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One-carbon units in the biosynthesis of hydrastine and berberine
Vol. 13, No. 2, 1963
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ORE-CARSON UNITS IN TEEBIOSYNTRESIS OFHYDRASTINEANDBERBERINE+ RamNath Gupta and Ian D. Spenser Department of Chemistry, McMsster University Hamilton, Ontario. Canada
Received
July
8, 1963
With the demonstration that two C6-C2 units, incorporated
into hydrastine
(Spenser and Gear, 196.h)
derived from tyrosine,
(Gear and Spenser, 1961,
and into narcotine
19631,
(Battersby
in the biosynthesis
into berberine
and McCaldin, 19621,
that dopamine (Spenser and Gear, 1962b) and norlaudanosoline McCaldin, 1962) are utilized
are
(Battersby
and
and
of this group of compounds, a
source of all but one of the carbon atoms in the nucleus of membersof the pthalideisoquinoline
and protoberberine
alkaloids
has been established.
The remaining carbon atom in the nucleus of these bases, i.e., csrbonyl carbon of hydrastine berberine,
the lactone-
and of narcotine , and the so-called bridge-carbon
has long been postulated to arise from a one-carbon unit
In agreement with this classical from labelled
hypothesis it
from the lactone-carbonyl
group of narcotine
(Robinson, 1955)
has been shown that radioactivity
administered to Papaver somniferum
formate,
(Battersby
plants,
vas recovered
and McCaldin, 1962).
In the present communication evidence is presented which shows that the lactone-carbonyl
carbon of hydrastine,
0- and N-attached extra-nuclear
the bridge-carbon of berberine and the
one-carbon substituents
of both bases are
derived from one-carbon precursors , and that the methyl group of methioniae is incorporated
into these sites more efficiently
than formate.
In a series of experiments sodium formate-C 14 and methionine methyl-Cl4 were administered to plants of Hydra&is root or by infusion
into the stem (c.f. -
canadensis &., either
by way of the
Gear and Spenser, 1963).
Labelled
* Supported by a grant from the Ontario Research Foundation. 115
of
Vol. 13, No. 2, 1963
hydrastine
BIOCHEMICAL
AND BlOPHYSlCAL
and berberine were isolated
RESEARCH COMMUNICATIONS
from the roots and rhizomes.
and radiochemical yields are recorded in Table 1. activity
Chemical
In terms of the specific
of the products, as well as in terms of recovery in the products of
administered tracer,
methionine appears to be more efficiently
incorporated
then
formate. The labelled degraded (after
ssmples of hydrastine dilution
one-carbon sites.
when necessary) to isolate
with carrier,
Oxidation
and berberine from each experiment were
of hydrsstine
(CH202, 2 x OC$, NC$, CO) gave
(CH202, NCH3) and opianic acid (2 x CC$, CO), which wss isolated
hydrastinine
Exhaustive methylation
as the anil.
trapped as the picrate,
of hydrastinine
and hydrsstsl,
gave trimethylamine
which was further
piperonylic
acid (CH202). In one case the latter
piperonylic
acid and then hydrolysed to yield
veratral,
which was converted to veratric
acid with hydrogen iodide gave inactive which was isolated
was reduced to 6-ethyl-
inactive
6-ethylprotocatechtic
activity
of each of the different
within experimental error, indicating
of berberine
formaldehyde ((X202),
picrate
activities
with the specific
the enil of
Treatment of veratric
acid and methyl iodide
(2 x oCH3). The specific
of the individual
activity
sites agreed,
of the original
hydrastine,
(CH202, 2 x OCH3, bridge) with sulphuric
acid gave
the self-consistency
Hydrolysis
protocatechuic
to yield
suspected one-carbon sites was thus determined
The sum of the specific
separately.
acid (2 x OC$).
as tetramethylamsoniu
(NC&,),
converted to 6-vinyl-
Opianic acid anil (2 x OC$, CO) was decarboxylated
acid.
the predicted
of the results.
isolated
as the dimethone, and a phenolic product which on
treatment with hydrogen iodide gave methyl iodide,
recovered as tetramethyl-
ammoniumpicrate
of the bridge-carbon
(2 x OCH3). Direct determination
as yet incomplete, and its From the specific
activity
activities
was therefore
calculated
by difference.
of the degradation products of hydrastine
berberine obtained in each of the five experiments, the contribution of the one-carbon sites makes to the specific was calculated
(Table 2).
atom is
The results
clearly
116
activity indicate
of the original
and
which each molecule
that the lactone-carbonyl
'u
5
4
3
2
1
-s-
(2.51) ,I
methi3 nine
cl4
1,
I,
(4.81)
HC1400-Na+
x 10-7)
activity count.9 min-l
Precursor (total
wick
root
wick
root
root
6
6
6
6
3
Mode and duration of feeding (days)
460
250
3o3
190
184
Yym
2.68
15.83
1.14
0.67
0.39
specific activity counte mill-1 mN-1 x 10-b
3.22
10.3
0.90
0.33
0.19
total activity counts min-1 x10-4
and Radiochemical
Hydrarptine
Chemical
TABLE:1
749
344
481
208
220
m ytelg;
Yields
3.90
1.54
0.89
x 10-4
4.63
20.50
a-1
specific activity count6 mid
Berberine
8.50
17.3
4.60
0.79
0.48
x 10-4
total activity counts mid
0.47
1.10
0.12
0.023
0.014
% recovery of activity
Vol. 13, No. 2, 1963
BIOCHEMICAL
AND BIOPHYSICAL
group, as well as the methylenedioxy, hydrastine
RESEARCH COMMUNICATIONS
the N-methyl and the O-methyl groups of
are derived from one-carbon precursors.
The results
show further,
that the methylenedioxy and the O-methyl groups of berberine are similarly derived.
Although the one-carbon origin
not demonstrated directly, total
activity
it
is shown that approximately
It has been shown earlier
Sear and Spenser, 1963) that two tyrosine iato the nucleus, accounting for all
its
of the nucleus must therefore
atom, i.e.,
at the bridge-carbon.
carbon for direct
This indicates
units are specifically
incorporated
carbon atoms, but one.
The present
Ekperiments designed to isolate
It is evident
each site makes to the total
(and presumably also of berberine)
that all
one-carbon sites in hydrastine
derived from the sameimmediate precursor.
resulted.
in either
specific magnitude.
(and berberine)
are
For if one of the one-carbon sites
order of magnitude of the specific
the other sites,
(Table 2) that
is of similar
had been derived from some other immediate precursor than all different
the bridge-
are in progress.
inferences may be drawn.
in every experiment the Contribution of hydrastine
(Spenser and Sear, 1%2a,
be concentrated at the remaining carbon
assay of radioactivity
A number of further
activity
one-quarter of the
of berberine derived from one-carbon precursors resides in the
nucleus of the molecule.
activity
of the bridge-carbon of berberine is
activity
other sites,
a
at that site compared to
the formate or the methionine experiments, would have
It follows that the lactone-carbonyl
bridge carbon of berberine)
carbon of hydrastine
(and the
are derived from the sameprecursor as the extra-
nuclear one-carbon units. Since the incorporation efficient
than the incorporation
methyl and formate give rise, ultimate
of methionine-methyl
one-carbon precursor,
of formate, it
into both alkaloids follows either
was more
that methionine-
by independent pathways, to one and the same the former at a faster
rate than the latter,
or
that formate and methionine each represent stages on a commonroute to the ultimate
one-carbon precursor.
On the basis of established pathways of one-
carbon metabolism, the second alternative likely
that the ultimate
is the more attractive,
end it
one-carbon precursor is S-adenosylmethionine, 118
is
and that
2 OCH
GO
41 t 3
18 ,+ 4
18 2 6
2122 18 2 1
Sumof specific
activity
activity
Specific
Specific
by difference
2
3
4 5
a
b
C
d
of intact
of intact
,+ 3
3
berberine = 100
hydrastine
sites = 100
2222
101 ,+ 2
one-carbon
25 ,+ 2-
37 ,+ 3
26 k 3
31 ,+ 3
3
CCH
of
52 2 2 53 f 2
41 ,+ 2
5023
47 ,+ 2
2
distribution
bridged
Relative
103 t 3
102 2 8
103 t 6
92 ,+ 7
Total t%jb
= 100
24 2 1 29 2 1
16 2 2
20 f 2
20
NCH
(%la
of individual
19 f .l 17 2 1
24 f 2
2122
2122
cH202
of activity
Berberine
of Or+%-carbonUnite into H@ra@tine and Berberine
Hydrastine
activities
36 2 2 35 ,+ 1
42 ,+ 5
43 ,+ 5
17 t 5
3
distribution
Relative
1
Exp.
Incorporation
TABLE2
,+ 2
(%I’
23 ,+ 2 25 ,+ 1
2221
23 2 i
21
cH202
activity
H v,
8 E f 7i 3
H
fi ks P
9 d %
8
z
2
8
.! 3;
Vol. 13, No. 2, 1963
all
BIOCHEMICAL
one-carbon sites originate
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
by tranemethylation
precedent in other systems for the introduction
proceases.
There is ample
of 0- and N-methyl groups in
this manner. That the formation of the methylenedioxy group requires euch a step is very likely,
since it was conclusively
demon&rated (Barton,
that such a group arises by oxidative present results
are consistent
every caee the specific experimental
error,
cyclization
1962) 'Ihe
They show (Table 2) that in
of the methylenedioxy group is identical,
with the specific
activity
assuming that these are equally labelled, 0-substituents
of an +!ethoxyphenol.
with such a process.
activity
Kirby and Taylor,
within
of each of the two methoxy groups,
suggesting that both types of
arise from the sameprecursor and that they are introduced into
the skeleton in close succession. The origin
of the lactone carbonyl group of hydrastine
carbon of berberine) glance more unusual. take place either
by a route involving
Such a methyl transfer
onto, e.g.,
at the nitrogen atom or at the requisite
catechol nucleus, to yield Starting
a transmethylation
from either
(and the bridge step is at first
norlaudanosoline might carbon
an N- or a C-methylated intermediate, of these, hypothetical
lead to the endproducts in a plausible merits of these alternatives,
atom of the
respectively.
sequences can be written
which
manner. Rather than speculate on the
we prefer to await the outcome of further
experimen
References
Barton, D. H. R., Kirby, 0. W. end Taylor, J. B. (1962). Rroc. Chem. SOC. 340. Battersby, A. R. and McCaldin, D. J. (1962). Proc. Chem. Sot. 365. Gear, J. R. and Spencer, I. D. (1961). Nature 191, 1393. Gear, J. R. and Spenser, I. D. (1963). Can. J. Chem. 9, 783. Robinson, B. (1955). The Structural Relations of Natural Products, p. 86. Clarendon F'resa, Oxford. Spencer, I. D. and Gear, J. R. (1962a). Proc. Chem. Sot. 228. Spencer, I. D. and Gear, J. R. (1962b). J. Am. Chem. Sot. 84, 1059.
120
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ORE-CARSON UNITS IN TEEBIOSYNTRESIS OFHYDRASTINEANDBERBERINE+ RamNath Gupta and Ian D. Spenser Department of Chemistry, McMsster University Hamilton, Ontario. Canada
Received
July
8, 1963
With the demonstration that two C6-C2 units, incorporated
into hydrastine
(Spenser and Gear, 196.h)
derived from tyrosine,
(Gear and Spenser, 1961,
and into narcotine
19631,
(Battersby
in the biosynthesis
into berberine
and McCaldin, 19621,
that dopamine (Spenser and Gear, 1962b) and norlaudanosoline McCaldin, 1962) are utilized
are
(Battersby
and
and
of this group of compounds, a
source of all but one of the carbon atoms in the nucleus of membersof the pthalideisoquinoline
and protoberberine
alkaloids
has been established.
The remaining carbon atom in the nucleus of these bases, i.e., csrbonyl carbon of hydrastine berberine,
the lactone-
and of narcotine , and the so-called bridge-carbon
has long been postulated to arise from a one-carbon unit
In agreement with this classical from labelled
hypothesis it
from the lactone-carbonyl
group of narcotine
(Robinson, 1955)
has been shown that radioactivity
administered to Papaver somniferum
formate,
(Battersby
plants,
vas recovered
and McCaldin, 1962).
In the present communication evidence is presented which shows that the lactone-carbonyl
carbon of hydrastine,
0- and N-attached extra-nuclear
the bridge-carbon of berberine and the
one-carbon substituents
of both bases are
derived from one-carbon precursors , and that the methyl group of methioniae is incorporated
into these sites more efficiently
than formate.
In a series of experiments sodium formate-C 14 and methionine methyl-Cl4 were administered to plants of Hydra&is root or by infusion
into the stem (c.f. -
canadensis &., either
by way of the
Gear and Spenser, 1963).
Labelled
* Supported by a grant from the Ontario Research Foundation. 115
of
Vol. 13, No. 2, 1963
hydrastine
BIOCHEMICAL
AND BlOPHYSlCAL
and berberine were isolated
RESEARCH COMMUNICATIONS
from the roots and rhizomes.
and radiochemical yields are recorded in Table 1. activity
Chemical
In terms of the specific
of the products, as well as in terms of recovery in the products of
administered tracer,
methionine appears to be more efficiently
incorporated
then
formate. The labelled degraded (after
ssmples of hydrastine dilution
one-carbon sites.
when necessary) to isolate
with carrier,
Oxidation
and berberine from each experiment were
of hydrsstine
(CH202, 2 x OC$, NC$, CO) gave
(CH202, NCH3) and opianic acid (2 x CC$, CO), which wss isolated
hydrastinine
Exhaustive methylation
as the anil.
trapped as the picrate,
of hydrastinine
and hydrsstsl,
gave trimethylamine
which was further
piperonylic
acid (CH202). In one case the latter
piperonylic
acid and then hydrolysed to yield
veratral,
which was converted to veratric
acid with hydrogen iodide gave inactive which was isolated
was reduced to 6-ethyl-
inactive
6-ethylprotocatechtic
activity
of each of the different
within experimental error, indicating
of berberine
formaldehyde ((X202),
picrate
activities
with the specific
the enil of
Treatment of veratric
acid and methyl iodide
(2 x oCH3). The specific
of the individual
activity
sites agreed,
of the original
hydrastine,
(CH202, 2 x OCH3, bridge) with sulphuric
acid gave
the self-consistency
Hydrolysis
protocatechuic
to yield
suspected one-carbon sites was thus determined
The sum of the specific
separately.
acid (2 x OC$).
as tetramethylamsoniu
(NC&,),
converted to 6-vinyl-
Opianic acid anil (2 x OC$, CO) was decarboxylated
acid.
the predicted
of the results.
isolated
as the dimethone, and a phenolic product which on
treatment with hydrogen iodide gave methyl iodide,
recovered as tetramethyl-
ammoniumpicrate
of the bridge-carbon
(2 x OCH3). Direct determination
as yet incomplete, and its From the specific
activity
activities
was therefore
calculated
by difference.
of the degradation products of hydrastine
berberine obtained in each of the five experiments, the contribution of the one-carbon sites makes to the specific was calculated
(Table 2).
atom is
The results
clearly
116
activity indicate
of the original
and
which each molecule
that the lactone-carbonyl
'u
5
4
3
2
1
-s-
(2.51) ,I
methi3 nine
cl4
1,
I,
(4.81)
HC1400-Na+
x 10-7)
activity count.9 min-l
Precursor (total
wick
root
wick
root
root
6
6
6
6
3
Mode and duration of feeding (days)
460
250
3o3
190
184
Yym
2.68
15.83
1.14
0.67
0.39
specific activity counte mill-1 mN-1 x 10-b
3.22
10.3
0.90
0.33
0.19
total activity counts min-1 x10-4
and Radiochemical
Hydrarptine
Chemical
TABLE:1
749
344
481
208
220
m ytelg;
Yields
3.90
1.54
0.89
x 10-4
4.63
20.50
a-1
specific activity count6 mid
Berberine
8.50
17.3
4.60
0.79
0.48
x 10-4
total activity counts mid
0.47
1.10
0.12
0.023
0.014
% recovery of activity
Vol. 13, No. 2, 1963
BIOCHEMICAL
AND BIOPHYSICAL
group, as well as the methylenedioxy, hydrastine
RESEARCH COMMUNICATIONS
the N-methyl and the O-methyl groups of
are derived from one-carbon precursors.
The results
show further,
that the methylenedioxy and the O-methyl groups of berberine are similarly derived.
Although the one-carbon origin
not demonstrated directly, total
activity
it
is shown that approximately
It has been shown earlier
Sear and Spenser, 1963) that two tyrosine iato the nucleus, accounting for all
its
of the nucleus must therefore
atom, i.e.,
at the bridge-carbon.
carbon for direct
This indicates
units are specifically
incorporated
carbon atoms, but one.
The present
Ekperiments designed to isolate
It is evident
each site makes to the total
(and presumably also of berberine)
that all
one-carbon sites in hydrastine
derived from the sameimmediate precursor.
resulted.
in either
specific magnitude.
(and berberine)
are
For if one of the one-carbon sites
order of magnitude of the specific
the other sites,
(Table 2) that
is of similar
had been derived from some other immediate precursor than all different
the bridge-
are in progress.
inferences may be drawn.
in every experiment the Contribution of hydrastine
(Spenser and Sear, 1%2a,
be concentrated at the remaining carbon
assay of radioactivity
A number of further
activity
one-quarter of the
of berberine derived from one-carbon precursors resides in the
nucleus of the molecule.
activity
of the bridge-carbon of berberine is
activity
other sites,
a
at that site compared to
the formate or the methionine experiments, would have
It follows that the lactone-carbonyl
bridge carbon of berberine)
carbon of hydrastine
(and the
are derived from the sameprecursor as the extra-
nuclear one-carbon units. Since the incorporation efficient
than the incorporation
methyl and formate give rise, ultimate
of methionine-methyl
one-carbon precursor,
of formate, it
into both alkaloids follows either
was more
that methionine-
by independent pathways, to one and the same the former at a faster
rate than the latter,
or
that formate and methionine each represent stages on a commonroute to the ultimate
one-carbon precursor.
On the basis of established pathways of one-
carbon metabolism, the second alternative likely
that the ultimate
is the more attractive,
end it
one-carbon precursor is S-adenosylmethionine, 118
is
and that
2 OCH
GO
41 t 3
18 ,+ 4
18 2 6
2122 18 2 1
Sumof specific
activity
activity
Specific
Specific
by difference
2
3
4 5
a
b
C
d
of intact
of intact
,+ 3
3
berberine = 100
hydrastine
sites = 100
2222
101 ,+ 2
one-carbon
25 ,+ 2-
37 ,+ 3
26 k 3
31 ,+ 3
3
CCH
of
52 2 2 53 f 2
41 ,+ 2
5023
47 ,+ 2
2
distribution
bridged
Relative
103 t 3
102 2 8
103 t 6
92 ,+ 7
Total t%jb
= 100
24 2 1 29 2 1
16 2 2
20 f 2
20
NCH
(%la
of individual
19 f .l 17 2 1
24 f 2
2122
2122
cH202
of activity
Berberine
of Or+%-carbonUnite into H@ra@tine and Berberine
Hydrastine
activities
36 2 2 35 ,+ 1
42 ,+ 5
43 ,+ 5
17 t 5
3
distribution
Relative
1
Exp.
Incorporation
TABLE2
,+ 2
(%I’
23 ,+ 2 25 ,+ 1
2221
23 2 i
21
cH202
activity
H v,
8 E f 7i 3
H
fi ks P
9 d %
8
z
2
8
.! 3;
Vol. 13, No. 2, 1963
all
BIOCHEMICAL
one-carbon sites originate
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
by tranemethylation
precedent in other systems for the introduction
proceases.
There is ample
of 0- and N-methyl groups in
this manner. That the formation of the methylenedioxy group requires euch a step is very likely,
since it was conclusively
demon&rated (Barton,
that such a group arises by oxidative present results
are consistent
every caee the specific experimental
error,
cyclization
1962) 'Ihe
They show (Table 2) that in
of the methylenedioxy group is identical,
with the specific
activity
assuming that these are equally labelled, 0-substituents
of an +!ethoxyphenol.
with such a process.
activity
Kirby and Taylor,
within
of each of the two methoxy groups,
suggesting that both types of
arise from the sameprecursor and that they are introduced into
the skeleton in close succession. The origin
of the lactone carbonyl group of hydrastine
carbon of berberine) glance more unusual. take place either
by a route involving
Such a methyl transfer
onto, e.g.,
at the nitrogen atom or at the requisite
catechol nucleus, to yield Starting
a transmethylation
from either
(and the bridge step is at first
norlaudanosoline might carbon
an N- or a C-methylated intermediate, of these, hypothetical
lead to the endproducts in a plausible merits of these alternatives,
atom of the
respectively.
sequences can be written
which
manner. Rather than speculate on the
we prefer to await the outcome of further
experimen
References
Barton, D. H. R., Kirby, 0. W. end Taylor, J. B. (1962). Rroc. Chem. SOC. 340. Battersby, A. R. and McCaldin, D. J. (1962). Proc. Chem. Sot. 365. Gear, J. R. and Spencer, I. D. (1961). Nature 191, 1393. Gear, J. R. and Spenser, I. D. (1963). Can. J. Chem. 9, 783. Robinson, B. (1955). The Structural Relations of Natural Products, p. 86. Clarendon F'resa, Oxford. Spencer, I. D. and Gear, J. R. (1962a). Proc. Chem. Sot. 228. Spencer, I. D. and Gear, J. R. (1962b). J. Am. Chem. Sot. 84, 1059.
120