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The constitution and configuration of the theaflavin pigments of black tea
Tetrahedron Letters No.60, Pp. 5237-5240, 1970. Pergamon Press.
Printed in Great Britain.
THE CONSI’IlUTION AND CONFIGURATION OF THE THEAFLAVIN FIGMENTS OF BLACK TEA D. T. Coxon, A. Holmes, W. D. Ollis, * and V. C. Vora Department of Chemistry, The University, Sheffield S3 7HF (Received in UK 7 October 1970; accepted for publication
25 November 1970)
The pigments characteristic of black tea infusions are of two general types’ associated with the theaflavin fraction and the thearubigin fraction. proanthocyanidins . 2
The thearubigins have been recognised as polymeric
The theaflavin fraction contains a considerable number of compounds which are
benxtropolone derivatives including theaflavm 3’4 (I; ca. 8%). theaflavin-3-gallate (II; ca. 3%). the&h&n-3’-gallate
(III; ca. 20%), theaflavin-3.3’-digallate
theaflavic and epitheaflavic acids6
(IV; ca. 4%).
isotheaflavin5 (4%). and
the approximate relative proportions given by our present
fractionation procedures are indicated.
We now report upon the elucidation of the constitutions snd
configurations of the galloyl esters (II, III, and IV) by spectroscopic methods, and the confirmation of these configurations by synthesis. Chromatography [Sephadex LH 20; propan-2-01, acetic acid, water (4 : I: 4)] of the thesflavin fraction7 gave three well separated bands.
The first consisted mainly of theaflavin (I) and isotheaflavin,
the second yielded a mixture of the monogallates (II and III), and the third gave the digallate (IV).
The
IR spectra of the galloyl esters were very similar to that of theaflavin, but there were additional bands w 1690 cm -1. ) which could be assigned to gallates derived from alcoholic hydroxyl groups. (%o absorption spectra (Table 1) of the compounds (II, III, and IV) were extremely similar to that of
‘Ihe
theailavm (I) and their ORD curves closely corresponded with the ORD curve of theaflavin (I). ‘Ihe molecular formulae of the compounds (II), (III), and (IV) were established by high resolution mass spectrometric examination of their methyl ethers (methylation with dimethyl sulphate and potassium carbonate in boiling acetone). (VI and VII)
[Found: M, 856.2939.
The monogallates (II and III) gave the decamethyl ethers
C36H1806(0Me)lo requires M, 856.29431;
similarly characterised as the tridecamethyl ether (VIII) requires M, 1050.352].
[Found: M, 1050.353.
the digallate (IV) was C43H1907(OMe)13
Their absorption spectra (Table 1) and their mass spectral fragmentation
patterns summarised in the Scheme showed a striking relationship to that of theaflavm heptamethyl ether3 (V) and indicated particularly that the gallates (II, III, and IV) were galloyl derivatives involving either the 3- or the 3’-hydroxyl groups of theaflavin (I). (i) cleavage (-212 a.m.u.)
involving the trimethoxybenxoyloxy residues and (ii) retro-Diels-Alder
reactions (-166 a.m.u.). *
The fragmentations were clearly dominated by
To whom correspondence should be addressed. 5237
5
No.60
OR1
X = Galloyl(3,4.5-trlhydronybenroyl);
(IV).
of the NMR spectra
Although the monogallates
these three galloyl configurations
chloride
H
(III)
H
(Iv)
H
approximate
the assignments the indication
in pyridine gave,
(v)
Me
(VI)
Me
(VII)
Me
(VIII)
Me
1
hmax (Emax) in ethanol
supported the constitutions
by chromatography ratio 3
from their ORD curves
Having established
the Nh4R spectra
the constitutions
that they had corresponding
llmaflavin
after chromatographic
ether (VIII) and a mixture of theaflavin-3-
as a single fraction,
(II), (III), and
: 2) and the derived mixture of decamethyl
given in Table 2.
(II, III, and IV) was settled by synthesis.
methoxybenzoyl tridecamethyl
esters,
H
given in Table 2 clearly
were isolated
obtained on this mixture (II + III; ethers (VI + VII) permitted
spectra
(1) (IQ
R3 =
Y = 3,4,5-trimethoxybenzoyl
TABLE Absor@on
Comparison
R2 =
R1 =
_
heptamethyl
fractionation,
and -3’-monogallate
of
absolute
ether (V) and 3,4, S-tritheaflavin3,3:digallate
decamethyl
ethers
(VI and
VII). The theaflavln oxldative
process
is not involved,
pigments do not occur
during the fermentation then the probable
and (-)-epigallocatechin
gallate],
in the green leaf,
but are formed
of green leaf to give black tea.
precursors’
are as indicated:
theaflavin-3’-gallate
(III)
theaflavln
[(-)-epicatechin
by an enzyme-catalysed If hydrolysis 3-gallate
of gallate esters (II)
[(-)-epicatechln
gallate and (-)-epigallo-
2 G?
a
b
C
d
Chemical
5”
2.00
7”
-4.84
-4.88
Tropolone -OH
3.05
3.12
MHz.)a
3”
2.02
1.98
-4.76
(100
6, 8; 6’, 8’ ma 2.46 1.99
1.98
-4.81
Spectra
4., 4’ ma 3.97-3.99 2.41
1.99
2.01
NMR
ma 7.07 3.85-4.01 2.41
2.01
2.35
2.
5.54d 6.92 3.85-4.01
2.26
2.79
TABLE
4.25
I 5.36 6.92 3.82-3.89
4.05
5.86
7.09
7.09
3.72-3.91
3.72-3.91
4.05
4.05
2.96
2.96
2.52
2.43
2.36
6.04,
6.45
6.27-6.30
Methoxyl
6.04-6.24,
6.45
6.12, 2.92
6.04 -6.24,
6.50
2.92 2.90
6.35,
2.88,
6.14-6.22,
2.96,
HC g
5.46d 4.20 4.20 6.91
3.79-3.96
I
4.97 4.20 4.01 4.17 7.12
4.30 4.66
2.95
3’
4.66 5.54 3.90
/ 5.76
2’
4.90 4.17 4.50
2
4.52 5.52 4.66 4.30
4.02
3.01
5.38 5.04 5.86
3.74-3.87
I
5.33
7.02
is quoted.
the signals given in Table 2 are singlets,
4.48
indicated,
4.27
Unless otherwise
4.48
scale.
4.96
shifts are given on the f
(V) - (VIII).
= m, and either the range of the multiplet or the centre of broad multiplets CDC13 for compounds
Multiplets
(I) - (IV);
et a1.8 __
atoms of either galloyl or 3,4, S-trimethoxybenzoyl
for compounds
(CD3)2C0
to the 2,6-hydrogen
differ from those of P. D. Collier
groups
Solvents: Hg refers
These assignments
3240
No.60
catechin]
and theaflavin-3,
Our configurations
I’-digallate
(IV)
[(-)-epicatechin
(II, Ill, and IV) are different
has kindly informed us that these earlier
gallate and (-)-epigallocatechin
gallate]
.
from those already reported, 8 but Dr. Collier
formulations8
are in error
and that they should be amended
to become identical with ours.
W)
tw w w
cw
-
(a
-
l(V)-]
(-166)~
m/e 496
T6,
(-166)b
m/e 330
m,e 478
m/e 644
m/e 672
m/e 626
OMe
OMe (IX)
(-166)~
m/e 212
(X)
m/e 460
OMe
m/e 195
(XI)
OMe
m/e 191
(XII)
m/e 167
REFERENCES
(1) E. A. H. Roberts,
in The Chemistry of Flavonoid Compounds (edited by T. A. Geissman), p. 468, Pergamon Press, Oxford (1962); D. J. Millin and D. W. Rustidge, Process Biochem. (6), 2, 9 (1967); D. J. Millin, D. Swaine, and P. L. Dix, J.Sci.Food Agric. 22, 296 (1969).
Nature 221, 742 (1969); (2) A. G. Brown, W. B. Eyton, A. Holmes, and W. D. Ollis, -Phytochemistrp
5, 2333 (1969).
(3)
Y. Takino,
A. Ferretti,
V. Flanagan, M. Gianturco,
(4)
A. G. Brown, C. P. Falshaw, 1193 (1966).
E. Haslam,
and M. Vogel,
A. Holmes,
Tetrahedron
and W. D. Ollis,
Tetrahedron
(5)
D. T. Coxon, A. Holmes,
and W. D. Ollis,
Tetrahedron
Letters
(1970).
(6)
D. T. Coxon, A. Holmes,
and W. D. Ollis,
Tetrahedron
Letters
(1970).
(7)
E. A. H. Roberts and M. Myers,
(8)
T. Bryce, P. D. Collier, Letters 2789 (1970).
J.Sci.Food
I. Fowlis,
Agric.
P. E. Thomas,
Letters
4019 (1965).
Letters
Lo=, 176 (1959). D. Frost,
and C. K. Wilkins,
Tetrahedron
Printed in Great Britain.
THE CONSI’IlUTION AND CONFIGURATION OF THE THEAFLAVIN FIGMENTS OF BLACK TEA D. T. Coxon, A. Holmes, W. D. Ollis, * and V. C. Vora Department of Chemistry, The University, Sheffield S3 7HF (Received in UK 7 October 1970; accepted for publication
25 November 1970)
The pigments characteristic of black tea infusions are of two general types’ associated with the theaflavin fraction and the thearubigin fraction. proanthocyanidins . 2
The thearubigins have been recognised as polymeric
The theaflavin fraction contains a considerable number of compounds which are
benxtropolone derivatives including theaflavm 3’4 (I; ca. 8%). theaflavin-3-gallate (II; ca. 3%). the&h&n-3’-gallate
(III; ca. 20%), theaflavin-3.3’-digallate
theaflavic and epitheaflavic acids6
(IV; ca. 4%).
isotheaflavin5 (4%). and
the approximate relative proportions given by our present
fractionation procedures are indicated.
We now report upon the elucidation of the constitutions snd
configurations of the galloyl esters (II, III, and IV) by spectroscopic methods, and the confirmation of these configurations by synthesis. Chromatography [Sephadex LH 20; propan-2-01, acetic acid, water (4 : I: 4)] of the thesflavin fraction7 gave three well separated bands.
The first consisted mainly of theaflavin (I) and isotheaflavin,
the second yielded a mixture of the monogallates (II and III), and the third gave the digallate (IV).
The
IR spectra of the galloyl esters were very similar to that of theaflavin, but there were additional bands w 1690 cm -1. ) which could be assigned to gallates derived from alcoholic hydroxyl groups. (%o absorption spectra (Table 1) of the compounds (II, III, and IV) were extremely similar to that of
‘Ihe
theailavm (I) and their ORD curves closely corresponded with the ORD curve of theaflavin (I). ‘Ihe molecular formulae of the compounds (II), (III), and (IV) were established by high resolution mass spectrometric examination of their methyl ethers (methylation with dimethyl sulphate and potassium carbonate in boiling acetone). (VI and VII)
[Found: M, 856.2939.
The monogallates (II and III) gave the decamethyl ethers
C36H1806(0Me)lo requires M, 856.29431;
similarly characterised as the tridecamethyl ether (VIII) requires M, 1050.352].
[Found: M, 1050.353.
the digallate (IV) was C43H1907(OMe)13
Their absorption spectra (Table 1) and their mass spectral fragmentation
patterns summarised in the Scheme showed a striking relationship to that of theaflavm heptamethyl ether3 (V) and indicated particularly that the gallates (II, III, and IV) were galloyl derivatives involving either the 3- or the 3’-hydroxyl groups of theaflavin (I). (i) cleavage (-212 a.m.u.)
involving the trimethoxybenxoyloxy residues and (ii) retro-Diels-Alder
reactions (-166 a.m.u.). *
The fragmentations were clearly dominated by
To whom correspondence should be addressed. 5237
5
No.60
OR1
X = Galloyl(3,4.5-trlhydronybenroyl);
(IV).
of the NMR spectra
Although the monogallates
these three galloyl configurations
chloride
H
(III)
H
(Iv)
H
approximate
the assignments the indication
in pyridine gave,
(v)
Me
(VI)
Me
(VII)
Me
(VIII)
Me
1
hmax (Emax) in ethanol
supported the constitutions
by chromatography ratio 3
from their ORD curves
Having established
the Nh4R spectra
the constitutions
that they had corresponding
llmaflavin
after chromatographic
ether (VIII) and a mixture of theaflavin-3-
as a single fraction,
(II), (III), and
: 2) and the derived mixture of decamethyl
given in Table 2.
(II, III, and IV) was settled by synthesis.
methoxybenzoyl tridecamethyl
esters,
H
given in Table 2 clearly
were isolated
obtained on this mixture (II + III; ethers (VI + VII) permitted
spectra
(1) (IQ
R3 =
Y = 3,4,5-trimethoxybenzoyl
TABLE Absor@on
Comparison
R2 =
R1 =
_
heptamethyl
fractionation,
and -3’-monogallate
of
absolute
ether (V) and 3,4, S-tritheaflavin3,3:digallate
decamethyl
ethers
(VI and
VII). The theaflavln oxldative
process
is not involved,
pigments do not occur
during the fermentation then the probable
and (-)-epigallocatechin
gallate],
in the green leaf,
but are formed
of green leaf to give black tea.
precursors’
are as indicated:
theaflavin-3’-gallate
(III)
theaflavln
[(-)-epicatechin
by an enzyme-catalysed If hydrolysis 3-gallate
of gallate esters (II)
[(-)-epicatechln
gallate and (-)-epigallo-
2 G?
a
b
C
d
Chemical
5”
2.00
7”
-4.84
-4.88
Tropolone -OH
3.05
3.12
MHz.)a
3”
2.02
1.98
-4.76
(100
6, 8; 6’, 8’ ma 2.46 1.99
1.98
-4.81
Spectra
4., 4’ ma 3.97-3.99 2.41
1.99
2.01
NMR
ma 7.07 3.85-4.01 2.41
2.01
2.35
2.
5.54d 6.92 3.85-4.01
2.26
2.79
TABLE
4.25
I 5.36 6.92 3.82-3.89
4.05
5.86
7.09
7.09
3.72-3.91
3.72-3.91
4.05
4.05
2.96
2.96
2.52
2.43
2.36
6.04,
6.45
6.27-6.30
Methoxyl
6.04-6.24,
6.45
6.12, 2.92
6.04 -6.24,
6.50
2.92 2.90
6.35,
2.88,
6.14-6.22,
2.96,
HC g
5.46d 4.20 4.20 6.91
3.79-3.96
I
4.97 4.20 4.01 4.17 7.12
4.30 4.66
2.95
3’
4.66 5.54 3.90
/ 5.76
2’
4.90 4.17 4.50
2
4.52 5.52 4.66 4.30
4.02
3.01
5.38 5.04 5.86
3.74-3.87
I
5.33
7.02
is quoted.
the signals given in Table 2 are singlets,
4.48
indicated,
4.27
Unless otherwise
4.48
scale.
4.96
shifts are given on the f
(V) - (VIII).
= m, and either the range of the multiplet or the centre of broad multiplets CDC13 for compounds
Multiplets
(I) - (IV);
et a1.8 __
atoms of either galloyl or 3,4, S-trimethoxybenzoyl
for compounds
(CD3)2C0
to the 2,6-hydrogen
differ from those of P. D. Collier
groups
Solvents: Hg refers
These assignments
3240
No.60
catechin]
and theaflavin-3,
Our configurations
I’-digallate
(IV)
[(-)-epicatechin
(II, Ill, and IV) are different
has kindly informed us that these earlier
gallate and (-)-epigallocatechin
gallate]
.
from those already reported, 8 but Dr. Collier
formulations8
are in error
and that they should be amended
to become identical with ours.
W)
tw w w
cw
-
(a
-
l(V)-]
(-166)~
m/e 496
T6,
(-166)b
m/e 330
m,e 478
m/e 644
m/e 672
m/e 626
OMe
OMe (IX)
(-166)~
m/e 212
(X)
m/e 460
OMe
m/e 195
(XI)
OMe
m/e 191
(XII)
m/e 167
REFERENCES
(1) E. A. H. Roberts,
in The Chemistry of Flavonoid Compounds (edited by T. A. Geissman), p. 468, Pergamon Press, Oxford (1962); D. J. Millin and D. W. Rustidge, Process Biochem. (6), 2, 9 (1967); D. J. Millin, D. Swaine, and P. L. Dix, J.Sci.Food Agric. 22, 296 (1969).
Nature 221, 742 (1969); (2) A. G. Brown, W. B. Eyton, A. Holmes, and W. D. Ollis, -Phytochemistrp
5, 2333 (1969).
(3)
Y. Takino,
A. Ferretti,
V. Flanagan, M. Gianturco,
(4)
A. G. Brown, C. P. Falshaw, 1193 (1966).
E. Haslam,
and M. Vogel,
A. Holmes,
Tetrahedron
and W. D. Ollis,
Tetrahedron
(5)
D. T. Coxon, A. Holmes,
and W. D. Ollis,
Tetrahedron
Letters
(1970).
(6)
D. T. Coxon, A. Holmes,
and W. D. Ollis,
Tetrahedron
Letters
(1970).
(7)
E. A. H. Roberts and M. Myers,
(8)
T. Bryce, P. D. Collier, Letters 2789 (1970).
J.Sci.Food
I. Fowlis,
Agric.
P. E. Thomas,
Letters
4019 (1965).
Letters
Lo=, 176 (1959). D. Frost,
and C. K. Wilkins,
Tetrahedron