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Equilibrated structures of oolongtheanins
Tetrahedron Letters 57 (2016) 2067–2069
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Equilibrated structures of oolongtheanins Sayumi Hirose a, Kazuki Ogawa b, Emiko Yanase a,b,⇑ a b
United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan 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
Article history: Received 25 January 2016 Revised 24 March 2016 Accepted 29 March 2016 Available online 1 April 2016 Keywords: Catechin Oolongtheanin Equilibrated structure
a b s t r a c t Oolongtheanins, catechin dimers derived from oolong tea leaves, were isolated by Hashimoto et al. in 1988. Later, Tanaka et al. revised the oolongtheanin structure. In this study, it was clarified that oolongtheanins in organic solvent like acetone or methanol have the structures proposed by Tanaka et al. Based on the hydrogen/deuterium exchange reaction in aqueous solution, it was suggested that they exist as an equilibrium mixture of the structures proposed by Hashimoto and Tanaka. Ó 2016 Elsevier Ltd. All rights reserved.
Introduction
Results and discussion
Tea (Camellia sinensis) is one of the more popular beverages worldwide, and has attracted increasing attention from researchers because of its various health benefits. Tea is classified into green tea, oolong tea, and black tea based on the difference in their processing methods. In oolong tea and black tea, the components of the leaves are changed during the fermentation process. Catechins, major polyphenols present in tea leaves, are oxidized by the enzyme polyphenoloxidase, generating typical dimers such as theaflavins, theasinensins, and other polymers.1 Oolongtheanins (1–4a), dimers of pyrogallol-type catechins present in oolong tea and black tea, were isolated and their structures elucidated by Hashimoto et al. in 1988,2 and recently, their structures were revised as 1–4a by Tanaka et al.3 Oolongtheanins have been predicted to exert various bioactive effects.4,5 However, detailed studies on oolongtheanins have been limited because such components are found as complicated mixtures and are difficult to isolate. To clarify the formation mechanism of oolongtheanins, we have investigated oxidation products of catechins. Previously, we had reported the three-step formation of oolongtheanins from pyrogallol-type catechins, in which dehydrotheasinensins and pro-oolongtheanins were the intermediates.6 In this study, we investigated the reaction products to find that oolongtheanins isomerize in aqueous solution, and exist as a mixture of two isomers which corresponds to the originally reported one and the previously revised one (Fig. 1).
Desgalloyl pro-oolongtheanin (6), which is the intermediate of desgalloyl oolongtheanin (1a) synthesis, was prepared from () epigallocatechin (5) via two steps.7 Compound 6 is converted to desgalloyl oolongtheanin (1a) by hydrolysis of the lactone moiety. When this reaction was analyzed using high-performance liquid chromatography (HPLC), two peaks X and Y were observed (Fig. 2), which were separated by HPLC and were then analyzed by 1H NMR in acetone-d6. However, 1H NMR spectra of compounds X and Y showed that both have structure 1a. After NMR analysis, the NMR samples in acetone-d6 were re-analyzed by HPLC; this time, both samples showed only peak X. Furthermore, when the reaction solution of 6 was concentrated, dissolved in methanol, and analyzed by HPLC, it showed only peak X. Therefore, it was suggested that the peak X corresponds to 1a, and peak Y corresponds to the intermediate, hydrate, or equilibrium, and not the byproduct. Assuming that compound Y was the intermediate of this reaction, compound 6 was reacted under heating condition for a long time. Had it been an intermediate, peak intensity of X would have increased and that of peak Y would have decreased with time in the HPLC analysis. However, the intensity ratio of peaks X and Y remained constant, suggesting that compound Y is not an intermediate. Since 1a has a ketone moiety at the C4000 position, compound Y could be assumed to be the hydrate of 1a, formed by addition of the reaction solvent, water. Therefore, a 2-Da mass shift introduced by the 18O is expected when 1a is treated with H218O instead of H2O. Compound 6 was treated with H218O, and the reaction solution was analyzed by HPLC. The resultant HPLC chromatogram
⇑ Corresponding author. Tel./fax: +81 58 293 2914. E-mail address: [email protected] (E. Yanase). http://dx.doi.org/10.1016/j.tetlet.2016.03.092 0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.
2068
S. Hirose et al. / Tetrahedron Letters 57 (2016) 2067–2069
Upper unit OH
OH
R1 O HO O
4''' H
HO
A' OH
3''' 2''' 1''' 5''' O
2'' O
O
A
OH O OH
OH OR2
OH
G = galloyl
A
OH
HO
O
Lower unit 1a: Desgalloyl oolongtheanin R1=R 2 = H 2a: Oolongtheanin-3'- O-gallate R1=R2 = G 3a: Oolongtheanin R1=H, R2=G 4a: Oolongtheanin-3- O-gallate R1=G, R2=H
OH OH
O
A
HO
O
OH
8
HO 6
OH
OH
OH
1'''
OH
B
5''' H
8a O 2
A'
4a
OH O 4'''
2'''
2''
4' 1'
6'
4 3 OH
OH
O OH
5' OH
5: (-)- Epigallocatechin ((-)-EGC) 6: Desgalloyl pro-oolongtheanin
Figure 1. Chemical structures of 1–4a, 5, 6.
carbon atom bearing –OH group shows different chemical shifts in 13C NMR from the one bearing –OD group, it is possible to distinguish the presence and absence of –OH group by comparing 13C NMR spectrum in D2O and D2O/H2O. Therefore, to verify the structure of 1b, 13C NMR analyses were performed in D2O and D2O/H2O, and the resultant spectra were compared (Table 1). Two types of signals, derived from 1a and 1b were observed in both solutions. The sp2 carbon signal of 1b at dc 174.2 in D2O, assigned to the –OH-group-bearing C-3000 in 1a, did not show any chemical shift
Figure 2. HPLC chromatogram of reaction (25% MeOH 1% AcOH in H2O).
Table 1 H (600 MHz) and
1
13
Position
showed two peaks, X and Y, corresponding to those recorded in H2O. Reaction solutions in H2O and H218O were analyzed by electrospray ionization mass spectrometry (ESI-MS). ESI-MS of both reaction solutions showed peaks at m/z 603 [M+Na]+ and 619 [M+K]+; no effect of 18O was observed. Therefore, it was suggested that compound Y is not the hydrate of 1a. Next, we investigated compound Y by NMR analysis, because the above results suggest that Y might be an equilibrium structure of compound 1a. The reaction mixture of 6 was passed through an HP20SS column, and the fraction containing compound Y was collected and concentrated. The 1H NMR of this fraction in acetone-d6 showed only the signals of compound 1a. Furthermore, the NMR sample was analyzed by HPLC to confirm that it corresponded with 1a. The NMR sample was concentrated, and 1H NMR spectrum was obtained again, using D2O as the solvent. The resultant NMR spectrum showed the mixture of two compounds, and HPLC chromatogram of this NMR sample showed the peaks X and Y. These results showed that compound Y exists in an equilibrium mixture of 1a in aqueous solution. The 1H NMR spectrum in D2O showed that the two compounds were in a 2:1 ratio. In comparison with the chemical shifts of 1a in acetone-d6, the chemical shifts of the A-rings of the major component in D2O were shifted upfield. Therefore, it was suggested that Y, the equilibrium structure of 1a, is the major component in D2O, instead of pure 1a. The 13C NMR, heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond coherence (HMBC) analyses were performed using D2O as the solvent, and the chemical structure of Y was assigned as 1b. The oolongtheanin structures were revised by Tanaka et al. based on the hydrogen/deuterium exchange reaction.3 Since the
C NMR (150 MHz) of 1b in D2O or D2O/H2O (5:95 v/v) 1b (D2O) 13
1
13
2 3
5.19 (s) 4.23 (br s)
78.22 67.76
5.00 (s) 4.04 (br s)
78.20 67.86
4 4a 5
2.68–2.91
31.71 102.00 157.70
2.50–2.80
31.76 102.02 157.85
6 7
5.59 (s)
— 157.93
5.44 (s)
— 158.07
8 8a 10 20 30 40
5.86 (s)
— 102.00 129.74 117.88 149.12 132.57
5.67 (s)
— 102.02 129.69 117.81 149.15 132.72
H
50
C
H
149.24
149.36
7.04 (s) 4.57 (s) 4.51 (br s)
112.74 75.45 64.68
6.88 (s) 4.39 (s) 4.3 (s)
112.79 75.50 64.77
400 400 a 500
2.68–2.91
30.67 101.82 157.83
2.50–2.80
30.71 101.84 157.98
600 700
5.80 (s)a
— 158.38
5.63 (s)b
— 158.50
800 800 a 1000 2000 3000 4000
5.92 (s)a
— 102.79 174.17 131.04 203.10 110.06
5.75 (s)b
— 102.75 174.17 131.03 203.10 110.15
6.56 (s)
56.11
6.37 (s)
Ddcc
C
60 200 300
5000 a,b
1b (D2O/H2O)
1
56.12
0.02 0.10 0.05 0.02 0.15 — 0.14 — 0.02 0.05 0.08 0.03 0.14 0.11 0.05 0.05 0.10 0.04 0.02 0.15 — 0.12 — 0.04 0.00 0.01 0.00 0.09 0.01
May be interchanged in the same column. Hydrogen/deuterium exchange shifts in 13C NMR spectra.; Ddc = 1b (D2O/ H2O)—1b (D2O). The Ddc value where the apparent change of chemical shifts has been detected are underlined. c
S. Hirose et al. / Tetrahedron Letters 57 (2016) 2067–2069
Figure 3. Chemical structures and key HMBC correlations of equilibrium 1b.
H O
1) Keto-enol tautomerization
O OH
2) ring-opening
O 3) Recyclization
O
H
OH
results, it was suggested that galloyl group on the upper unit is involved in the equilibrium reaction in aqueous solution by hindering the deprotonation of the –OH group at C-3000 or recyclization. Similar equilibrium was not observed in methanol or ethanol, and on increasing the ratio of methanol in aqueous solution, the ratio of the structures 1–4b decreased. Since structure a has –OH group at C-3000 , the nearly coplanar orientation of the ketone group at C-4000 , makes it possible to form a hydrogen bond between the –OH group at C-3000 and the ketone group at C-4000 . On the other hand, in structure b, the –OH group at C-4000 is not coplanar to the ketone group at C-3000 ; hence, no hydrogen bond is possible with the C-3000 ketone, which leaves the –OH group at C-4000 bare, and polarity of structure a increases. Therefore, it is suggested that oolongtheanins form structure b in highly polar solvents like water. Conclusion
O
H OH
a
2069
OH
b
OH
Scheme 1. Isomerization mechanism of a to b.
change, thereby indicating that no –OH group was bonded to this position. Furthermore, HMBC correlation was observed from H-200 to the signal at dc 174.2, which is absent in 1a. This carbon signal is similar to that for C-1000 (dc 171.6 in acetone-d6) of 6, and it was suggested that the sp2 carbon corresponding to dc 174.2 bonds to C-200 of the C-ring. Meanwhile, the carbon signal at dc 110.1 ppm of 1b in D2O showed change in chemical shift (Ddc 0.09) due to a hydrogen/deuterium exchange reaction, and was assigned to a hemiacetal carbon. From the above-mentioned NMR experiments, the structure of 1b was confirmed to be the same as the originally reported structure by Hashimoto et al. (Fig. 3). The relative stereochemistry of 1b was determined by its formation mechanism from 1a (Scheme 1). The other three types of oolongtheanins (2a, 3a, and 4a) were also investigated to confirm whether such equilibriums exist. First, oolongtheanin-30 -O-gallate (2a), one of the oolongtheanins bearing two galloyl groups, was synthesized, and its aqueous solution was analyzed by HPLC. The resultant chromatogram showed the two peaks corresponding to 2a and 2b. 1H NMR in D2O showed signals derived from these two compounds, and their ratio was 4:1. The lower-intensity signals showed upfield shift of the A-ring, which was similar to 1b, and it was confirmed as structure 2b by 2DNMR analyses. Next, mono-gallate-type oolongtheanins (3a and 4a) were analyzed similarly. Oolongtheanin 3a, which has galloyl group on the lower unit, showed a 3a/3b ratio of 4:5. On the other hand, 4a, which has galloyl group on the upper unit, showed a 4a/4b ratio of 3:1. Clearly, the amount of 4b was lower, similar to that of 2b. On the other hand, the ratio of 1a and 1b was 1:2, similar to that for 3a and 3b. The isomerization mechanism for 1–4a involved recyclization at the C-4000 position, after ring-opening by keto–enol tautomerization of the C-3000 position (Scheme 1). Based on these
In this study, it was clarified that oolongtheanins exist as structures 1–4a in organic solvents like acetone or methanol; in aqueous solution, they exist as an equilibrium mixture of 1–4a and 1–4b. The structures 1–4b were determined as the ones reported by Hashimoto et al. Furthermore, the ratio of the equilibrium mixture varies with the water content of the solvent and the type of oolongtheanins. Since the components of oolong tea leaves are dissolved in water when we consume it, the structures 1–4b are probably related to the bioactivities of oolong tea. Furthermore, the equilibrium between structures a and b could have occurred during the fermentation of the tea leaves, and structures b could also be the intermediates of oolong tea polymers. The results of this study can aid in elucidating the bioactivities of oolongtheanins and the formation mechanism of complex oolong tea polyphenols. Acknowledgments This work was supported by a JSPS Kakenhi Grant (15K07427) and by the Toyo Institute of Food Technology. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2016.03. 092. References and notes 1. Haslam, E. Phytochemistry 2003, 64, 61. 2. Hashimoto, F.; Nonaka, G.; Nishioka, I. Chem. Pharm. Bull. 1988, 36, 1676. 3. Matsuo, Y.; Tadakuma, F.; Shii, T.; Saito, Y.; Tanaka, T. Tetrahedron 2015, 71, 2540. 4. Akizawa, T.; Yahara, S.; Hashimoto, F.; Yamada, M.; Suma, S.; Kano, T.; Uchida, K.; Oshida, Y. Jpn Kokai Tokkyo Koho 2000. JP 2000226329 A. 5. Nakai, M.; Fukui, Y.; Asami, S.; Toyoda-Ono, Y.; Iwashita, T.; Shivata, H.; Mitsunaga, T.; Hashimoto, F.; Kiso, Y. J. Agric. Food Chem. 2005, 53, 4593. 6. Hirose, S.; Tomatsu, K.; Yanase, E. Tetrahedron Lett. 2013, 54, 7040. 7. Ogawa, K.; Hirose, S.; Yamamoto, H.; Shimada, M.; Nagaoka, S.; Yanase, E. Bioorg. Med. Chem. Lett. 2015, 25, 749.
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Equilibrated structures of oolongtheanins Sayumi Hirose a, Kazuki Ogawa b, Emiko Yanase a,b,⇑ a b
United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan 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
Article history: Received 25 January 2016 Revised 24 March 2016 Accepted 29 March 2016 Available online 1 April 2016 Keywords: Catechin Oolongtheanin Equilibrated structure
a b s t r a c t Oolongtheanins, catechin dimers derived from oolong tea leaves, were isolated by Hashimoto et al. in 1988. Later, Tanaka et al. revised the oolongtheanin structure. In this study, it was clarified that oolongtheanins in organic solvent like acetone or methanol have the structures proposed by Tanaka et al. Based on the hydrogen/deuterium exchange reaction in aqueous solution, it was suggested that they exist as an equilibrium mixture of the structures proposed by Hashimoto and Tanaka. Ó 2016 Elsevier Ltd. All rights reserved.
Introduction
Results and discussion
Tea (Camellia sinensis) is one of the more popular beverages worldwide, and has attracted increasing attention from researchers because of its various health benefits. Tea is classified into green tea, oolong tea, and black tea based on the difference in their processing methods. In oolong tea and black tea, the components of the leaves are changed during the fermentation process. Catechins, major polyphenols present in tea leaves, are oxidized by the enzyme polyphenoloxidase, generating typical dimers such as theaflavins, theasinensins, and other polymers.1 Oolongtheanins (1–4a), dimers of pyrogallol-type catechins present in oolong tea and black tea, were isolated and their structures elucidated by Hashimoto et al. in 1988,2 and recently, their structures were revised as 1–4a by Tanaka et al.3 Oolongtheanins have been predicted to exert various bioactive effects.4,5 However, detailed studies on oolongtheanins have been limited because such components are found as complicated mixtures and are difficult to isolate. To clarify the formation mechanism of oolongtheanins, we have investigated oxidation products of catechins. Previously, we had reported the three-step formation of oolongtheanins from pyrogallol-type catechins, in which dehydrotheasinensins and pro-oolongtheanins were the intermediates.6 In this study, we investigated the reaction products to find that oolongtheanins isomerize in aqueous solution, and exist as a mixture of two isomers which corresponds to the originally reported one and the previously revised one (Fig. 1).
Desgalloyl pro-oolongtheanin (6), which is the intermediate of desgalloyl oolongtheanin (1a) synthesis, was prepared from () epigallocatechin (5) via two steps.7 Compound 6 is converted to desgalloyl oolongtheanin (1a) by hydrolysis of the lactone moiety. When this reaction was analyzed using high-performance liquid chromatography (HPLC), two peaks X and Y were observed (Fig. 2), which were separated by HPLC and were then analyzed by 1H NMR in acetone-d6. However, 1H NMR spectra of compounds X and Y showed that both have structure 1a. After NMR analysis, the NMR samples in acetone-d6 were re-analyzed by HPLC; this time, both samples showed only peak X. Furthermore, when the reaction solution of 6 was concentrated, dissolved in methanol, and analyzed by HPLC, it showed only peak X. Therefore, it was suggested that the peak X corresponds to 1a, and peak Y corresponds to the intermediate, hydrate, or equilibrium, and not the byproduct. Assuming that compound Y was the intermediate of this reaction, compound 6 was reacted under heating condition for a long time. Had it been an intermediate, peak intensity of X would have increased and that of peak Y would have decreased with time in the HPLC analysis. However, the intensity ratio of peaks X and Y remained constant, suggesting that compound Y is not an intermediate. Since 1a has a ketone moiety at the C4000 position, compound Y could be assumed to be the hydrate of 1a, formed by addition of the reaction solvent, water. Therefore, a 2-Da mass shift introduced by the 18O is expected when 1a is treated with H218O instead of H2O. Compound 6 was treated with H218O, and the reaction solution was analyzed by HPLC. The resultant HPLC chromatogram
⇑ Corresponding author. Tel./fax: +81 58 293 2914. E-mail address: [email protected] (E. Yanase). http://dx.doi.org/10.1016/j.tetlet.2016.03.092 0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.
2068
S. Hirose et al. / Tetrahedron Letters 57 (2016) 2067–2069
Upper unit OH
OH
R1 O HO O
4''' H
HO
A' OH
3''' 2''' 1''' 5''' O
2'' O
O
A
OH O OH
OH OR2
OH
G = galloyl
A
OH
HO
O
Lower unit 1a: Desgalloyl oolongtheanin R1=R 2 = H 2a: Oolongtheanin-3'- O-gallate R1=R2 = G 3a: Oolongtheanin R1=H, R2=G 4a: Oolongtheanin-3- O-gallate R1=G, R2=H
OH OH
O
A
HO
O
OH
8
HO 6
OH
OH
OH
1'''
OH
B
5''' H
8a O 2
A'
4a
OH O 4'''
2'''
2''
4' 1'
6'
4 3 OH
OH
O OH
5' OH
5: (-)- Epigallocatechin ((-)-EGC) 6: Desgalloyl pro-oolongtheanin
Figure 1. Chemical structures of 1–4a, 5, 6.
carbon atom bearing –OH group shows different chemical shifts in 13C NMR from the one bearing –OD group, it is possible to distinguish the presence and absence of –OH group by comparing 13C NMR spectrum in D2O and D2O/H2O. Therefore, to verify the structure of 1b, 13C NMR analyses were performed in D2O and D2O/H2O, and the resultant spectra were compared (Table 1). Two types of signals, derived from 1a and 1b were observed in both solutions. The sp2 carbon signal of 1b at dc 174.2 in D2O, assigned to the –OH-group-bearing C-3000 in 1a, did not show any chemical shift
Figure 2. HPLC chromatogram of reaction (25% MeOH 1% AcOH in H2O).
Table 1 H (600 MHz) and
1
13
Position
showed two peaks, X and Y, corresponding to those recorded in H2O. Reaction solutions in H2O and H218O were analyzed by electrospray ionization mass spectrometry (ESI-MS). ESI-MS of both reaction solutions showed peaks at m/z 603 [M+Na]+ and 619 [M+K]+; no effect of 18O was observed. Therefore, it was suggested that compound Y is not the hydrate of 1a. Next, we investigated compound Y by NMR analysis, because the above results suggest that Y might be an equilibrium structure of compound 1a. The reaction mixture of 6 was passed through an HP20SS column, and the fraction containing compound Y was collected and concentrated. The 1H NMR of this fraction in acetone-d6 showed only the signals of compound 1a. Furthermore, the NMR sample was analyzed by HPLC to confirm that it corresponded with 1a. The NMR sample was concentrated, and 1H NMR spectrum was obtained again, using D2O as the solvent. The resultant NMR spectrum showed the mixture of two compounds, and HPLC chromatogram of this NMR sample showed the peaks X and Y. These results showed that compound Y exists in an equilibrium mixture of 1a in aqueous solution. The 1H NMR spectrum in D2O showed that the two compounds were in a 2:1 ratio. In comparison with the chemical shifts of 1a in acetone-d6, the chemical shifts of the A-rings of the major component in D2O were shifted upfield. Therefore, it was suggested that Y, the equilibrium structure of 1a, is the major component in D2O, instead of pure 1a. The 13C NMR, heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond coherence (HMBC) analyses were performed using D2O as the solvent, and the chemical structure of Y was assigned as 1b. The oolongtheanin structures were revised by Tanaka et al. based on the hydrogen/deuterium exchange reaction.3 Since the
C NMR (150 MHz) of 1b in D2O or D2O/H2O (5:95 v/v) 1b (D2O) 13
1
13
2 3
5.19 (s) 4.23 (br s)
78.22 67.76
5.00 (s) 4.04 (br s)
78.20 67.86
4 4a 5
2.68–2.91
31.71 102.00 157.70
2.50–2.80
31.76 102.02 157.85
6 7
5.59 (s)
— 157.93
5.44 (s)
— 158.07
8 8a 10 20 30 40
5.86 (s)
— 102.00 129.74 117.88 149.12 132.57
5.67 (s)
— 102.02 129.69 117.81 149.15 132.72
H
50
C
H
149.24
149.36
7.04 (s) 4.57 (s) 4.51 (br s)
112.74 75.45 64.68
6.88 (s) 4.39 (s) 4.3 (s)
112.79 75.50 64.77
400 400 a 500
2.68–2.91
30.67 101.82 157.83
2.50–2.80
30.71 101.84 157.98
600 700
5.80 (s)a
— 158.38
5.63 (s)b
— 158.50
800 800 a 1000 2000 3000 4000
5.92 (s)a
— 102.79 174.17 131.04 203.10 110.06
5.75 (s)b
— 102.75 174.17 131.03 203.10 110.15
6.56 (s)
56.11
6.37 (s)
Ddcc
C
60 200 300
5000 a,b
1b (D2O/H2O)
1
56.12
0.02 0.10 0.05 0.02 0.15 — 0.14 — 0.02 0.05 0.08 0.03 0.14 0.11 0.05 0.05 0.10 0.04 0.02 0.15 — 0.12 — 0.04 0.00 0.01 0.00 0.09 0.01
May be interchanged in the same column. Hydrogen/deuterium exchange shifts in 13C NMR spectra.; Ddc = 1b (D2O/ H2O)—1b (D2O). The Ddc value where the apparent change of chemical shifts has been detected are underlined. c
S. Hirose et al. / Tetrahedron Letters 57 (2016) 2067–2069
Figure 3. Chemical structures and key HMBC correlations of equilibrium 1b.
H O
1) Keto-enol tautomerization
O OH
2) ring-opening
O 3) Recyclization
O
H
OH
results, it was suggested that galloyl group on the upper unit is involved in the equilibrium reaction in aqueous solution by hindering the deprotonation of the –OH group at C-3000 or recyclization. Similar equilibrium was not observed in methanol or ethanol, and on increasing the ratio of methanol in aqueous solution, the ratio of the structures 1–4b decreased. Since structure a has –OH group at C-3000 , the nearly coplanar orientation of the ketone group at C-4000 , makes it possible to form a hydrogen bond between the –OH group at C-3000 and the ketone group at C-4000 . On the other hand, in structure b, the –OH group at C-4000 is not coplanar to the ketone group at C-3000 ; hence, no hydrogen bond is possible with the C-3000 ketone, which leaves the –OH group at C-4000 bare, and polarity of structure a increases. Therefore, it is suggested that oolongtheanins form structure b in highly polar solvents like water. Conclusion
O
H OH
a
2069
OH
b
OH
Scheme 1. Isomerization mechanism of a to b.
change, thereby indicating that no –OH group was bonded to this position. Furthermore, HMBC correlation was observed from H-200 to the signal at dc 174.2, which is absent in 1a. This carbon signal is similar to that for C-1000 (dc 171.6 in acetone-d6) of 6, and it was suggested that the sp2 carbon corresponding to dc 174.2 bonds to C-200 of the C-ring. Meanwhile, the carbon signal at dc 110.1 ppm of 1b in D2O showed change in chemical shift (Ddc 0.09) due to a hydrogen/deuterium exchange reaction, and was assigned to a hemiacetal carbon. From the above-mentioned NMR experiments, the structure of 1b was confirmed to be the same as the originally reported structure by Hashimoto et al. (Fig. 3). The relative stereochemistry of 1b was determined by its formation mechanism from 1a (Scheme 1). The other three types of oolongtheanins (2a, 3a, and 4a) were also investigated to confirm whether such equilibriums exist. First, oolongtheanin-30 -O-gallate (2a), one of the oolongtheanins bearing two galloyl groups, was synthesized, and its aqueous solution was analyzed by HPLC. The resultant chromatogram showed the two peaks corresponding to 2a and 2b. 1H NMR in D2O showed signals derived from these two compounds, and their ratio was 4:1. The lower-intensity signals showed upfield shift of the A-ring, which was similar to 1b, and it was confirmed as structure 2b by 2DNMR analyses. Next, mono-gallate-type oolongtheanins (3a and 4a) were analyzed similarly. Oolongtheanin 3a, which has galloyl group on the lower unit, showed a 3a/3b ratio of 4:5. On the other hand, 4a, which has galloyl group on the upper unit, showed a 4a/4b ratio of 3:1. Clearly, the amount of 4b was lower, similar to that of 2b. On the other hand, the ratio of 1a and 1b was 1:2, similar to that for 3a and 3b. The isomerization mechanism for 1–4a involved recyclization at the C-4000 position, after ring-opening by keto–enol tautomerization of the C-3000 position (Scheme 1). Based on these
In this study, it was clarified that oolongtheanins exist as structures 1–4a in organic solvents like acetone or methanol; in aqueous solution, they exist as an equilibrium mixture of 1–4a and 1–4b. The structures 1–4b were determined as the ones reported by Hashimoto et al. Furthermore, the ratio of the equilibrium mixture varies with the water content of the solvent and the type of oolongtheanins. Since the components of oolong tea leaves are dissolved in water when we consume it, the structures 1–4b are probably related to the bioactivities of oolong tea. Furthermore, the equilibrium between structures a and b could have occurred during the fermentation of the tea leaves, and structures b could also be the intermediates of oolong tea polymers. The results of this study can aid in elucidating the bioactivities of oolongtheanins and the formation mechanism of complex oolong tea polyphenols. Acknowledgments This work was supported by a JSPS Kakenhi Grant (15K07427) and by the Toyo Institute of Food Technology. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2016.03. 092. References and notes 1. Haslam, E. Phytochemistry 2003, 64, 61. 2. Hashimoto, F.; Nonaka, G.; Nishioka, I. Chem. Pharm. Bull. 1988, 36, 1676. 3. Matsuo, Y.; Tadakuma, F.; Shii, T.; Saito, Y.; Tanaka, T. Tetrahedron 2015, 71, 2540. 4. Akizawa, T.; Yahara, S.; Hashimoto, F.; Yamada, M.; Suma, S.; Kano, T.; Uchida, K.; Oshida, Y. Jpn Kokai Tokkyo Koho 2000. JP 2000226329 A. 5. Nakai, M.; Fukui, Y.; Asami, S.; Toyoda-Ono, Y.; Iwashita, T.; Shivata, H.; Mitsunaga, T.; Hashimoto, F.; Kiso, Y. J. Agric. Food Chem. 2005, 53, 4593. 6. Hirose, S.; Tomatsu, K.; Yanase, E. Tetrahedron Lett. 2013, 54, 7040. 7. Ogawa, K.; Hirose, S.; Yamamoto, H.; Shimada, M.; Nagaoka, S.; Yanase, E. Bioorg. Med. Chem. Lett. 2015, 25, 749.