.22). In the 1H NMR of P1, one singlet benzene signal was observed at H
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7.15 as the corresponding signal (H 7.12) of P2. However, another singlet benzene signal
was significantly shifted to a higher magnetic field (H 7.78) in comparison with the signal
(H 8.22) of P2 (Table 3). These observations indicated that P1 is a structural isomer of P2 in
which the phenazine moiety is bound to a different position of the B-ring of the original
EGCg quinone dimer (Figure 8). Compounds P1 and P2 should be produced from quinone
equivalents (9a and 9b) of dehydrotheasinensin A,28, 29 respectively (Figure 8). These results
indicate that oxidative dimerization of EGCg quinone rapidly occurs in an aqueous buffer
solution, but not in the 1-octanol phase. This is one of the reasons for differences in yield of 2
between reaction systems in the absence and presence of 1-octanol. In the present study, the
phenazine derivative of EGCg quinone could not be detected; however, it is so hydrophobic
compared to 8 as to be preferentially distributed into the 1-octanol phase. Such a water-
immiscible solvent might lower the polarization of 8 and EGCg quinones, inhibiting the
Michael addition reaction required for their dimerization. On the other hand, catechol
quinones in aprotic solvents are known to be relatively stable but not completely stop the
quinone-induced oxidation.23, 24 Therefore, this suggests that the highly reactive EC quinone
enables its condensation with EGCg quinone either in the buffer or 1-octanol phase, resulting
in synthesis of 2.
Preventive effect of the 1-octanol/buffer biphasic system against EC quinone-induced
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oxidative degradation of 2
To investigate whether the preferential partition of 2 into the 1-octanol phase prevents its
oxidative degradation caused by the attack of EC quinone, tyrosinase was added into the
mixture of 2 and 5 in the absence or presence of 1-octanol. As a result, both 2 and 5 more
rapidly diminished in the absence of 1-octanol than in the 1-octanol/buffer biphasic system
(Figure 9), whereas without tyrosinase these two polyphenols were stable under the
experimental conditions used (data not shown). It is known that tyrosinase does not directly
use 2 as a substrate.22 Therefore, it is clear that EC quinone, the product of enzymatic
oxidation of 5, is involved in the degradation of 2.4, 22 Considering that the decreases of 2 and
5 were not completely inhibited even in the 1-octanol phase (Figure 9), the degraded 2 in the
buffer phase might have been compensated for by 2 from the other phase to keep its partition
equilibrium. Although it is unclear why degradation of 2 was not noticeable in the 1-
octanol/buffer biphasic system containing 8, the latter may be more susceptible to EC
quinone-induced oxidation than 2.
Using a 1-octanol/buffer biphasic system is a promising approach to synthesize 2 in good
yield by the tyrosinase-catalyzed reaction with 5 and 8. Water-immiscible solvent-water
biphasic systems are attractive for other enzymatic condensation reactions, giving products
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with no dissociating groups.33-38 The expected effects of organic solvents on product yields
are dependent on the partition coefficients of substrates and products. Furthermore, these
solvents can play a role in limiting the reverse hydrolysis of products by lowering water
activity.36-38 The present study for the first time elucidated the utility of a 1-octanol/buffer
biphasic system for the tyrosinase-catalyzed synthesis of 2, suggesting that the solvent
restricts undesirable non-enzymatic reactions and enhances product yield. On the other hand,
this system is not applicable to synthesis of the other theaflavins, because the distribution of
combined 5 and 7 into the aqueous phase is not effective for 1, and the excessive localization
of 6 into the 1-octanol phase prevents it from enzymatic oxidation, leading to very slow
synthesis of 3 and 4 (data not shown). Therefore, further study is needed to find other
methods for enzymatic syntheses of 1, 3, and 4. Screening other highly volatile solvents that
not only have similar effects of 1-octanol on the synthesis of 2, but are also available in the
food industry is the next step for applying synthesized theaflavins into health foods,
pharmaceuticals, and to study their various bioactivities.
Abbreviations used
EC: (-)-epicatechin; EGC: (-)-epigallocatechin; ECg: (-)-epicatechin gallate; EGCg: (-)-
epigallocatechin gallate; log Pow: 1-octanol–water partition coefficient
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Acknowledgement
This work was supported by JSPS KAKENHI Grant Number JP17K07823 and JP18K05525,
and a Grant from the Cross-Ministerial Strategic Innovation Promotion Program (SIP), Urgent
Project for Development and Diffusion of Innovative Technology towards Realization of the
Aggressive Agriculture, Forestry, and Fisheries.
Supporting Information
The HR-ESI-TOF MS spectra of P1 and P2 (Figure S1). This material is available free of
charge via the Internet at http://pubs.acs.org.
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3) Tanaka T.; Inoue K.; Betsumiya Y.; Mine C.; Kouno I. Two types of oxidative
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Figure captions
Figure 1 Mechanism of tyrosinase-catalyzed synthesis of theaflavins (1-4) from catechins (5-
8) in aqueous conditions. R1 and R2 are H or a galloyl group (G).
Figure 2 Expected reactions in tyrosinase-catalyzed synthesis of 2 in the 1-octanol/buffer
biphasic system. Additional Q means the quinone form of catechins.
Figure 3 Tyrosinase-catalyzed reactions with (A) 5 only; (B) 8 only; and (C) a mixture of 5 and
8 in buffer solution without 1-octanol. The concentrations of 2 (□), 5 (○), and 8 (△) are
indicated. Data are means ± SD (n=3).
Figure 4 Tyrosinase-catalyzed reactions with (A) single 5 only; (B) single 8 only; and (C) 1-
octanol; (D) buffer phase of a mixture of 5 and 8; in a 1-octanol/buffer biphasic system
containing tyrosinase at 0.025 mg/mL. For 2 (□, ■), 5 (○, ●) and 8 (△, ▲), closed and
open symbols indicate the concentrations in 1-octanol and buffer phases, respectively. Data are
means ± SD (n=3).
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Figure 5 Tyrosinase-catalyzed reactions with a mixture of 5 and 8; (A) 1-octanol; (B) buffer
phase; in a 1-octanol/buffer biphasic system containing tyrosinase at 0.05 mg/mL. For 2 (□,
■), 5 ( ○, ●) and 8 ( △, ▲), closed and open symbols indicate the concentrations in 1-
octanol and buffer phases, respectively. Data are means ± SD (n=3).
Figure 6 HPLC chromatograms of o-phenylenediamine-treated samples after an enzymatic
reaction for 30 min in buffer solution without 1-octanol. Each aliquot of (A) 5 only; (B) 8 only;
and (C) a mixture of 5 and 8 was treated with o-phenylenediamine and analyzed (signal output
= 1.0 AU/1000 mV).
Figure 7 HPLC chromatograms of o-phenylenediamine-treated samples. After an enzymatic
reaction for 30 min in the 1-octanol/buffer biphasic system, each aliquot of 1-octanol phases of
(A) 5 only; (B) 8 only; and (C) a mixture of 5 and 8 was treated with o-phenylenediamine and
analyzed (signal output = 1.0 AU/1000 mV).
Figure 8 Derivatization scheme of EGCg quinone dimers by o-phenylenediamine,27,28 and
chemical structures of P1 and P2.
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Figure 9 Tyrosinase-catalyzed reactions with a mixture of 2 and 5 in the absence (dotted lines)
or presence (solid lines) of 1-octanol containing tyrosinase at 0.025 mg/mL. For 5 (○ and ●
in A) and 2 (□ and ■ in B), closed and open symbols indicate the concentrations in 1-octanol
and buffer phases, respectively. Data are means ± SD (n=3).
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Table 1 Solution Composition (mL) for Reaction Systems.
5 only
8 only
Mixture of
5 and 8
1-Octanol (–)
(+)
0.2
-
(–)
(+)
-
(–)
0.2
0.2
-
(+)
0.2
0.2
-
5 mM of 5 solutiona
5 mM of 8 solutiona
0.2
-
-
0.2
0.2
-
0.2
0.2
0.4
50 mM phosphate buffer, pH 6.0 0.2
0.2
0.4
1-Octanol
-
-
0.4
1 mg/mL Tyrosinaseb
0.01 0.01 0.01 0.01 0.01 0.01
a) Catechins are dissolved in 50 mM sodium phosphate buffer, pH 6.0.
b) Final concentration of tyrosinase in buffer is 0.025 mg/mL.
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Table 2 log Pow Values of Catechins and Theaflavins.
Catechins
log Pow
Theaflavins
log Pow
5
6
7
8
0.15 ± 0.03
1.66 ± 0.04
—0.43 ± 0.02
1.04 ± 0.05
1
2
3
4
0.09 ± 0.01
0.90 ± 0.01
1.31 ± 0.05
2.29 ± 0.09
Initial concentrations of catechins and theaflavins in water were 1 mM and 0.5
mM, respectively. Data are means ± SD (n=3).
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Table 3. NMR Spectroscopic Data of P1 and P2 in Acetone-d6
P2
P1
1H
1H
13C
position
2
3
5.30 (1H, s)
5.22 (1H, s)
5.59 (1H, br d, J = 4.0 Hz)
76.6
67.8
5.73 (1H, s, J = 4.0 Hz)
2.69 (1H, dd, J = 4.4 Hz, 17.6 Hz)
3.02 (1H, d, J = 17.6 Hz)
2.63 (1H, dd, J = 4.6 Hz, 17.4 Hz)
3.04 (1H, d, J = 17.2 Hz)
4
27.1
157.3 b)
96.1
5
6
6.06 (1H, d, J = 2.2 Hz)
6.05 (1H, d, J = 2.2 Hz)
6.12 (1H, s, J = 2.2 Hz)
6.08 (1H, s, J = 2.2 Hz)
7
157.9
95.9
8
4a
8a
1'
2'
3'
4'
5'
6'
2''
3''
98.6
157.1
143.2
116.5
144.3 c)
135.7
152.1
119.5
76.4
7.78 (1H, s)
8.22 (1H, d, J = 0.8 Hz)
4.71 (1H. s)
4.79 (1H, s)
5.33 (1H, br d, J = 2.6 Hz)
1.93 (1H, dd, J = 4.9 Hz, 17.6 Hz)
2.45 (1H, d, J = 17.2 Hz)
5.44 (1H, ddd, J = 0.9 Hz, 1.8 Hz, 4.4 Hz)
2.34 (1H, dd, J = 4.4 Hz, 17.6 Hz)
2.77 (1H, d, J = 17.2 Hz)
68.6
4''
27.1
157.5 b)
96.9
5''
6''
5.92 (1H, d, J = 2.6 Hz)
5.88 (1H, d, J = 2.2 Hz)
5.95 (1H, d, J = 2.2 Hz)
5.90 (1H, d, J = 2.2 Hz)
7''
157.6
96.4
8''
4a''
98.7
8a''
157.3
112.4
128.5
1'''
2'''
144.0 c)
3'''
4'''
134.0
5'''
146.5
6'''
7.15 (1H, s)
7.12 (1H, s)
6.98 (2H, s)
108.5
1''''
121.4
7.12 (2H, s) a)
2'''', 6''''
3'''', 5''''
4''''
109.8
145.9
138.9
CO
1'''''
166.4
122.0
7.11 (2H, s) a)
2''''', 6'''''
3''''', 5'''''
4'''''
7.09 (2H, s)
110.0
145.9
138.7
CO'
166.1
7.89–7.95 (2H, m)
8.18–8.22 (2H, m)
7.91–7.94 (2H, m)
8.17–8.20 (1H, m)
8.23–8.26 (1H. m)
131.4, 131.8
130.3
phenazine
130.2
142.3, 144.9
a–c) Assignments may be interchanged
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Catechins
Theaflavins
1
2
R1
H
G
R2
H
G
EGC
EGCg
5
6
EC
ECg
7
8
Non-
enzymatic
redox
Non-enzymatic
condensation
reaction
R1 R2
H H
TF2A G H
TF2B H G
2
TF1
1
2
3
4
1
Quinone-induced
TF degradation
TF3
G G
(G: galloyl)
Self-dimerization
Figure 1
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