polyphenol

1. MAGNETIC RESONANCE IN CHEMISTRY, VOL. 34, 887-890 (1996)
'H and 13CNMR Assignments of Some Green
Tea Polyphenols
Adrienne L. Davis,"Ya Cai, Alan P. Davies and J. R. Lewis
Unilever Research, Colworth House, Sharnbrook, Bedford MK44 lLQ, UK
Complete and unequivocal 'H and "C NMR assignments for a range of green tea polyphenols lepiafzelechin,
catechin, epicatechin, gallocatechin, epigallocatechin, catechin-3-O-gallate,epicatechin-3-O-gallate, gallocatechin-
3-O-gallate, epigallocatechin-3-0-gallateand epigallocatechin-3-0-(3'-O-methyl)-gallate] in acetone-d, solvent
have been achieved using the 2D proton-carbon correlation experiments HMQC and HMBC and also 1D NOE
differencespectroscopy.
KEY WORDS NMR; 'H NMR; NMR; flavan-3-01;catechin; green tea polyphenols
INTRODUCTION
There are numerous types of polyphenols contained in
tea leaves, the most important of which are the flavan-
3-01s and their gallates, often referred to as 'catechins'
It has been recognized that the catechins and their oxi-
dation products (theaflavins) are very important in gov-
erning the organoleptic properties of tea such as taste
and colour. Over the last few decades a large number of
catechins have been isolated and their structures char-
acterized, mainly by the technique of NMR spectros-
copy. However, the NMR data are widely scattered in
the literature (particularly in the case of 'H data) and
are often found to be in~ornpletel-~
or to contain incor-
rect a~signments.4.~
Therefore, in the course of our
studies of tea polyphenols, we have undertaken the task
* Author to whom correspondence should be addressed.
of obtaining a complete set of correctly assigned NMR
data for a representative set of the catechin complexes
found in green tea: catechin (l), gallocatechin (2),
catechin-3-0-gallate (3), gallocatechin-3-0-gallate (4),
epicatechin (5), epigallocatechin (6), epicatechin-3-0-
gallate (7),epigallocatechin-3-0-gallate (8),epiafzelechin
(9) and epigallocatechin-3-O-(3'-O-methyl)gallate(10).
The structures of these compounds and the numbering
scheme employed are shown in Scheme 1.
The NMR data obtained in this work will be of use
in the characterization of new polyphenol compounds
and in studies of the interactions of catechins with other
species of interest.
EXPERIMENTAL
Isolation
Instant green tea was purchased from Ceytea of Sri
Lanka. The tea (1.0 kg) was dissolved in water (1.0 1)
Compound R R'
1 H H
2 H OH
3 G H
4 G OH
R R ' R"
H H OH
5
7 G H OH
G OH OH
8
H H H
9
10 GMe OH OH
6 H on OH
G = GMe =
b H bH
Scheme 1. Structures and numbering schemeof the green tea polyphenolsstudied.
CCC 0749-1581/96/110887-04
01996by John Wiley & Sons, Ltd.
Received 12 February 1996
Accepted (reuised) 17 June 1996

2. 888 A. L. DAVIS ET AL.
and was extracted with ethyl acetate (6 x 1.0 1). The
ethyl acetate extract (200 g) was dissolved in ethanol
(200 ml) and the solution was loaded on a Sephadex
LH-20 column (25 x 12 cm id.). The column was eluted
with ethanol, and 20 fractions (500 ml each) were col-
lected. The fractions containing catechins were com-
bined into four fractions after TLC and HPLC analysis.
The yield of these fractions were 11, 35, 45 and 70 g,
respectively.
These four fractions were then repeatedly purified on
a Sephadex LH-20 column (45 x 5 cm i.d.), and the
column was eluted with aqueous acetone (0-25%).
Evaporation of the appropriate fractions in uucuo fol-
lowed by freeze-drying gave pure epiafzelechin (0.35 g),
catechin (1.10g), epicatechin (1.52 g), gallocatechin (0.24
g), epigallocatechin (2.55 g), catechin-3-0-gallate (0.11
g), epicatechin-3-0-gallate (2.64 g), gallocatechin-3-0-
gallate (0.18 mg), epigallocatechin-3-0-gallate (4.21 g)
and epigallocatechin-3-0-(3'-0-methyl)gallate(38 mg).
NMR spectroscopy
NMR spectra were measured on a Bruker AMX400
spectrometer operating at a probe temperature of 303 K
using either a dual 'H/13C 5 mm probe or a multinu-
clear 5 mm inverse probe as appropriate. The solvent
used was acetone-d, and spectra were referenced rela-
tive to internal TMS. Sample concentrations were typi-
cally in the range 1-10 mg per 0.5 ml.
NOE difference experiments6 were typically acquired
with 8K data points covering a spectral width of ca.
3500 Hz and with a 2 s presaturation time. Spectra at
each presaturation position were interleaved in groups
of 16 scans to minimize artifacts due to instrument
instabilities and processed with a 0.5 Hz exponential
line broadening to reduce subtraction artifacts. Samples
were not degassed for the NOE measurements. HMQC
experiments7'* were acquired with a 400 ms delay after
the BIRD pulse, a dephasing/refocusing delay of 3.571
ms and a relaxation delay of 0.5 s between scans;
GARP decoupling was applied during acquisition.
HMBC experiment^',^ were recorded with a 3 ms delay
for the low-pass J filter, a 60 ms delay for evolution of
long-range couplings and a relaxation delay of 0.6 s.
For each spectrum 512 or 1024 increments of 2K data
points were collected covering a typical spectral width
of 3000 Hz in F, and 15000 Hz in F,. The HMQC was
recorded phase sensitive using TPPI and processed with
742 shifted sine-bell windows in both dimensions. The
HMBC spectra were processed with a Gaussian window
function in both dimensions and were collected in the
absolute value mode.
RESULTS
AND DISCUSSION
The current work serves to give a set of unambiguous
NMR assignments for a representative series of green
tea polyphenols (Scheme 1). 'H NMR data for com-
pounds 1-10 are given in Tables 1 and 2 and 13CNMR
data in Table 3.
13C NMR data for the majority of the compounds
shown in Scheme 1have been reported previously in the
literature;" however, assignments are generally incom-
plete, particularly with respect to the C-5, C-7 and C-8a
resonances. We are not aware of any published 13C
NMR data for 4 and the published data for 10 is incom-
plete.' 'H NMR data are more difficult to locate in the
literature. A complete and reliable 'H NMR assignment
for 1 in acetone-d, is available." We also note that
unambiguous 'H and 13CNMR assignments have been
published for 1 and 5 in DMSO-d, solvent.12However,
it is well known that solvent differences can cause sig-
nificant chemical shift changes (see below); therefore, a
complete 'H and 13Cassignment of 1 and 5 in acetone-
d, solvent is included in this work. 'H NMR data for 10
have been reported but were not fully
assigned.
The 'H and 13C NMR assignments obtained in this
work were achieved primarily by the means of proton-
carbon correlation methods, specifically the HMQC
experiment for direct correlations and the HMBC
experiment for long-range correlations.
The long-range proton-carbon correlations for com-
pound 4 are given in Table 4. These data are largely
typical of the long-range correlations observed for
Table 1. 'H NMR chemical shift data for green tea polyphenols"
Proton 1 2 3 4 5 6 7 8 9 10
2 4.563 4.512 5.153 5.135 4.882 4.820 5.134 5.067 4.933 5.100
3 3.994 3.970 5.401 5.402 4.209 4.190 5.555 5.562 4.221 5.512
40 2.912 2.870 2.888 2.824 2.740 2.727 2.925 2.909 2.749 2.948
4B 2.531 2.521 2.764 2.765 2.867 2.849 3.052 3.040 2.880 3.051
6 6.024 6.018 6.072 6.064 6.022 6.016 6.063 6.062 6.027 6.049
8 5.881 5.877 5.993 5.988 5.922 5.913 6.037 6.036 5.927 6.033
2 6.898 6.457 6.924 6.481 7.054 6.574 7.059 6.624 7.362 6.647
- 6.816
3
5
' 6.798 - 6.794 - 6.787 - 6.764 - 6.816
6 6.758 6.794 6.840 6.891 7.362
2" - - 7.045 7.050 - - 7.030 7.028 - 7.056
- 7.134
6"
- - 3.816
OME
-
- - - - - - -
-
__ - - -
- - - - - - -
a Spectra measured in acetone-d, at 303 K and referenced relative to internal TMS. Magnetically equivalent protons are tabulated only
once: dashes indicate that the given proton is not present in the molecule.

3. ASSIGNMENTS OF GREEN TEA POLYPHENOLS 889
Table 2. 'H NMR couplingconstant data for green tea polyphenols"
J 1 2 3 4 5 6 7 8 9 10
2.3
16.1
8.4
5.5
7.8
n.r.
n.r.
n.r.
n.r.
1.9
8.1
-
-
2.3
16.1
8.2
5.5
7.4
n.r.
n.r.
0.5
-
-
-
2.3
16.6
6.0
5.2
6.0
1.o
n.r.
0.7
0.7
n.m.
n.m.
-
2.3
16.7
5.5
5.2
5.3
n.r.
n.r.
n.r.
-
-
2.3
16.5
4.6
3.2
1.6
0.9
0.5
0.7
2.0
8.1
0.8
-
-
2.3
16.6
4.5
3.5
1.7
0.8
0.8
0.6
-
-
-
2.3
17.4
4.5
2.1
1.4
0.7
0.9
0.5
0.7
2.0
8.2
-
2.3
17.4
4.6
2.2
1.4
0.8
1.o
0.6
-
-
2.3
16.7
4.5
3.2
1.5
0.8
1.1
0.6
0.6
2.5b
8.4b
2.5b
-
2.3
17.4
4.4
2.8
1.6
n.r.
n.r.
0.6
-
-
1.9
a Measuredin Hz and accurate to -+0.15Hz;n.r.= not resolved, n.m. = not measured. Magneticallyequivalentnucleiare listed only once.
Calculated from the [AX], spin system of this molecule,J(2.5') =J(3'6') = 0.4 Hz.
Table 3. I3CNMR chemical shift data for green tea polyphenols"
Carbon
2
3
4
4a
6
8
5
7
8a
1'
2
3
4
5'
6
1"
2
3"
4
5
6"
co
OMe
1
82.76
68.38
28.84
100.69
96.19
95.50
157.23
157.77
156.95
132.27
115.27
145.66
145.73
115.74
120.08
-
-
-
-
-
-
-
-
2
82.80
68.35
28.46
100.63
96.15
95.49
157.24
157.74
156.90
131.62
107.25
146.30
133.34
-
-
-
-
-
-
-
-
3
78.80
70.46
24.20
99.24
96.47
95.61
157.25
158.08
156.32
131.47
114.30
145.82b
145.8gb
115.99
119.13
121.79
109.98
146.00
138.93
166.04
-
4
78.64
70.36
23.58
99.14
96.40
95.57
157.23
158.02
156.24
130.95
106.20
146.57
133.37
121.80
109.99
146.03
138.97
166.15
-
5
79.46
66.97
29.01
99.85
96.22
95.75
157.61
157.60
157.19
132.32
115.31
145.42
145.31
115.51
119.41
-
-
-
-
-
-
-
-
6
79.46
67.02
28.82
99.92
96.18
95.73
157.57
157.59
157.14
131.56
106.97
146.17
132.97
-
-
-
-
-
-
-
-
7
78.11
69.36
26.63
99.03
96.56
95.87
157.50
157.81
157.14
131.42
114.95
145.60
145.53
115.67
119.25
121.87
109.99
145.94
138.83
166.04
-
8
78.10
69.28
26.64
99.09
96.54
157.46
157.78
157.12
130.77
106.81
146.27
133.17
95.87
121.92
110.04
145.90
138.79
166.07
-
9
79.53
66.90
29.16
99.79
96.25
95.78
157.63
157.66
157.24
131.55
129.17
115.50
157.69
-
-
-
-
-
-
-
-
10
78.01
69.83
26.36
96.50
95.79
157.45
157.06
106.69
146.35
133.17
98.99
157.88
130.85
121.81
106.00
148.47
139.80
145.79
111.67
166.16
56.54
a Spectra measured in acetone-d, at 303 K and referenced relative to internal TMS. Equivalent carbons are tabulated only once; dashes
indicatethat the carbon atom is not present in the molecule.
bAssignmentsinterchangeable.
Table 4. Long-range proton4arbon correlations observed for 4
using the HMBC experiment"
Proton Carbon
2"
2
6
8
2
3
4
,
4
,
a Measuredat 303 K in acetone-d,.
1", 3". 4", co
2, l',
3
'
,4
4a, 5, 7, 8
4a, 6, 7, 8a
3, 4, 11,2, 8a
4a. CO
2, 3, 4a, 5, 8a
2, 3, 4a, 5, 8a
flavan-3-01 molecules and assignments using these data
are straightforward. However, in compounds with cis
stereochemistry (i.e. epicatechin-like) the H-2 to C-8a
correlation is weak and in some of the molecules
studied (6, 7, 9 and lo), this correlation could not be
observed. Consequently, C-8a, C-5 and C-7 could not
be distinguished from each other and H-6 and H-8
could not be assigned. However, in all cases ambiguity
in the assignment of H-6 and H-8 could be resolved by
NOE difference spectroscopy since H-8 is observed to
exhibit an NOE from H-2 and/or H-2' in these com-
pounds. This knowledge, combined with the HMBC
connectivities, allows a complete 13C assignment to be

4. 890 A. L. DAVIS ET AL.
made and all the protons to be assigned with the excep-
tion of H-4a and H-41. NOE difference spectroscopy
was used in order to distinguish between H-4a and
H-41, with the proton showing an NOE from H-2 being
assigned as H-41, the quasi-axial proton. Therefore, a
complete 'H and 13C NMR assignment was possible
for compounds 1-10.
The data obtained in this work demonstrate that, in
acetone-d, ,H-6 resonates at higher frequency than H-8
and that C-6 is at higher frequency than C-8 regardless
of the presence of a galloyl ester group on C-3 or the
stereochemistry at this carbon. H-6 was also found to
be at higher frequency than H-8 in both 1 and 5 in
DMSO-d, solvent.' However, we have observed that
in MeOH-d, solvent the H-6 and H-8 resonances of 8
are coincident at 400 MHz, whereas for 10, H-8 was
found to resonate at higher frequency than H-6 in this
solvent. In all but one case (5), the data in Table 3 indi-
cate that for this group of compounds, C-7 is at higher
frequency than C-5, which in turn is at higher frequency
than C-8a: in 5, the C-5 and C-7 resonances are very
close together and their positions are reversed, as was
observed to be the case in DMSO-d, .12
The presence of galloyl ester substituent at C-3 causes
the chemical shift of the H-8 resonance to increase by
ca. 0.1 ppm, whilst H-6 remains relatively unaffected. By
contrast, both C-6 and C-8 shift to higher frequency in
the presence of a galloyl ester at C-3, but C-6 is the
more sensitive of the two. H-3 is extremely sensitive to
the presence of a galloyl ester at C-3, showing an
increase in chemical shift of ca. 1.5 ppm; the chemical
shift of H-2 also increases on gallation at C-3, although
to a lesser extent (ca. 0.3-0.5 ppm). We note that the
presence of a galloyl ester group significantly affects the
magnitude of the epimeric shielding difference of C-2,
C-3 and C-4. On going from trans to cis in the
ungallated species (i.e. 1 -+ 5 and 2 +6), the mean
shieldings are + 3.3, + 1.4and - 0.3 ppm, respectively;
however, in the corresponding gallates (3+7 and
4 + 8), the mean shieldings are +0.6, +1.1 and -2.7
ppm. These differences suggest that the presence of a
galloyl ester group at C-3 alters the conformation of the
heterocyclic ring, as indeed do the variations in the
magnitudes of J(4, ,3), J(4, ,3) and 5(3,2) within this
group of compounds (see Table 2).
CONCLUSION
The proton and carbon chemical shifts and NMR
assignments of a range of important green tea poly-
phenols have been determined using the HMQC,
HMBC and NOE difference experiments. These data
will be useful in further work on tea polyphenols in
terms of both structure elucidation and the study of the
interactions of these molecules with other species.
REFERENCES
1. G. I. Nonaka, 0. Kawahara and I. Nishioka, Chem. Pharm.
2. R. Saijo,Agric. Biol. Chem.46,1969 (1982).
3. A. M. Balde, L. A. Pieters, A. Gergely, H. Kolodziej, M. Claeys
4. S. Morimoto, G. Nonaka, I. Nishioka, N. Ezaki and T. Taki-
5. T. Tanaka, G. I. Nishioka and I. Nishioka, Phytochemisfry 22,
6. J. K. Saunders and B. K. Hunter, Modern NMR Spectroscopy,
Bull. 31,3906 (1983).
and A. J. Vlietinck, Phytochemisrry 30, 337 (1991).
zawa, Chem.Pharm.Bull. 33, 2281 (1985).
2575 (1983).
Chapt. 6. Oxford University Press, Oxford (1987).
7. A. Bax and S. Subramanian, J. Magn. Reson. 67, 565 (1986).
8. A. Bax and M. F. Summers, J. Am. Chem. SOC.108, 2093
9. A. Bax and L.Lerner,Carbohydr.Res. 166, 35 (1987).
(1986).
10. P. K. Agrawal, M. C. Bansal, L.J. Porter and L. Yeap Foo, in
Carbon-13 NMR of Flavonoids, edited by P. K. Agrawal,
Chapt. 8, and referencestherein. Elsevier, Amsterdam (1989).
11. E.Kiehlmann and A. S.Tracy, Can.J. Chem.64,1998 (1 986).
12. C. C. Shen, Y. S. Chang and L. K. Ho, Phytochemisrry 34, 843
(1993).