BIBSYNTHESIS OF MUSTARD OIL GLUCBSIDES

V. FORMATION OF GLUCONASTURTIIN FROM L-Y-PHEMYLBUTYRINE-C~~-N'~

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IN WATERCRESS1

E . W* UNDERBILL
Prairie Regisnak Laboratory, National Research Council, SasRatoon, Saskatchewan
Received August 12, 1 9 4

Abstract
Cl"-Labelled compounds were fed to watercress (Nastzertium oficinale R. Br.)
and their eficiency as precursors of the aglycone portion of gluconasturtiin
compared. Phenylalanine-2- and -3-C14 and sodium acetate-2-C" were eficient
precursors of the aglycone but neither compound was as efficient a precursor as
y-pheaaylbutyrine (2-amino-4-phenyibutyric acid). Approximately 40YQ of the
C14 from -pphenyibut rine-2- or -3-C14 was incorporated into the aglycone side
chain. Results of studiles with doubly labelled L-r-phenylbutyrine-C14-Nl~
show
that the amino nitrogen is incorporated directly into the thisglucsside. Evidence
that indicates that wr-phenylbutyrine is converted t o its L-isomer by the plant
is also presented. The resuits of an isotope competition experiment provide some
evidence for the existence of a chain-lengthening pathway (analogous to the
formation of leucine from valine) for the biosynthesm of 7-phenylbutyrine from
phenylalanine and acetate in watercress.

Introduction
In a previous publication (1) it was shown that both phenylalanine and
acetate can be utilized by watercress (Nasturtium oficinale W. Br.) for the
synthesis of the aglycone moiety (11) of gluconasturtiin (I). The carboxyl
carbons of both phenylalanine and acetate were not incorporated into the
thioglucoside aglycone, but the methyl carbon of acetate and the alpha and
beta carbons of phenylalanine were specifically incorporated into carbons 1, 2,
and 3 of @-phenylethyl isotkiocyanate respectively. The methyl carbon of
acetate was also shown to be an efficient precursor of the corresponding
'isothiocymate9 carbon in the thioglucoside sinigrin (I).

The similarities between the carbon and nitrogen structures of some amino
acids and certain thioglucosides have led to the suggestion that amino acids
may be the direct precursors of the aglycone portion of a number of these
'Issued as N.R.C. No. 8230.
Canadhn Journal of Bbchemfstny. Volume 43 (1965)

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180

CANADIAN JOURNAL OF BIOCHEMISTRY. %DL. 43, 1965

thiog'tucosides (2). This hypothesis has been verified for glucstropaeolin, where
both the carbon (except for the earboxyl) and the nitrogen of phenylalanine
have been shown to be incorporated directly into this glucoside (3). Since the
amins acid structurally related to gluconasturtiin, y-phenylbutyrine (2-an-aino4-phenylbutyric acid), has not been reported to occur naturally, it was of
interest to investigate more fully this apparent difference in the biosynthesis
of mustard oil glucoaides.

Experimental Methods
Synthesis of Compozknds
The labelled compounds employed in this investigation were obtained from
commercial sources, except for cinnamic acid-3-C14 which was a gift from Dr.
J. E. Watkin and y - p h e n y l b ~ t y r i n e - C ~ ~which
- N ~ ~ was prepared as follows.
Freshly distilled benzaldehyde (1 mmole) in 1 ml of 25% methanol containing
10 mg of sodium hydroxide was reacted with either sodium pyruvate-2-CBqor
sodium pyruvate-3-Cu ((0.5 mmole). After the reaction mixture was stirred
for 4 hours under an atmosphere of nitrogen and a t room temperature, thc
excess of benzaldehyde was extracted into ether. Cinnamsylformic acid-2- or
-3-C14 was recovered after acidification of the mixture and extraction into
ether. ~ ~ - y - P h e n y l b u t y r i n e - 2 - C ~was
~ - Nprepared
~
according to the nlethod
of du Vigneaud et al. (4) by reductive amination of ci~anamoylformicacid-2-C14
with a r n r n ~ n i a - Nthat
~ ~ had been liberated from amanonium chloride. DL-yPhenylbutyrine-3-C"4 was prepared in a similar manner from cinnamoylformic acid-3-C14 with ordinary ammonia. The amino acid was purified by
ion-exchange chromatography (5) and was recrystallized from water.
The L-isomer of ~ - p h e n y l b u t y r i n e - 2 - C ~ ~was
- N ~obtained
~
by treating the
racemate with a preparation of D-amino acid oxidase from Trigol;8opsisuariabil'is
(6). ~ ~ - ~ - P h e n j y l b u t y r i n e - 2 - C(140
~~-N
pmoles)
~~
that was dissolved in 25 ml
of phosphate buffer (0.65
pH 8.0) was divided into 10 Warburg vessels
containing 0,1 ml of enzyme, 2 yg of crystalline catalase, and potassium hydroxide in the center well. The reaction was carried out a t 30' and the theoretical
amount of oxygen was consumed in 20 minutes. The contents of the flasks were
combined, and the L-isomer was recovered by ion-exchange chromatography
(5) and crystallized from water (yield 51 pnloles, 75%).
Nonlabelled 8-benzylmalic acid was prepared from benzylmalonic acid and
chloral according to the method described by Doebner and Kerste~a (7).

-w

Administration of Compozknds
Watercress was grown in gravel culture under fluorescent and tungsten
lights and the plants were used for feeding experiments when both flowers
and fruit were present. Labelled compounds were administered to four to six
shoots (fresh weight 60-75 g) by immersing the cut end of the plant stems into
an aqueous solution (2-10 ml) sf the tracer. When most of the solution was
absorbed, distilled water (1-2 ml) was added; when this water was absorbed,
the "washing in" process was repeated. Absorption of the compounds by the

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UNDERWILL: MUSTARD OIL GLUCOSIBES. V

181

shoots was complete within 6 hours, as was evidenced by the lack of radioactivity in a 10% aliquot taken from the solution remaining in the beaker. At
the end of the absorption time the shoots were transferred to a larger beaker
containing about 50 ml of water and were maintained under continuous light
for the rest of the metabolic period (24 hours from the start of feeding).

Isohtion a d Degradation of Products

The fresh plant material was ground with an equal weight of 0.2 11%citrate
buffer (pH 4.0) in a Waring Blendor and incubated with myrosinase a t room
temperature for 3 hours to hydrolyze the thioglucoside. The liberated aglycone,
@-phenylethylisothiocyanate, was steam-distilled into an excess of ammonia
to form the thiourea derivative (I I I), which was subsequently recrystallized
from water (1). The distribution of CB4in the side-chain carbons of the aglycone
was determined as previously described (I) by degradation of 0-phenylethylthiourea.
Isotope Analysis
Compounds were combusted (8) to carbon dioxide for C1"analysis by a
vibrating reed electrometer (Dynacon, Nuclear Chicago). This method can
detect 0.1 mpc sf CB4.Nitrogen samples were prepared in duplicate according
to the method of Sprinson and Rittenberg (9) and assayed with an Associated
Electrical Industries Ltd. mass spectrometer type his-3.

Results and Discussion

The data in Table 1 summarize the results of feeding experiments in which
the incorporation of C14 into gluconasturtiin aglycone was determined. The
percentage of C14 incorporated is the total C1*present in the aglycone carbons
of the thioglucoside expressed as a percentage of the C14absorbed by the watercress shoots. The dilution, defined as the specific activity of the compound fed
divided by the specific activity of the aglycone isolated, is also a measure of
the relative effectiveness of the compounds fed as precursors of gluconasturtiin
aglycone. Compounds showing small dilution values are considered more
efficient precursors than those showing high values. The assumptions made in
the use of this criterion of con~poundefficiency, and some of the limitations to
its use, have been discussed by Brown and Neish (10) and Watkin and Neish
(11). However, in this work and particularly in Table I11 where N1%nd CB4
dilutions are involved, the use of such values allows for a clearer presentation
of the results.
Little of the CB4from the compounds fed in the first experiment was incorporated into the aglycone, except from phenylafanine and acetate-2-C14. The
fact that the C14 from formate was not efficiently utilized in the synthesis of
the aglycone would suggest the 'iisothiocyanate' carbon is not formed via a
"one-carbon" compound. When the amino acid possessing the same carbon to
nitrogen skeleton as is found in gluconasturtiin was fed in experiments I%,
111, and IV, the percentage conversion of C14 was approximately 20 times

Compound administered

DL-Phenylalanine-204
Cinnamic acid-3-CI4
Sodium acetate-1-C1"
Sodium acetate-2-CI4
Calcium glycolate-2-C14
Sodium formate-C14

Expt.

Amount
fed
(pmoles)

Specific
activity
(pc/mmole>

Arnount
isolated
(mmole)

Specific
activity
(~lc/mmoje>

%I Cl4
incorporated

Comparison of CU-labelled compounds as precursors of the gluconasturtiin aglycone*

TABLE I

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Dilution

183

UNDERHILL: MUSTARD OIL GLUCCBIDES. V

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greater than that when phenylalanine was used. Furthern~ore,the C1* from
DL-y-phenylbutyrine was incorporated into the aglycone with an efficiency
equal to that when the L-isomer was fed in experiment IV.
The results in Table 11 show that gluconasturtiin aglycone formed from
TABLE II
Diatributior~sf CI4 in gluconasturtiin aglycone

yodistribution sf
Ph-CHp

-CH2-*

Compound administered
~~-~-Phenyl$utyrine-3-C~~
DL-Phenylalanine-2-C1'
DL-7-Phenylbutyrine-2-61'
Sodium acetate-2-Cl4

614

in:

-C

/"
\

N-

0.3
0.4
8.1
8.3

98.4
99.5'
0.6
3.5

1.3
8.1
99.3
88.2

*By calculation; see ref. 1.

phenylalanine-2-C14 and y-phenyibutyrine-3-C14 was labelled almost entirely

in the carbon alpha to the 'isothiocyanatey carbon, whereas y-phenylbutyrine2-C14 and acetate-2-Cf4 gave rise to the aglycone labelled primarily in the
5sothiocyanate' carbon. These results, when they are combined with the high
eficiencgr sf C14 incorporation from y-phenylbutyrine as indicated in Table I,
strongly suggest that all of the carbons of the amino acid, except for the
carboxyl, are incorporated as a unit into the aglycone. These results are
entirely analogous to the formation of glucotropaeolin (the next lower homologue to gluconasturtiin), where the Ca-C2 carbon skeleton of the aglycone is
formed from phenylalanine by loss of its terminal carbon (1).
Shown in Table 111 are the results of experiments that employed doubly
labelled compounds and which were designed to determine if the amino nitrogen
of y-phenylbutyrine was incorporated directly into the thioglucoside aglycone.
Since the carboxyl carbon of phenylalanine is not converted to the thioglucoside aglycone (I), the molar specific activity of the C1* of L-phenylalanineG-C14-N15was multiplied by eight-ninths to obtain the molar specific activity
of that portion used in the synthesis of the aglyc~neand to calculate the
appropriate C14/hT1%ratio and C14 dilution value. As was expected, when
L-phenylalanine-G-C14-N1"
was fed, the dilution values for C1' and NI5 in the
aglycone were found to differ (by a factor of 4) ; these values indicate that both
the carbon and nitrogen of this amino acid were not incorporated as a unit into
the aglycone. In experiments VI, VII, and VIII, it can be seen that, while the
CI4 utilization was the same for both racemic and L-y-phenylbutyrine, the NI5
incorporation from DL-y-phenylbutyrine was only one-half of that when the
L-isomer was used. One explanation to account for these results is that 13-7phenylbutyrine was converted to the corresponding a-keto acid (the pool size

113
~-Pheny%alanine-G-@14-W~
~ ~ - ~ - P h e n y l b u t y r i n e - 2 - @ ~ ~ - ~ ~192
"
~~-~-Phenylbutyrine-2-C"-N~~ 57
~ - ~ - P h e n y l b u t y r i n e - 2 - C ~ ~ - S ~ ~ 15.8

Compound administered

46
34
31
118.3

2.19
5.65
1 .S4
1.82

CP~/N"~

8.80
13.11
3.81
2.19

C14/N16t

in 8-phenylethyl isothiocyanate).

Atoms %
excess
N 15

*Isoktd as 0-phenylethylthiourea.
tC14/Nla = (specific activity of C" (~c/mrnole~)/(atom
OJo excess W5).
$Dilution Nl6 = (atoms 70 excess Nf5 in cornpolanid administered)/(atsm % excess W

VIII

V
VI
VHI

Expt.

Specific
activity
(pc/mmole)

337
15
17
15

C14

N15$

1,350
35
35
18

Dilution

@-Phenylethyl isotbiocyanate*

Incorporation of Cu-N16-labelled compounds into the aglycone* of gluconasturtiin

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--

J-+

!=

iZ

ii!

l2

20
!

o
p11

60E!

185

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UPSDEWHILL: MUSTARD OIL GLUCOSIDES. V

of which is assumed to be small) by a D-amino acid oxidase. Transamination

of the labelled keto acid with nonlabelled amino nitrogen by an L-amino acid
transaminase would then form the L-isomer labelled essentially with only CB4.
The incorporation of CI4 from the DL-amino acid into the aglycone would,
therefore, be the same as feeding the L-isomer alone, while the NI5 incorporation from the racernlate would only be one-half that from the L-isomer. The
results of experiment VIII, where similar dilutions were obtained for Ca4and
NX5in the aglycone derived from ~ - ~ - p h e n y l b n t y r i n e - 2 - C ~ ~clearly
- N I ~ ~show
that the amino nitrogen is incorporated directly into gluconasturtiin aglycone;
this result constitutes the second example of an amino acid that is the direct
precursor of a thioglucoside. At the present there is no experimental evidence
to suggest the mechanism whereby the amino nitrogen is oxidized to the stage
where it could form the N-sulfate ester of the thioglucoside.
The above results are similar to those obtained by du Vigneaud and Irish
(12), who fed L-y-phenylbutjrrine and D-y-phenylbutyrir-re separately to dogs
and isolated considerable and equal proportions of N-acetyl-L-r-phenylbutyrine
from the urine. In the case of the D-isomer, there was clear-cut evidence of its
conversion in the body to the L-isomer. The results are also consistent with
findings that the D-isomerides of several essential amino acids can support
the growth of rats (13).
Although y-phenylbutyrine is not known to be a naturally occurriilg compound, its formation from phenylalanine and acetate may be thought of as
occurrirag by a one-carbon chain-lengthening mechanism outlined in Fig. 1.

COOH

OCti2-CH-CROH-COOH

-Cq-

2H

3-

~ c H ~ - C H ~ - C - C O O H

I4

FIG. 1. Proposed pathway for the formation of r-phenylbutyrine and gluconasturtiin.

This chain-lengthening route is analogous to the pathway leading to the

formation of Ieucine from valine (14), of glutan~icacid from aspartic acid, and
to the pathway suggested for the biosynthesis of sinigrin from rnethionine and
acetate (15). The relative efficiencies of the compounds employed in this study
as precursors of the aglycone (Table I) and the distribution patterns of CI4
incorporated therefrom (Table I I) are consistent with such a pathway.

186

CANADIAN JOURNAL OF

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Dilution of

BIOCHEMISTRY. VOL.

43, 1985

'FABLE HV
into gluconasturtiin aglycone* from adnlinistration of labelled
compounds and 8-benzylmalic acid

614

Compound administered

.Amount
fed
(pmoles)

Specific
activity
(pc/mrnole)

Specific
activity
(pc/mrnole)

Dilution

Sodium acetate-2-Cu
Sodium acetate-2-C14
8-Benzylanalic acid
~%-Phenylalanine-3-C~~
~~-Phenylalanine-3-P
8-Benzylmalic acid
~%-Phenylalanine-3-@'~
DL-Phenylalanine-3-C1"
p-Benzylmalic acid

Presented in Table Ik7 are the results of an isotope competition experiment

in which the dilution of CB4into the aglycone is given when acetate-2-C14 and
phenylalanine-3-@I4were fed alone as well as in admixture with nonlabelled
8-benzylmalic acid. If such a pathway exists in watercress and if P-benz~rlmalic
acid were a precursor of gluconasturtiin, then one would expect a greater
dilution of CI4 in the aglycone when the nonlabelled P-benzylnnalic was fed
with the labelled precursors than when the labelled precursors were fed 3, 1one.
In each instance when nonlabelled P-benzylmalic acid was fed together with
labelled phenylalanine or acetate, the dilution sf CM into the aglycone was
greater than when the labelled precursor was fed alone. While these results
provide some support for the proposed pathway, they cannot be taken as
evidence for its existence, since the dilution values obtained may be due to
secondary and unrelated effects caused by the addition of the four isomers of
P-benzylmalic acid. Furthermore, a sufficient number of experiments has not
beell carried out to permit statistical treatment of the results.
I t s h o ~ ~ be
l d noted that P-henzyltnalic acid, reisolated from plants to which
it was fed along with the labelled csn~pound,was found to be radioactive. The
acid, isolated by solvent extraction, nyas nlethylated with diazo~nethaneand
separated from the other plant acids by gas-liquid chromatograpl~y.%TThile
the
purity of the acid ester was not rigorously proved prior to CI4assay, its specific
activity was equal to or slightly higher than the specific activity of the agiycone
recovered. I t is of interest that a compound whose structure was similar to
a- and 8-benzylmalic acids has been isolated from a plant source. Buckle ef al.
(16) report the isolation of piscidic acid (P-[P-hydroxybenzy1l-P-hydroxq.malic
acid) from the bark of Piscidia erythrina L. The synthesis of C14-labelled aand @-benzyIrnalicacids is currently being undertaken and their participation

UNDERIIILL: MUSTARD OIL GLUCOSIDES. V

187

in the above pathway Beading to the formation of gluconasturtiin aglycowe

will be the subject of a future paper.

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Acknowledgments
The author thanks
oxidase prepared from
assistance during this
analyses, and J. Dyck

Dr. T. LaRue for the generous gift of D-amino acid

Trigonopsis ntctriabilis, D. F. Kirkland for his technical
investigation, &I. h4azurek for carbon and hydrogen
for carbon-14 and nitrogen-15 assays.

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5.