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Biosynthesis of mustard oil glucosides III. Formation of glucotropaeolin from L-phenylalanine-C14-N15
Vol. 14, No. 5, 1964
BIOSYNTHESISOF MUSTARD OIL GLTJCOSIDRS III.
14-&s*
FORMATIONOF GLUCOTROPAEOLIN FROML-V-C .E. W. Underhill
and M. D. Chisholm
Prairie Regional Laboratory National Research Council Saskatoon, Saskatchewan Canada
Received
6,1%3
December
Glucotropaeolin
(I),
one of several mustard oil
or isothio-
cyanate glucosides (Kjaer, 1960), when hydrolyzed by myrosinase yields potassium bisulphate, thiocyanate
(II),
glucose and a steam volatile
aglycone, benzyl iso-
which is isolated as benzylthiourea
of the isothiocyanate type intramolecular
(III).
aglycone upon enzymic hydrolysis rearrangement (Rttlinger
Formation
involves a Lossen-
and Lundeen, 1956) which
also occurs in hydroxsmic acids.
KHS04 ,S-c6Hl105 ,C
CH2-
\
N-O-i-0
P
CH2 - N=C=S II NH3 / Is CH2-NH-C-NH2
III
* Issued as N.R.C. No.
CSH12OS
-K+
0
I
+
MYROSINASE
770.
425
Vol. 14, No. 5, 1964
BIOCHEMICAL
The structural
AND BIOPHYSICAL
similarities
RESEARCH COMMUNICATIONS
between someisothiocyanate
glu-
cosides and amino acids has led to the speculation that these acids may be the precursors of the thioglucosides publication
(Underhill
phenylalanine-2-
In a previous 14 1962) it was shown that the C from
14 as well as from phenyllactic
acid-2-CL4 was
randomization into the aglycone moiety of gluco-
with high efficiency.
glucotropaeolin
1954).
and -3-C
incorporated without tropaeolin
et al.,
(Kjaer,
The conversion of phenylalanine to
has also been reported by Benn (1962).
This paper reports the source of the glucoside nitrogen to be the nitrogen
of phenylslanine and describes attempts to elucidate
nature of the intermediates
the
between phenylalanine and glucotropaeolin.
MXTHODS ANDMATFXALS Carbon-14 and nitrogen-15 from commercial sources.
labelled
phenylalanine were obtained
The remaining C14-labelled compoundswere
synthesized in this laboratory.
Labelled compoundswere administered to the aerial portion
of
Tropaeolum majus L. (commongarden nasturtium) by immersing the cut end of the plant stems in an aqueous solution of the tracer. were allowed to metabolize under continuous light
for 24 hours.
plant material was treated with myrosinase to hydrolyze side and the isothiocyanate resulting
benzylthiourea
was steam distilled
was recrystallized
The plants The
the thiogluco-
into ammonia. The
from water (Underhill
et al.,
1962). Compoundswere converted to carbon diotide 2"
for measurementof
in a Nuclear Chicago, model 6000 Dynacon Electrometer.
Nitrogen
samples were prepared according to the method of Sprinson and Rittenberg (1949) and assayed in duplicate using an Associated Electrical. Ltd. mass spectrometer type MS-3. 426
Industries
BIOCHEMICAL
Vol. 14, No. 5, 1964
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
l3FSJLTSANDDISCUSSION Table I shows the results of three feeding experiments using doubly-labelled
L-phenylalanine-U-C 14-N15.
Since the carboxyl
carbon of
phenylalanine is not converted to the thioglucoside et al.,
1962), the molar specific
U-C1"4P activity calculate
was multiplied
aglycone (Underhill of the Cl4 of L-phenylalanine-
activity
by eight-ninths
to obtain the molar specific
of that portion used in the synthesis of the aglycone the appropriate Cl"/Nl'
ratio.
In addition,
excess d5 in the aglycone, benzyl isothiocyanate, the N15 content of benzylthiourea
and to
the atoms per cent
was calculated
from
by applying appropriate corrections
the nitrogen atom derived from the ammoniaused in preparation
for
of the
thiourea derivative.
TABLE1 Conversion of L-phenylalanine-u- cF4-N15 into glucotropaeolin Fresh w-k.. of plant cd
L-Phenylalanine-U-C14-$5
Eenzyl isothiocyanate**
Specific activity aJ+N
Amt. fed bd
W
73 4-o
15.7
160
8.9
106
46.0 46.0
3.09 2.06
52
7.9
119
46.0
2.30
* Specific activity atom - see text.
aglycone
Specific
4.10 4.08 2.34
Atoms $ Cl4 $5
1.380 1.978 0.976
2.97 2.06 2.40
of phenylalanine corrected for loss of one carbon
** Calculated from benzylthiourea
- see text.
The C14/N15 ratios for phenylalanine and the glucoside aglycone remained unchanged within the three experiments.
the accuracy of the analyses in each of
Therefore,
nitrogen of the thioglucoside
it is evident that the carbon and
aglycone are derived as a unit from phenyl-
alanine and that the intermediates must be nitrogenous.
Furthermore,
it would appear that the aminoritrogen must be oxidized to the -1
427
Vol. 14, No. 5, 1964
oxidation
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
state (eg. HO-N=) to pemnit the formation of the glucoside
N-sulphate ester. of oxidation
Compoundsin which nitrogen is present at this level
have been known in biology for several years.
Stumpf -et al.
(1951) have demonstrated the formation of glutamohydroxsmic acid from glutamine and hydroxylamine by glutamotransferase
isolated
from pumpkin.
Evidence for the formation of other hydroxamic acids by a similar transferase mechanismalso exists
The occurrence of alpha-oximino acids has also been reported
1959 1.
(Ysmafuji et al.,
1950).
However, we are unaware of previous experi-
mental evidence for the oxidation indicated
of the intact
amino nitrogen
compounds,most of which are analogous to
occurring products of amino acid metabolism, were administered
to Tropaeolum majus (70 to 150 g fresh weight) and their precursors of the thioglucoside 2-2".
as
in the experiments reported here. Carbon-14 labelled
naturally
(Kimura, 1959; Jones and Elliott,
efficiency
as
aglycone comparedwith phenylalanine-
The data in Table II summarieethe results of feeding experi-
ments. Although the compoundswere administered to plants of similar weight and maturity
for an individual
between experiments is reflected poration of phenylalanine~
experiment, variation
by the differences
Relative
in per cent incor-
to phenylalanine,
nitrogenous compoundsfed were efficient
in plants
none of the
precursors of the thioglucoside
aglycone.
poration
Somereservations must be held concerning the lack of incorof C14 from phenylpgruvic acid ox!me and its apparent failure
to serve as a precursor of the aglycone. shown that alpha-keto acid oties dioxide on warming in dilute tion is possible, it
Ahmadand Spenser (1951) have
give the lower nitrile
aqueous solution.
is unlikely
Although such a reac-
that it would proceed to completion
during the time required for uptake of phenylpyruvic A more important consideration
and carbon
acid oxime-Z- cl".
concerns the conformation around the 428
BIOCHEMICAL
Vol. 14, No. 5, 1964
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLEII Comparison of compoundsas precursors of glucotropaeolin
aglycone*
Benzylthiourea Compound administered
@Pt. I
II
Amount fed (mg) $l/zi'
DL-Phenylalanine-Z-Cl4 2 Phenylethylamine 1 Cl" Phenylacetsmide-l-~lii
16.2 16.2 6.0
DL Phenylalanine 2 Cl" Be~zylc~de-l-~14
17.1 3.8
* Isolated
208
1580 4 Ill
8.58 0.05 0.03
105 188
2340 10
8.97 0.09
194 136 271
3850 3 212
12.10 0.02 0.53
7’;
III
I cl4 incorp.
$$$$
as benzylthiourea
oxime double bond in the alpha-o ldmino acid and glucotropaeolin. configuration recently
around the CZN double bond of the mustard oil
sinigrin,
confirms the indirect
configuration
of the migrating
the case of glucotropaeolin) infrared
spectrum of phenylpyruvic
that the a-isomer corporation
was in fact
phenylpyruvic
14 acid oxime-2-C with that assumed
administered.
(benzyl-
-OH) indicates
Hence, the lack of inof the incorrect anti-
acid oxime are presently being pursued. that both the nitrogen-15
L-phenylalanine-U-C 14-& thesis of the thioglucoside
and the carbon-14 of
were diluted to the sameextent in the synwould suggest that phenylalanine and its
corresponding keto acid were not readily
unlabelled
Comparison of the
Methods for the synthesis of C14-$5-labelled
The finding
smination.
side-chain (benzyl in
may have been due to the administration isomer.
pat-
evidence (Kjaer,
and the sulphate group.
by Ahmadand Spenser (1961) to be the E-isomer
geometric
glucosides,
determined (Waser and Watson, 1963) by X-ray diffraction
terns of crystalline
The
interchangeable
through trans-
Such interchange of nitrogen from phenylalanine with an %itrogen
benzyl isothiocyanate
poole would have resulted in C'"/$' of higher values. 429
ratios
for
Vol. 14, No. 5, 1964
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS The authors are indebted to D. F. Kirkland for his technical assistance during this investigation and nitrogen-15
and to J. Dyck for the carbon-14
assays.
REPEREXCES Ahmad, A. and Spenser, I.D. Benn, M.H. Ettlinger,
Chem. and Ind.
Can. J. Chem.2, (London) 1907 (1962).
M.G. and Lundeen, A.J.
Jones, KM. and Elliott,
4172 (1956).
W.H. Biochem. J. 22, 706 (1959).
Kjaer, A.
Acta Chem. Stand. 8, Ill0
Kjaer, A.
Fortschr.
Sklmpf, PK.,
(1954).
Chem. org. Naturstoffe
Sprinson, D.B. and Rittenberg,
Underhill,
J. Am. Chem. Sot. z,
J. Biochem. (Tokyo) 46, 1133 (1959).
Kimura, T.
2,
1340 (1961).
D.
J. Biol.
l-8, 122 (1960). Chem. 180, 707 (1949).
Loomis, W.D. and Michelson, C. Arch. Biochem. Biophys.
126 (1951). E.W., Chisholm, M.D. and Wetter,
and Physiol.
&,
L.R.
Can. J. Biochem.
1505 (1962).
Waser, J. and Watson, W.H. Nature 198, 1297 (1963). Yamafuji,
K., Kond, H. and Omura, H. Enzymologia I&
430
153 (1950).
BIOSYNTHESISOF MUSTARD OIL GLTJCOSIDRS III.
14-&s*
FORMATIONOF GLUCOTROPAEOLIN FROML-V-C .E. W. Underhill
and M. D. Chisholm
Prairie Regional Laboratory National Research Council Saskatoon, Saskatchewan Canada
Received
6,1%3
December
Glucotropaeolin
(I),
one of several mustard oil
or isothio-
cyanate glucosides (Kjaer, 1960), when hydrolyzed by myrosinase yields potassium bisulphate, thiocyanate
(II),
glucose and a steam volatile
aglycone, benzyl iso-
which is isolated as benzylthiourea
of the isothiocyanate type intramolecular
(III).
aglycone upon enzymic hydrolysis rearrangement (Rttlinger
Formation
involves a Lossen-
and Lundeen, 1956) which
also occurs in hydroxsmic acids.
KHS04 ,S-c6Hl105 ,C
CH2-
\
N-O-i-0
P
CH2 - N=C=S II NH3 / Is CH2-NH-C-NH2
III
* Issued as N.R.C. No.
CSH12OS
-K+
0
I
+
MYROSINASE
770.
425
Vol. 14, No. 5, 1964
BIOCHEMICAL
The structural
AND BIOPHYSICAL
similarities
RESEARCH COMMUNICATIONS
between someisothiocyanate
glu-
cosides and amino acids has led to the speculation that these acids may be the precursors of the thioglucosides publication
(Underhill
phenylalanine-2-
In a previous 14 1962) it was shown that the C from
14 as well as from phenyllactic
acid-2-CL4 was
randomization into the aglycone moiety of gluco-
with high efficiency.
glucotropaeolin
1954).
and -3-C
incorporated without tropaeolin
et al.,
(Kjaer,
The conversion of phenylalanine to
has also been reported by Benn (1962).
This paper reports the source of the glucoside nitrogen to be the nitrogen
of phenylslanine and describes attempts to elucidate
nature of the intermediates
the
between phenylalanine and glucotropaeolin.
MXTHODS ANDMATFXALS Carbon-14 and nitrogen-15 from commercial sources.
labelled
phenylalanine were obtained
The remaining C14-labelled compoundswere
synthesized in this laboratory.
Labelled compoundswere administered to the aerial portion
of
Tropaeolum majus L. (commongarden nasturtium) by immersing the cut end of the plant stems in an aqueous solution of the tracer. were allowed to metabolize under continuous light
for 24 hours.
plant material was treated with myrosinase to hydrolyze side and the isothiocyanate resulting
benzylthiourea
was steam distilled
was recrystallized
The plants The
the thiogluco-
into ammonia. The
from water (Underhill
et al.,
1962). Compoundswere converted to carbon diotide 2"
for measurementof
in a Nuclear Chicago, model 6000 Dynacon Electrometer.
Nitrogen
samples were prepared according to the method of Sprinson and Rittenberg (1949) and assayed in duplicate using an Associated Electrical. Ltd. mass spectrometer type MS-3. 426
Industries
BIOCHEMICAL
Vol. 14, No. 5, 1964
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
l3FSJLTSANDDISCUSSION Table I shows the results of three feeding experiments using doubly-labelled
L-phenylalanine-U-C 14-N15.
Since the carboxyl
carbon of
phenylalanine is not converted to the thioglucoside et al.,
1962), the molar specific
U-C1"4P activity calculate
was multiplied
aglycone (Underhill of the Cl4 of L-phenylalanine-
activity
by eight-ninths
to obtain the molar specific
of that portion used in the synthesis of the aglycone the appropriate Cl"/Nl'
ratio.
In addition,
excess d5 in the aglycone, benzyl isothiocyanate, the N15 content of benzylthiourea
and to
the atoms per cent
was calculated
from
by applying appropriate corrections
the nitrogen atom derived from the ammoniaused in preparation
for
of the
thiourea derivative.
TABLE1 Conversion of L-phenylalanine-u- cF4-N15 into glucotropaeolin Fresh w-k.. of plant cd
L-Phenylalanine-U-C14-$5
Eenzyl isothiocyanate**
Specific activity aJ+N
Amt. fed bd
W
73 4-o
15.7
160
8.9
106
46.0 46.0
3.09 2.06
52
7.9
119
46.0
2.30
* Specific activity atom - see text.
aglycone
Specific
4.10 4.08 2.34
Atoms $ Cl4 $5
1.380 1.978 0.976
2.97 2.06 2.40
of phenylalanine corrected for loss of one carbon
** Calculated from benzylthiourea
- see text.
The C14/N15 ratios for phenylalanine and the glucoside aglycone remained unchanged within the three experiments.
the accuracy of the analyses in each of
Therefore,
nitrogen of the thioglucoside
it is evident that the carbon and
aglycone are derived as a unit from phenyl-
alanine and that the intermediates must be nitrogenous.
Furthermore,
it would appear that the aminoritrogen must be oxidized to the -1
427
Vol. 14, No. 5, 1964
oxidation
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
state (eg. HO-N=) to pemnit the formation of the glucoside
N-sulphate ester. of oxidation
Compoundsin which nitrogen is present at this level
have been known in biology for several years.
Stumpf -et al.
(1951) have demonstrated the formation of glutamohydroxsmic acid from glutamine and hydroxylamine by glutamotransferase
isolated
from pumpkin.
Evidence for the formation of other hydroxamic acids by a similar transferase mechanismalso exists
The occurrence of alpha-oximino acids has also been reported
1959 1.
(Ysmafuji et al.,
1950).
However, we are unaware of previous experi-
mental evidence for the oxidation indicated
of the intact
amino nitrogen
compounds,most of which are analogous to
occurring products of amino acid metabolism, were administered
to Tropaeolum majus (70 to 150 g fresh weight) and their precursors of the thioglucoside 2-2".
as
in the experiments reported here. Carbon-14 labelled
naturally
(Kimura, 1959; Jones and Elliott,
efficiency
as
aglycone comparedwith phenylalanine-
The data in Table II summarieethe results of feeding experi-
ments. Although the compoundswere administered to plants of similar weight and maturity
for an individual
between experiments is reflected poration of phenylalanine~
experiment, variation
by the differences
Relative
in per cent incor-
to phenylalanine,
nitrogenous compoundsfed were efficient
in plants
none of the
precursors of the thioglucoside
aglycone.
poration
Somereservations must be held concerning the lack of incorof C14 from phenylpgruvic acid ox!me and its apparent failure
to serve as a precursor of the aglycone. shown that alpha-keto acid oties dioxide on warming in dilute tion is possible, it
Ahmadand Spenser (1951) have
give the lower nitrile
aqueous solution.
is unlikely
Although such a reac-
that it would proceed to completion
during the time required for uptake of phenylpyruvic A more important consideration
and carbon
acid oxime-Z- cl".
concerns the conformation around the 428
BIOCHEMICAL
Vol. 14, No. 5, 1964
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLEII Comparison of compoundsas precursors of glucotropaeolin
aglycone*
Benzylthiourea Compound administered
@Pt. I
II
Amount fed (mg) $l/zi'
DL-Phenylalanine-Z-Cl4 2 Phenylethylamine 1 Cl" Phenylacetsmide-l-~lii
16.2 16.2 6.0
DL Phenylalanine 2 Cl" Be~zylc~de-l-~14
17.1 3.8
* Isolated
208
1580 4 Ill
8.58 0.05 0.03
105 188
2340 10
8.97 0.09
194 136 271
3850 3 212
12.10 0.02 0.53
7’;
III
I cl4 incorp.
$$$$
as benzylthiourea
oxime double bond in the alpha-o ldmino acid and glucotropaeolin. configuration recently
around the CZN double bond of the mustard oil
sinigrin,
confirms the indirect
configuration
of the migrating
the case of glucotropaeolin) infrared
spectrum of phenylpyruvic
that the a-isomer corporation
was in fact
phenylpyruvic
14 acid oxime-2-C with that assumed
administered.
(benzyl-
-OH) indicates
Hence, the lack of inof the incorrect anti-
acid oxime are presently being pursued. that both the nitrogen-15
L-phenylalanine-U-C 14-& thesis of the thioglucoside
and the carbon-14 of
were diluted to the sameextent in the synwould suggest that phenylalanine and its
corresponding keto acid were not readily
unlabelled
Comparison of the
Methods for the synthesis of C14-$5-labelled
The finding
smination.
side-chain (benzyl in
may have been due to the administration isomer.
pat-
evidence (Kjaer,
and the sulphate group.
by Ahmadand Spenser (1961) to be the E-isomer
geometric
glucosides,
determined (Waser and Watson, 1963) by X-ray diffraction
terns of crystalline
The
interchangeable
through trans-
Such interchange of nitrogen from phenylalanine with an %itrogen
benzyl isothiocyanate
poole would have resulted in C'"/$' of higher values. 429
ratios
for
Vol. 14, No. 5, 1964
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS The authors are indebted to D. F. Kirkland for his technical assistance during this investigation and nitrogen-15
and to J. Dyck for the carbon-14
assays.
REPEREXCES Ahmad, A. and Spenser, I.D. Benn, M.H. Ettlinger,
Chem. and Ind.
Can. J. Chem.2, (London) 1907 (1962).
M.G. and Lundeen, A.J.
Jones, KM. and Elliott,
4172 (1956).
W.H. Biochem. J. 22, 706 (1959).
Kjaer, A.
Acta Chem. Stand. 8, Ill0
Kjaer, A.
Fortschr.
Sklmpf, PK.,
(1954).
Chem. org. Naturstoffe
Sprinson, D.B. and Rittenberg,
Underhill,
J. Am. Chem. Sot. z,
J. Biochem. (Tokyo) 46, 1133 (1959).
Kimura, T.
2,
1340 (1961).
D.
J. Biol.
l-8, 122 (1960). Chem. 180, 707 (1949).
Loomis, W.D. and Michelson, C. Arch. Biochem. Biophys.
126 (1951). E.W., Chisholm, M.D. and Wetter,
and Physiol.
&,
L.R.
Can. J. Biochem.
1505 (1962).
Waser, J. and Watson, W.H. Nature 198, 1297 (1963). Yamafuji,
K., Kond, H. and Omura, H. Enzymologia I&
430
153 (1950).