Biochemical Pharmacology, Vol 24, pp. 929-932. Pergamon Press, 1975. Printed in Great Britain.
PRELIMINARY
ENZYMATIC
FORMALDEHYDE
DROFOLIC
ACID:
PRODUCTION
PRIOR
Pierre Department
COMMUNICATION
STEP
Laduron
5-METHYLTETRAHY-
TO ALKALOID
and Joske
of Neurobiochemistry, B-2340
FROM
Leysen
Janssen
Beerse,
FORMATION*
Pharmaceutics
Belgium
(Received 3 January 1975;accepted 31 J~IIUW 1975)
5-Methyltetrahydrofolic methylation enzymatic more
of dopamine reaction
general
nergic
in contrast
features
used
methyl
We tested
after crease zyme
reaction transfer
previously from
blank was run (Table of the assay,
properties
of this
enabling
us to attribute
involving
a
only the adre-
In fact that after
in the blank without
Part of this work July 1974
substrate
the corresponding
were
or
obtained
depending
suggested,
N-
or 5-hydroxy-
recently
markedly,
This
in this study:
The radioactivity with 5-Me
differed
Cl4
by
on the
that something
In contrast
other
than those
the amount
were
caused
from
re-
and two enzyme on how the
lower
than those
a tremendous
the blank containing for the blank without decreased
the substrate
at the IXth C. I. N. P.
929
in extracts
depending
of radioactivity
than in that where
rat kidney
3-hydroxyphenylethylamine)
to pH 10, to this,
one from
-H4-folate
markedly,
(4-methoxy,
was brought
was presented
measured
all the blank values
(even lower
evaporation,
results
differed
preparations
of substrate
mixture
values
Similar
First-
was involved.
Although
I).
this investigation.
N-methyltryptamine
products.
incubated
in the radioactivity. lower
6
rat kidney,
the addition
the incubation
gave
Various
we did not identify
blank values
pig brain.
isolated
4, 5
when tryptamine,
two enzyme
from
mixtures
fractions
the N-
us to undertake
as substrate.
of isolating
than a simple
action
to mediate
than a function
prompted
9 Secondly,
group.
and another
reported
2-7
been described,
studies
compounds were
procedure
1
in rat brain.
to this reaction
to other
0-methylated tryptamine
recently
8
system.
another
to epinine
have since
function
Two main ly,
acid was
in-
boiled
en-
substrate). much more
was added after
Congress,
Paris,
Preliminary Communication
930
TABLE
Radioactivity extracted from reaction mixtures incubated with 5-MeC14-H4-folate and purified enzyme fractions from rat kidney
I.
Total
extracted radioactivity (CPM x 103) Peak A 1 Peak B
Before evaporation Assay Blank Boiled enzyme Without substrate Substrate added after
19.1
33.4
1.4 2.1 8.4
2.5 3.8 18.8
0.3 8.4
0.2 7.6
fixation
After evaporation Blank Without substrate Substrate after fixation
Ammonium sulphate enzyme fraction (30 % to 60 %) from rat kidney was further purified on DEAE A50 Sephadex columns as described elsewhere. 9 An aliquot of two peaks (A and B) of enzyme activity was incubated with 0.1 pCi 5-MeC14-H4-folate (spec.act. 61 mCi/ mmol Radiochemical Centre Amersham) and the reaction products were extracted as previously described. 6
fixation,
suggested
formed.
Moreover,
a reaction
that,
during
this product
seemed
to occur
dance with this hypothesis, using
the dimedon
5-Me-H4-folate These
suggesting
condensation
by experiments amine
considerably
lowered
enzymatically
between
enzyme.
the amount
Here
with
amines.
11
like 4-methoxy, formed
aldehyde
formaldehydeC14
but without
incubated
was added to the incubation
reaction
where
demonstrated
of biogenic
of substrate
formaldehyde
In accor-
fixation.
mixtures
but in the absence
was
for the amines;
was recently
m reaction
if the amine
product
high affinity
production 10.
a volatile
added after
in the presence
The free
a possible
0” C with a biogenic
of the amine
enzyme
tremendously
This view is supported at
method,
now confirmed
3 -hydroxyphenylethylamine.
tures
have a very
formaldehyde
precipitation
found to decrease
must
period,
with the substrate
and rat kidney
data were
the incubation
was
mix-
and amine.
was only incubated again,
of formaldehyde
the presence precipitated
with dimedon. These
experiments
enzymatically but also
formed
approach
sented
in figure
2-methyl layer
from
that an additional
A first
clearly
5-Me-H reaction
in elucidating 1.
6, 7-hydroxy
chromatography
demonstrated 4
-folate
occurred
the nature
By incubating
epinine
not only that formaldehyde
as reported between
of this
1).
Similar
the aldehyde
condensation
with a pig brain
1, 2, 3, 4-tetrahydroisoquinoline (fig.
in detail
results
were
elsewhere,
11
and amine.
product
enzyme,
was identified obtained
was
is pre-
an alkaloid, by thin-
with other
amines
931
PreliminaryCommunication
Fig. 1 - Radiochromatograms of a reaction mixture incubated with 5-MeC14 -H4-folate, epinine and an ammonium sulphate enzyme fraction (30 to 65 per cent) from pig brain. Reaction products were isolated on A1203 columnsP An aliquot was spotted on (A) cellulose F and (B) silica gel 60 F plated. Solvent systems: (A) phenol-water-HCl 80/20/l (B) isobutyl alcohol-formic acidwater 150/30/20. Radioactivity was measured with a Berthold scanner and standards (E: epinine and Mi: 2-methyl 6,7 -hydroxy 1, 2, 3, 4-tetrahydroisoquinoline) were detected in U.V.
MI
t
like
tryptamine,
amine
using
respective mass
E
N-methyltryptamine
five
different
reaction
plains
the earlier
not be obtained the indole
as it will
but does
detected rence
13
postulated.
parkinsonian
results
A similar
already
described.
12
elsewhere.
and This
ex-
of indoleamines
of a possible
being
treated
14
As
enzyme seem
shown
could
could
methylation
more
of
in fig.
similar
2, the formation
it has not yet for
aceof been
the occur-
in numerous
to those
as
with
in the urine
evidence
is present
by the
formaldehyde
in vivo although
of the
in viva
of dopamine
provide
which or less
with
reaction
13
that alkaloids
be formed
amines
L-Dopa
results
tissues
of an enzyme
of certain
alkaloids
the Fig. 2 - Model illustrating alkaloid formation from biogenic amines through a one-carbon transfer from 5-Me-H4-folate with formaldehyde as intermediate compound.
ENZYME
scsa+:m-c”, E
group
demonstrated
with our
Furthermore,
of which
the view
condensation
5-Me-HI-FOLLIE
1 H‘+X#JE
of the
chromatography
N-methylation
support
has been
of a formaldehyde-forming
and the properties
in detail
of corresponding
salsolinol)
patients
in brain.
the identity
by gas
the suggestion
or the j3 -carboline
condensation
(yielding
be reported
not confirm
Moreover
proved
6, 9 in which
the foregoing
tetrahydroisoquinoline
taldehyde
further
3-hydroxyphenylethyl-
9
nitrogen.
Pictet-Spengler
12
systems.
was
experiments
Therefore,
recently
solvent
products
fragmentography
and 4-methoxy,
_
::a+ MI
Preliminary Communication
932
would involve
a two-step
and the second from
one by a condensation
indoleamines,
gens while ported
most
certain
corresponding
alkaloids
derived
phenylethylamines
be elucidated.
Nevertheless, in abnormal
in schizophrenic
derived 15
their
action
the alkaloids
derived
from
dopamine
were
and of catecholamine As these might
reuptake
compounds
be much more
16
are pro-
amines. from
are really
catecholamines, synthesized
we can hypothesize
amounts
enzymatically
are known to be potent hallucino-
metabolism
by MAO,
tonin and other
be produced
Among
only at high concentrations.
not degraded
these
one occurring
reaction.
and harmine
of neuroamine
than that of their Whether
the first
tetrahydroisoquinolines
in both cases
probably
longed
harmaline
as inhibitors
although
phenomenon,
in pathological
tryptamine, in viva,
that these conditions,
sero-
remains
compounds
to
might
and perhaps
even
disorders.
This work was partially supported by a grant from IRSIA. The skillful technical assistance of W. Gommeren, S. Aerts and M. Verwimp is greatly appreWe also thank H. Verhoeven for synthesis of compounds. ciated,
REFERENCES 1. 2. 3.
4. 5. 6. 7. 8. 9. 10.
11. 12. 13. 14. 15. 16.
P. Laduron, Nature New Biol. 238, 212 (1972). P. Laduron, In: Frontiers in Catecholamine Research (E. Usdin and S. Snyder eds.) pp. 121-128, Pergamon Press, New York (1973). In: Advances in Bjochemical Pharmacology J. Leysen and P. Laduron, (Sandler, Costa and Gessa eds.) Vol. II, pp. 65-74, Raven Press, New York (1974). L. L. Hsu and A. J. Mandell, Life Sci. 12, 847 (1973). S.P. Banerjee and S.H. Snyder, Science 182, 74 (1973). Biochem. Pharmacol. G, P. Laduron, W. Gommeren and J. Leysen, I 599 (1974). P.C. Waldmeier and L. Mai’tre, Experientia 30, 456 (1974). Arch.int.Physiol.Biochim. 81, 975 (1973). P. Laduron and J. Leysen, Res. Commun. Chem. Pathol. Pharmacol. R. L. Lin and N. Narasimhachari, &, 535 (1974). W.R. Fri,sell and C.G. MacKenzie, In: Methods of Biochemical Analysis (D. Glick ed.) Vol. 6, pp. 63-77, Interscience Publishers Inc., New York (1958). J. Leysen and P. Laduron, FEBS Letters 41. 299 (1974). H. Verhoeven, P. Laduron and M. Claeys, W. Lauwers, J. Leysen, Biomed. Mass Spectrom., in press (1974). M. Sandler, S. Bonham Carter, K.R. Hunter and G. M. Stern, Nature 241, 439 (1973). K. 0. Donaldson and J. C. Keresztesy, Biochem.Biophys.Res. Commun. 2, 289 (1961). A. C. Collins, J.L. Cashaw and V.E. Davis, Biochem. Pharmacol. 2337 (1973). J. Pharmacol. exp. Ther. R. Heikkila, G. Cohen and D. Dembee, 250 (1971).
22,
179,
PRELIMINARY
ENZYMATIC
FORMALDEHYDE
DROFOLIC
ACID:
PRODUCTION
PRIOR
Pierre Department
COMMUNICATION
STEP
Laduron
5-METHYLTETRAHY-
TO ALKALOID
and Joske
of Neurobiochemistry, B-2340
FROM
Leysen
Janssen
Beerse,
FORMATION*
Pharmaceutics
Belgium
(Received 3 January 1975;accepted 31 J~IIUW 1975)
5-Methyltetrahydrofolic methylation enzymatic more
of dopamine reaction
general
nergic
in contrast
features
used
methyl
We tested
after crease zyme
reaction transfer
previously from
blank was run (Table of the assay,
properties
of this
enabling
us to attribute
involving
a
only the adre-
In fact that after
in the blank without
Part of this work July 1974
substrate
the corresponding
were
or
obtained
depending
suggested,
N-
or 5-hydroxy-
recently
markedly,
This
in this study:
The radioactivity with 5-Me
differed
Cl4
by
on the
that something
In contrast
other
than those
the amount
were
caused
from
re-
and two enzyme on how the
lower
than those
a tremendous
the blank containing for the blank without decreased
the substrate
at the IXth C. I. N. P.
929
in extracts
depending
of radioactivity
than in that where
rat kidney
3-hydroxyphenylethylamine)
to pH 10, to this,
one from
-H4-folate
markedly,
(4-methoxy,
was brought
was presented
measured
all the blank values
(even lower
evaporation,
results
differed
preparations
of substrate
mixture
values
Similar
First-
was involved.
Although
I).
this investigation.
N-methyltryptamine
products.
incubated
in the radioactivity. lower
6
rat kidney,
the addition
the incubation
gave
Various
we did not identify
blank values
pig brain.
isolated
4, 5
when tryptamine,
two enzyme
from
mixtures
fractions
the N-
us to undertake
as substrate.
of isolating
than a simple
action
to mediate
than a function
prompted
9 Secondly,
group.
and another
reported
2-7
been described,
studies
compounds were
procedure
1
in rat brain.
to this reaction
to other
0-methylated tryptamine
recently
8
system.
another
to epinine
have since
function
Two main ly,
acid was
in-
boiled
en-
substrate). much more
was added after
Congress,
Paris,
Preliminary Communication
930
TABLE
Radioactivity extracted from reaction mixtures incubated with 5-MeC14-H4-folate and purified enzyme fractions from rat kidney
I.
Total
extracted radioactivity (CPM x 103) Peak A 1 Peak B
Before evaporation Assay Blank Boiled enzyme Without substrate Substrate added after
19.1
33.4
1.4 2.1 8.4
2.5 3.8 18.8
0.3 8.4
0.2 7.6
fixation
After evaporation Blank Without substrate Substrate after fixation
Ammonium sulphate enzyme fraction (30 % to 60 %) from rat kidney was further purified on DEAE A50 Sephadex columns as described elsewhere. 9 An aliquot of two peaks (A and B) of enzyme activity was incubated with 0.1 pCi 5-MeC14-H4-folate (spec.act. 61 mCi/ mmol Radiochemical Centre Amersham) and the reaction products were extracted as previously described. 6
fixation,
suggested
formed.
Moreover,
a reaction
that,
during
this product
seemed
to occur
dance with this hypothesis, using
the dimedon
5-Me-H4-folate These
suggesting
condensation
by experiments amine
considerably
lowered
enzymatically
between
enzyme.
the amount
Here
with
amines.
11
like 4-methoxy, formed
aldehyde
formaldehydeC14
but without
incubated
was added to the incubation
reaction
where
demonstrated
of biogenic
of substrate
formaldehyde
In accor-
fixation.
mixtures
but in the absence
was
for the amines;
was recently
m reaction
if the amine
product
high affinity
production 10.
a volatile
added after
in the presence
The free
a possible
0” C with a biogenic
of the amine
enzyme
tremendously
This view is supported at
method,
now confirmed
3 -hydroxyphenylethylamine.
tures
have a very
formaldehyde
precipitation
found to decrease
must
period,
with the substrate
and rat kidney
data were
the incubation
was
mix-
and amine.
was only incubated again,
of formaldehyde
the presence precipitated
with dimedon. These
experiments
enzymatically but also
formed
approach
sented
in figure
2-methyl layer
from
that an additional
A first
clearly
5-Me-H reaction
in elucidating 1.
6, 7-hydroxy
chromatography
demonstrated 4
-folate
occurred
the nature
By incubating
epinine
not only that formaldehyde
as reported between
of this
1).
Similar
the aldehyde
condensation
with a pig brain
1, 2, 3, 4-tetrahydroisoquinoline (fig.
in detail
results
were
elsewhere,
11
and amine.
product
enzyme,
was identified obtained
was
is pre-
an alkaloid, by thin-
with other
amines
931
PreliminaryCommunication
Fig. 1 - Radiochromatograms of a reaction mixture incubated with 5-MeC14 -H4-folate, epinine and an ammonium sulphate enzyme fraction (30 to 65 per cent) from pig brain. Reaction products were isolated on A1203 columnsP An aliquot was spotted on (A) cellulose F and (B) silica gel 60 F plated. Solvent systems: (A) phenol-water-HCl 80/20/l (B) isobutyl alcohol-formic acidwater 150/30/20. Radioactivity was measured with a Berthold scanner and standards (E: epinine and Mi: 2-methyl 6,7 -hydroxy 1, 2, 3, 4-tetrahydroisoquinoline) were detected in U.V.
MI
t
like
tryptamine,
amine
using
respective mass
E
N-methyltryptamine
five
different
reaction
plains
the earlier
not be obtained the indole
as it will
but does
detected rence
13
postulated.
parkinsonian
results
A similar
already
described.
12
elsewhere.
and This
ex-
of indoleamines
of a possible
being
treated
14
As
enzyme seem
shown
could
could
methylation
more
of
in fig.
similar
2, the formation
it has not yet for
aceof been
the occur-
in numerous
to those
as
with
in the urine
evidence
is present
by the
formaldehyde
in vivo although
of the
in viva
of dopamine
provide
which or less
with
reaction
13
that alkaloids
be formed
amines
L-Dopa
results
tissues
of an enzyme
of certain
alkaloids
the Fig. 2 - Model illustrating alkaloid formation from biogenic amines through a one-carbon transfer from 5-Me-H4-folate with formaldehyde as intermediate compound.
ENZYME
scsa+:m-c”, E
group
demonstrated
with our
Furthermore,
of which
the view
condensation
5-Me-HI-FOLLIE
1 H‘+X#JE
of the
chromatography
N-methylation
support
has been
of a formaldehyde-forming
and the properties
in detail
of corresponding
salsolinol)
patients
in brain.
the identity
by gas
the suggestion
or the j3 -carboline
condensation
(yielding
be reported
not confirm
Moreover
proved
6, 9 in which
the foregoing
tetrahydroisoquinoline
taldehyde
further
3-hydroxyphenylethyl-
9
nitrogen.
Pictet-Spengler
12
systems.
was
experiments
Therefore,
recently
solvent
products
fragmentography
and 4-methoxy,
_
::a+ MI
Preliminary Communication
932
would involve
a two-step
and the second from
one by a condensation
indoleamines,
gens while ported
most
certain
corresponding
alkaloids
derived
phenylethylamines
be elucidated.
Nevertheless, in abnormal
in schizophrenic
derived 15
their
action
the alkaloids
derived
from
dopamine
were
and of catecholamine As these might
reuptake
compounds
be much more
16
are pro-
amines. from
are really
catecholamines, synthesized
we can hypothesize
amounts
enzymatically
are known to be potent hallucino-
metabolism
by MAO,
tonin and other
be produced
Among
only at high concentrations.
not degraded
these
one occurring
reaction.
and harmine
of neuroamine
than that of their Whether
the first
tetrahydroisoquinolines
in both cases
probably
longed
harmaline
as inhibitors
although
phenomenon,
in pathological
tryptamine, in viva,
that these conditions,
sero-
remains
compounds
to
might
and perhaps
even
disorders.
This work was partially supported by a grant from IRSIA. The skillful technical assistance of W. Gommeren, S. Aerts and M. Verwimp is greatly appreWe also thank H. Verhoeven for synthesis of compounds. ciated,
REFERENCES 1. 2. 3.
4. 5. 6. 7. 8. 9. 10.
11. 12. 13. 14. 15. 16.
P. Laduron, Nature New Biol. 238, 212 (1972). P. Laduron, In: Frontiers in Catecholamine Research (E. Usdin and S. Snyder eds.) pp. 121-128, Pergamon Press, New York (1973). In: Advances in Bjochemical Pharmacology J. Leysen and P. Laduron, (Sandler, Costa and Gessa eds.) Vol. II, pp. 65-74, Raven Press, New York (1974). L. L. Hsu and A. J. Mandell, Life Sci. 12, 847 (1973). S.P. Banerjee and S.H. Snyder, Science 182, 74 (1973). Biochem. Pharmacol. G, P. Laduron, W. Gommeren and J. Leysen, I 599 (1974). P.C. Waldmeier and L. Mai’tre, Experientia 30, 456 (1974). Arch.int.Physiol.Biochim. 81, 975 (1973). P. Laduron and J. Leysen, Res. Commun. Chem. Pathol. Pharmacol. R. L. Lin and N. Narasimhachari, &, 535 (1974). W.R. Fri,sell and C.G. MacKenzie, In: Methods of Biochemical Analysis (D. Glick ed.) Vol. 6, pp. 63-77, Interscience Publishers Inc., New York (1958). J. Leysen and P. Laduron, FEBS Letters 41. 299 (1974). H. Verhoeven, P. Laduron and M. Claeys, W. Lauwers, J. Leysen, Biomed. Mass Spectrom., in press (1974). M. Sandler, S. Bonham Carter, K.R. Hunter and G. M. Stern, Nature 241, 439 (1973). K. 0. Donaldson and J. C. Keresztesy, Biochem.Biophys.Res. Commun. 2, 289 (1961). A. C. Collins, J.L. Cashaw and V.E. Davis, Biochem. Pharmacol. 2337 (1973). J. Pharmacol. exp. Ther. R. Heikkila, G. Cohen and D. Dembee, 250 (1971).
22,
179,
