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The biosynthesis of 2,5,6-trihydroxy-9-phenylphenalenone by Lachnanthes tinctoria. Incorporation of 1-13C-phenylalanine
Tetrahedron Letters No. 51, pp 4471 - 4474, 1977.
THE
BIOSYNTHESIS
BY LACHNANTHES
OF 2,5,6-TRIHYDROXY-9-PHENYLPHENALENONE INCORPORATION
TINCTORIA.
Alan D. Harmon School
of Pharmacy,
PergarnonPress. Printed in Great Britain.
University
OF 1-13C-PHENYLALANINE.1
and J. M. Edwards*
of Connecticut,
Storrs,
06268 USA
Conn.
R. J. Highet* Laboratory National
Heart,
(Received in
USA
Lung,
of Chemistry
and Blood
Institute,
pigment
isolated
In 1961,
(Haemodoraceae).' 9-phenylphenalenones
might
combination
of one mole
acetic
(Scheme);
acid
(3j4 would
precursors
phenylalanine, haemocorin observed
tyrosine,
aglycone
of 4, an observation biosynthetic
suggested
proceed
through
cyclisation
to the natural have
USA
and acetate
incorporation
which
lent
of lachnanthoside
of Lachnanthes
a sequence
involving
and tyrosine, of the
products.
established
aglycone
tinctoria
that the biosynthesis
each of phenylalanine
(4) by Haemodorum
the exclusive
(1) is the
from the root-system
Thomas3
oxidative
lead directly
14 C-labelled
MD 20014,
1 September 1977; received in UX for publication 31 October 1977)
2,5,6-Trihydroxy-9-phenylphenalenone (21, the major
Bethesda,
the ready
of the
the unique
and one mole
intermediate
biarylheptanoid
Biosynthetic
studies
incorporation
into 1 by 5. tinctoria,5
label
considerable
from 2support
using
of
and into
corymbos,,m ;6 significantly, of
of
14 C-tyrosine
Thomas
6
into C-5
to the proposed
scheme.
3
kOOH
1 R=OH,
R’-- I
2 R=OH,R’ 4 R=OCH,,R’
4471
B ose I
4472
No.
The preliminary a dramatic with the
(3.8%)
lack
biosynthetic
studies
incorporation
of suitable
techniques
thought
and carbons
7, 8 and 91, led us to an investigation
from 1-13C-glycine
with
et al,9 --
in 36% yield.
solution
isomeric
plants
showed
up by the plants.
26%
of 10.9%
discrepancy
scintillation
counting
Examiaation abundance
34%
128.8
occurs with
C-7 by single
most
deshielded
to
substituent, incorporation
between and MS
had been
proton
ratio
deductions
the
of the
had been
and converted
This
incorporation
acid together
analysis
described5
There
and
taken into the
(29.5 mg,
result
indicated
into the
isolated
into 6 indicated is no explanation
data
obtained
the for
by liquid
analysis. noise
decoupled
showed
sample.
CMR spectra
a significant
We have
decoupling
of 6, however,
experiments.
were
was calculated observed could
at 128.8
be made
peak
of 6 at natural
peak
been unable
in the PMR spectrum
the enriched
amino
incorporated
species.
prepared
and Michael,'
3 days,
was isolated,
(2 = 160 Hz) when undecoupled,
of 320% was
(see Figure);
access
fed to 36 prewashed,
of the 13C incorporation
of the CMR signals
and a peak height
Complementary
substrate
analysis
resonance
heights
After
incorporation").
in the CMR spectrum
as a doublet
enhancement
coupled
by MS71 was
(51 mg) of the phenylalanine
13C-enrichment,
resonances
The peak
the roots.
aglycone
specific
ppm in the enriched
all the
upon the
of Kirby
(5 and 6) as previously
of the proton
and with
13C determined
(4.6 x lo7 dpm) was
of the mono-labelled
the apparent
5
that
trimethylethers
spectrometric
presence
based
of the methods
through
6 mg of the 13C-labelled Mass
allow
(viz. the 9-phenyl
150 mg of the 13C-enriched
Lachnanthoside
1.3 x 107 dpm/mnole,
1.
excess
by a combination
Lachnanthes
feeding
that
(89 atom%
0.06 mg of l-l4 C-phenylalanine
growing,
two
from phenylalanine
would
This,
showed
substrate.
l-13C-Phenylalanine
Strange,
into 1.
which
the carbons
of a 13C-labelled
with L. tinctoria5
of l-14 C-phenylalanine
degradative
to arise
undertaken
51.
enhancement
to assign
was
readily
The proton
and resonates
normalized
correlated at C-7
of C-4
(relative
spectra
peak height
is
the
(118.5
(Table).
ppm in the 13C-enriched from the
ppm, which
at 8.57 ppm.2*11
to that
for each resonance
unequivocally
at 128.8
that
at
of the
An 6.
isomeric
increase
ppm),
ether
500%) was at
4473
No. 51
118.5 Natural-Abundance CMR Spectrum of Trimethyl Ether (5); 15 mg in 2.5 ml CDC13; 44816 Transients, 20° Pulse.
128.2
118.5
CMR Spectrum of 13C-Enriched Trimethyl Ether (5); 30° Pulse. 14.8 mg in 2.5 ml CDC13; 47919 Transients;
5
6
4474
No. 51
128.2 ppm which label port
is again
into carbon for Thomas'
consistant
with
7. We feel that these hypothesis
concerning
the specific observations
incorporation provide
the biosynthesis
of 13C
significant
sup-
of the plant
phenalenones. Table.
CMR of Biosynthetic
6.
Relative Chemical
Shift
Peak
??atural abundance/
Height
13
C-Enriched
99/100 zx
153/642
128:7 127.1 126.8 124.9 118.3 113.0 60.8 56.9 55.6
2181202 135/128 100/100 47/ 39 88/ 92
Determined
on a JEOL FX-60 nmr spectrometer at 15 MHz; over 1000 Hz were used with 1.2 set pulse repetition rate and 30 pulses. Both spectra were obtained on ca. 0.02 M solutions.
8~ data points
1.
REFERENCES Part VIII in the series "Pigments of L. tinctoria Eli.". Part J.A. zito, and J.M. Edwards, Lloyaia, 39, 192 (1976).
2.
J.M.
3.
R. Thomas,
4.
A.C.
Bazan,
5.
J.M.
Edwards,
6.
R. Thomas,
7.
I.M. Campbell,
8.
G.W. Kirby
9.
P.G.
Edwards
and U. Weiss,
Biochem.
J., 78,
J.M. Edwards, R.C.
Chem.
Phytochemistry,
Bioorganic
and J. Michael,
Tetrahedron
and U. Weiss,
739
(1974).
(1961).
and U.Weiss,
Schmidt,
Commun.,
807
l3, 1597
Letters,
Phytochemistry,
147
2,
Chemistry, J. Chem.
3,
386
and J.M. Edwards,
1717
(1972).
(1974).
Sot. Perkin
I, 115
(1973).
Strange, J. Staunton, H.R. Wiltshire, A.R. Battersby, and E.A. Havir, J. Chem. Sot. Perkin I, 2364 (1972).
R.J. Highet
(1977).
(1971).
K.R.
10. Specific incorporation is defined as the specific radioactivity in the metabolite divided by the specific radioactivity of the precursor that was fed. 11.
VII:
J. Mag.
Res.,
l7,
336 (1975).
Hanson,
found
THE
BIOSYNTHESIS
BY LACHNANTHES
OF 2,5,6-TRIHYDROXY-9-PHENYLPHENALENONE INCORPORATION
TINCTORIA.
Alan D. Harmon School
of Pharmacy,
PergarnonPress. Printed in Great Britain.
University
OF 1-13C-PHENYLALANINE.1
and J. M. Edwards*
of Connecticut,
Storrs,
06268 USA
Conn.
R. J. Highet* Laboratory National
Heart,
(Received in
USA
Lung,
of Chemistry
and Blood
Institute,
pigment
isolated
In 1961,
(Haemodoraceae).' 9-phenylphenalenones
might
combination
of one mole
acetic
(Scheme);
acid
(3j4 would
precursors
phenylalanine, haemocorin observed
tyrosine,
aglycone
of 4, an observation biosynthetic
suggested
proceed
through
cyclisation
to the natural have
USA
and acetate
incorporation
which
lent
of lachnanthoside
of Lachnanthes
a sequence
involving
and tyrosine, of the
products.
established
aglycone
tinctoria
that the biosynthesis
each of phenylalanine
(4) by Haemodorum
the exclusive
(1) is the
from the root-system
Thomas3
oxidative
lead directly
14 C-labelled
MD 20014,
1 September 1977; received in UX for publication 31 October 1977)
2,5,6-Trihydroxy-9-phenylphenalenone (21, the major
Bethesda,
the ready
of the
the unique
and one mole
intermediate
biarylheptanoid
Biosynthetic
studies
incorporation
into 1 by 5. tinctoria,5
label
considerable
from 2support
using
of
and into
corymbos,,m ;6 significantly, of
of
14 C-tyrosine
Thomas
6
into C-5
to the proposed
scheme.
3
kOOH
1 R=OH,
R’-- I
2 R=OH,R’ 4 R=OCH,,R’
4471
B ose I
4472
No.
The preliminary a dramatic with the
(3.8%)
lack
biosynthetic
studies
incorporation
of suitable
techniques
thought
and carbons
7, 8 and 91, led us to an investigation
from 1-13C-glycine
with
et al,9 --
in 36% yield.
solution
isomeric
plants
showed
up by the plants.
26%
of 10.9%
discrepancy
scintillation
counting
Examiaation abundance
34%
128.8
occurs with
C-7 by single
most
deshielded
to
substituent, incorporation
between and MS
had been
proton
ratio
deductions
the
of the
had been
and converted
This
incorporation
acid together
analysis
described5
There
and
taken into the
(29.5 mg,
result
indicated
into the
isolated
into 6 indicated is no explanation
data
obtained
the for
by liquid
analysis. noise
decoupled
showed
sample.
CMR spectra
a significant
We have
decoupling
of 6, however,
experiments.
were
was calculated observed could
at 128.8
be made
peak
of 6 at natural
peak
been unable
in the PMR spectrum
the enriched
amino
incorporated
species.
prepared
and Michael,'
3 days,
was isolated,
(2 = 160 Hz) when undecoupled,
of 320% was
(see Figure);
access
fed to 36 prewashed,
of the 13C incorporation
of the CMR signals
and a peak height
Complementary
substrate
analysis
resonance
heights
After
incorporation").
in the CMR spectrum
as a doublet
enhancement
coupled
by MS71 was
(51 mg) of the phenylalanine
13C-enrichment,
resonances
The peak
the roots.
aglycone
specific
ppm in the enriched
all the
upon the
of Kirby
(5 and 6) as previously
of the proton
and with
13C determined
(4.6 x lo7 dpm) was
of the mono-labelled
the apparent
5
that
trimethylethers
spectrometric
presence
based
of the methods
through
6 mg of the 13C-labelled Mass
allow
(viz. the 9-phenyl
150 mg of the 13C-enriched
Lachnanthoside
1.3 x 107 dpm/mnole,
1.
excess
by a combination
Lachnanthes
feeding
that
(89 atom%
0.06 mg of l-l4 C-phenylalanine
growing,
two
from phenylalanine
would
This,
showed
substrate.
l-13C-Phenylalanine
Strange,
into 1.
which
the carbons
of a 13C-labelled
with L. tinctoria5
of l-14 C-phenylalanine
degradative
to arise
undertaken
51.
enhancement
to assign
was
readily
The proton
and resonates
normalized
correlated at C-7
of C-4
(relative
spectra
peak height
is
the
(118.5
(Table).
ppm in the 13C-enriched from the
ppm, which
at 8.57 ppm.2*11
to that
for each resonance
unequivocally
at 128.8
that
at
of the
An 6.
isomeric
increase
ppm),
ether
500%) was at
4473
No. 51
118.5 Natural-Abundance CMR Spectrum of Trimethyl Ether (5); 15 mg in 2.5 ml CDC13; 44816 Transients, 20° Pulse.
128.2
118.5
CMR Spectrum of 13C-Enriched Trimethyl Ether (5); 30° Pulse. 14.8 mg in 2.5 ml CDC13; 47919 Transients;
5
6
4474
No. 51
128.2 ppm which label port
is again
into carbon for Thomas'
consistant
with
7. We feel that these hypothesis
concerning
the specific observations
incorporation provide
the biosynthesis
of 13C
significant
sup-
of the plant
phenalenones. Table.
CMR of Biosynthetic
6.
Relative Chemical
Shift
Peak
??atural abundance/
Height
13
C-Enriched
99/100 zx
153/642
128:7 127.1 126.8 124.9 118.3 113.0 60.8 56.9 55.6
2181202 135/128 100/100 47/ 39 88/ 92
Determined
on a JEOL FX-60 nmr spectrometer at 15 MHz; over 1000 Hz were used with 1.2 set pulse repetition rate and 30 pulses. Both spectra were obtained on ca. 0.02 M solutions.
8~ data points
1.
REFERENCES Part VIII in the series "Pigments of L. tinctoria Eli.". Part J.A. zito, and J.M. Edwards, Lloyaia, 39, 192 (1976).
2.
J.M.
3.
R. Thomas,
4.
A.C.
Bazan,
5.
J.M.
Edwards,
6.
R. Thomas,
7.
I.M. Campbell,
8.
G.W. Kirby
9.
P.G.
Edwards
and U. Weiss,
Biochem.
J., 78,
J.M. Edwards, R.C.
Chem.
Phytochemistry,
Bioorganic
and J. Michael,
Tetrahedron
and U. Weiss,
739
(1974).
(1961).
and U.Weiss,
Schmidt,
Commun.,
807
l3, 1597
Letters,
Phytochemistry,
147
2,
Chemistry, J. Chem.
3,
386
and J.M. Edwards,
1717
(1972).
(1974).
Sot. Perkin
I, 115
(1973).
Strange, J. Staunton, H.R. Wiltshire, A.R. Battersby, and E.A. Havir, J. Chem. Sot. Perkin I, 2364 (1972).
R.J. Highet
(1977).
(1971).
K.R.
10. Specific incorporation is defined as the specific radioactivity in the metabolite divided by the specific radioactivity of the precursor that was fed. 11.
VII:
J. Mag.
Res.,
l7,
336 (1975).
Hanson,
found