Biosynthesis of vanillin via ferulic acid in vanilla planifolia

Article abstract of DOI:10.1021/jf901204m

¹⁴C-Labeled phenylalanine, 4-coumaric acid, 4- hydroxybenzaldehyde, 4-hydroxybenzyl alcohol, ferulic acid, and methionine were applied to disks of green vanilla pods 3 and 6 months after pollination (immature and mature pods), and the conversion of these compounds to vanillin or glucovanillin was investigated. In mature green vanilla pods, radioactivities of 11, 15, 29, and 24% from ¹⁴C-labeled phenylalanine, 4-coumaric acid, ferulic acid, and methionine, respectively, were incorporated into glucovanillin within 24 h. In the incorporation processes of methionine and phenylalanine into glucovanillin, some of the ¹⁴C labels were also trapped by the unlabeled ferulic acid. However, ¹⁴C-labeled 4-hydroxybenzaldehyde and 4-hydroxybenzyl alcohol were not converted to glucovanillin. On the other hand, in immature green vanilla pods radioactivities of the above six compounds were not incorporated into glucovanillin. Although 4-coumaric acid, ferulic acid, 4-hydroxybenzaldehyde, and 4-hydroxybenzyl alcohol were converted to the respective glucose esters or glucosides and vanillin was converted to glucovanillin, their conversions were believed to be from the detoxication of the aglycones. These results suggest that the biosynthetic pathway for vanillin is 4-coumaric acid → → ferulic acid → → vanillin → glucovanillin in mature vanilla pods.

Full text of DOI:10.1021/jf901204m

9956 J. Agric. Food Chem. 2009, 57, 9956–9961
DOI:10.1021/jf901204m
Biosynthesis of Vanillin via Ferulic Acid in Vanilla planifolia
†,‡
§
O
SAMU
N
EGISHI,*,† KENJI
S
UGIURA
,
AND
Y
UKIKO
NEGISHI
†Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572,
Japan, and §Institute of Nutrition Sciences, Kagawa Nutrition University, Sakado, Saitama 350-0288,
Japan. ‡ Present address: Yuki Research Center, Bayer CropScience K.K., Yuki, Ibaraki 307-0001,
Japan.
14C-Labeled phenylalanine, 4-coumaric acid, 4-hydroxybenzaldehyde, 4-hydroxybenzyl alcohol,
ferulic acid, and methionine were applied to disks of green vanilla pods 3 and 6 months after
pollination (immature and mature pods), and the conversion of these compounds to vanillin or
glucovanillin was investigated. In mature green vanilla pods, radioactivities of 11, 15, 29, and 24%
from 14C-labeled phenylalanine, 4-coumaric acid, ferulic acid, and methionine, respectively, were
incorporated into glucovanillin within 24 h. In the incorporation processes of methionine and
phenylalanine into glucovanillin, some of the 14C labels were also trapped by the unlabeled ferulic
acid. However, 14C-labeled 4-hydroxybenzaldehyde and 4-hydroxybenzyl alcohol were not con-
verted to glucovanillin. On the other hand, in immature green vanilla pods radioactivities of the
above six compounds were not incorporated into glucovanillin. Although 4-coumaric acid, ferulic
acid, 4-hydroxybenzaldehyde, and 4-hydroxybenzyl alcohol were converted to the respective
glucose esters or glucosides and vanillin was converted to glucovanillin, their conversions were
believed to be from the detoxication of the aglycones. These results suggest that the biosynthetic
pathway for vanillin is 4-coumaric acid f f ferulic acid f f vanillin f glucovanillin in mature
vanilla pods.
KEYWORDS: Vanilla planifolia; biosynthesis; vanillin; glucovanillin; ferulic acid
INTRODUCTION
4-coumaric acid (C6-C3 compound) in cell-free extracts of Litho-
spermum erythrorhizon cell cultures. Furthermore, Podstolski
et al. (10) reported an enzyme involved in the reaction that converts
4-coumaric acid to 4-hydroxybenzaldehyde in tissue culture of vanilla.
However, the complete biosynthetic pathway of vanillin from
phenylpropanoids has not yet been demonstrated (4, 11). To clarify
the biosynthetic pathway for vanillin, we carried out pulse-chase
experiments with 14C-labeled compounds in disks of green vanilla
pods.
Vanillin is a compound with a sweet smell that has been used as
a flavor and fragrance since ancient times. Vanillin is accumulated
as a glucoside in the green pods of vanilla (Vanilla planifolia). The
pleasant aroma is released only after injury or by fermentation,
called “curing”, when the glucoside of vanillin, glucovanillin, is
hydrolyzed by glucosidase in vanilla pods (1). Vanillin was
isolated in 1858 (2) and the chemical structure determined in
1874 (3). Large-scale chemical synthesis of vanillin has been
achieved from materials such as guaiacol, eugenol, and safrole.
Nowadays, synthetic vanillin is prepared by the oxidation of
lignin contained in pulp wastes and is widely used in industry and
as a fragrance and medicine as well as flavor (4).
For the formation of vanillin in vanilla pods, Anwar (5) proposed
that vanillin is synthesized from coniferin, a precursor of lignin, by
shortening of the C3 side chain and hydrolysis of the glucoside,
whereas Zenk (6) proposed that vanillin is formed from ferulic acid
with shortening of the side chain by β-oxidation. Tokoro et al. (7) and
Kanisawa (8) proposed a pathway to form glucovanillin from the
glucoside of the 4-hydroxybenzyl alcohol after analysis of glucosides
in green vanilla pods. On the other hand, Yazaki et al. (9) reported the
existence of a nonoxidative reaction for shortening of the phenylpro-
panoid side chain and the formation of 4-hydroxybenzaldehyde from
MATERIALS AND METHODS
General Experimental Procedures. NMR spectra were measured
with a JNM-A400 spectrometer (JEOL, Japan). HPLC analysis was
performed with a Shimadzu LC-10A system liquid chromatograph
(Shimadzu Corp., Japan) and a reversed-phase C18 column (250 mmꢀ
4.6 mm i.d., 5 μm, TSK-gel ODS 80Ts) (Tosoh Corp., Japan). The
separation of compounds related to vanillin biosynthesis was carried out
according to our previous paper (12). Eluates were collected for every
1 min and mixed well with 4 mL of scintisol EX-H (Dojindo, Tokyo,
Japan), and their radioactivities were measured with a Beckmann LS-6500
scintillation counter. Bis[(β-
trate (glucoside A) and bis[(β-
tyl)tartrate (glucoside B) were supplied from Takasago International
Corp. [U-14C]- -Phenylalanine (Phe, 360 mCi/mmol), [U-14C]-
-tyrosine
(Tyr, 360 mCi/mmol), and [methyl-14C]-
-methionine (Met, 54 mCi/
mmol) were purchased from Moravek Biochemicals, Inc., Brea, CA.
14C]-Methyl iodide (7.6 mCi/mmol) was a product of DuPont, Boston,
MA. The purities of all 14C precursors were confirmed by HPLC.
D-glucopyranosyloxy)benzyl]-2-isopropyltar-
D-glucopyranosyloxy)benzyl]-2-(2-bu-
L
L
L
*Author to whom correspondence should be addressed
(telephone þ81-29-853-4933; fax þ81-29-853-4605; e-mail negishi@
sakura.cc.tsukuba.ac.jp).
[
pubs.acs.org/JAFC
Published on Web 10/09/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 57, No. 21, 2009 9957
Vanilla Pods. Green vanilla pods (V. planifolia) 3 and 6 months after
pollination, cultivated by a farmer in central Java, Indonesia, were
crystals had been dissolved in hot diethyl ether, the solution was cooled
and the resulting crystals were filtered. Recrystallization with
imported by Takasago International Corp.
ether-ligroin yielded 2.47 g of 2,3,4,6-tetraacetyl-β-
(c) Glucosylation (16). Acetyl-4-coumaroyl chloride (1.0 g) was
allowed to react with 2,3,4,6-tetraacetyl-β- -glucose (1.21 g) in a solution
of chloroform (2.6 mL) and pyridine (0.4 mL) at room temperature for
20 h. Afterward, the reaction mixture was washed with 2 N H2SO4 and 2 N
NaHCO3 and twice further with water. The chloroform layer was
evaporated and recrystallized from EtOH to obtain 1.63 g of acetyl-4-
D-glucose.
Syntheses of 14C-Precursors. (a) [U-14C]-4-Coumaric Acid.
14C-Labeled 4-coumaric acid was prepared by the deamination of
D
[U-14C]-
reaction mixture of [U-14C]-
L
-Tyr with phenylalanine ammonia lyase (PAL) as follows. The
L-Tyr (50 μCi/500 μL), 100 μL of 0.2 M TAPS-
KOH buffer (pH 8.5), 283 μL of water, and 27 μL of PAL from
Rhodotorula glutinis (0.33 unit/0.9 mL) (Sigma Chemicals) was incubated
at 30 °C for 3.5 h. After the addition of 10 μL of 3 N HCl, [U-14C]-4-
coumaric acid was extracted with 1 mL of diethyl ether 5 times. The diethyl
ether in the extract was removed, and the resulting 40 μCi of [U-14C]-4-
coumaric acid was 98% pure. The specific radioactivity of this compound
was diluted to 50 mCi/mmol by the addition of unlabeled 4-coumaric acid.
(b) [U-14C]-4-Hydroxybenzaldehyde. 14C-Labeled 4-hydroxy-
benzaldehyde was prepared by the ozonolysis of [U-14C]-4-coumaric acid
with a DMO-10BDF ozone generator (Ishimori Co., Ltd., Tokyo, Japan).
A dry methanol solution (500 μL) of [U-14C]-4-coumaric acid (50 μCi;
specific radioactivity = 50 mCi/mmol) was cooled to -78 °C in an
acetone-dry ice bath, and ozone-oxygen gas was introduced into the
solution for 30 s. After removal of the ozone from the solution by passing
dried nitrogen gas through the solution, the ozonides were destroyed by
the addition of 10 μL of methanol solution of dimethyl sulfide (10%). The
solution was concentrated on a rotary evaporator. The product was
purified by chromatography on a silica gel column (1 ꢀ 15 cm) by
chloroform/methanol (95:5) to obtain 38 μCi of [U-14C]-4-hydroxyben-
zaldehyde with a radiochemical purity of 95%.
(c) [U-14C]-4-Hydroxybenzyl Alcohol. This compound was
obtained by the reduction of [U-14C]-4-hydroxybenzaldehyde as follows.
[U-14C]-4-Hydroxybenzaldehyde (35 μCi) in 500 μL of anhydrous THF
was treated with a small amount of NaBH4 for 1 h at room temperature.
After the solution had been neutralized with 0.1 N HCl, it was concen-
trated and developed on a silica gel column (1ꢀ15 cm) by chloroform/
methanol (90:10) to obtain 21 μCi of [U-14C]-4-hydroxybenzyl alcohol
with a radiochemical purity of 96%.
coumaroyl-2,3,4,6-tetraacetyl-β-
(d) Deacetylation (17). The acetyl-4-coumaroyl-2,3,4,6-tetraacetyl-
-glucose (0.8 g) was dissolved in 15 mL of dry MeOH, and 0.1 N
D-glucose.
β-D
CH3ONa-MeOH solution (19.4 mL) was added dropwise over 15 min;
the reactionmixture was stirred under cooling with ice for 1 h. The reaction
mixture was passed through a Dowex 50W-H8 (Hþ) column (1ꢀ5 cm),
equilibrated with MeOH, and the 4-coumaroyl-β-D-glucose was eluted
with MeOH. The eluate was evaporated to dryness to obtain 0.43 g of
crude material. The crude material was washed with 9 mL of chloroform
and recrystallized from water to obtain 0.15 g of 4-coumaroyl-β-
D-glucose.
The structure was confirmed by NMR spectroscopy (18).
Feruloyl-β-
oyl-β- -glucose. Crude feruloyl-β-
deacetylation of acetyl-4-feruloyl-2,3,4,6-tetraacetyl-β-
D
-glucose was prepared in the same manner as 4-coumar-
-glucose (0.42 g) was obtained by the
-glucose (0.6 g).
D
D
D
Further purification by HPLC with a TSK-gel ODS-80Ts column
(4.6ꢀ250 mm) and elution with 18.5% MeOH gave 15 mg of feruloyl-β-
D-glucose.
Feeding Experiments of 14C-Labeled Compounds into Green
Vanilla Pods. [U-14C]-4-Coumaric acid (50 mCi/mmol), [O-meth-
yl-14C]-ferulic acid (7.6 mCi/mmol), [U-14C]-4-hydroxybenzaldehyde (50
mCi/mmol), [U-14C]-4-hydroxybenzyl alcohol (50 mCi/mmol), [U-14C]-
L-
Phe (50 mCi/mmol), and [methyl-14C]-
L-Met (50 mCi/mmol) were used as
14C-precursors in the feeding experiments. Green vanilla pods were
washed with 0.02% chloramphenicol and cut into 2 cm length pieces (2
g). Subsequently, the 2 cm pieces of green pods were further sliced into
2 mm lengths to form 10 disks. After the 10 disks had been dipped into
0.02% chloramphenicol solution and wiped with a paper towel, they were
separately placed on a Petri dish. Each disk was fed 10 μL of a water
solution of 14C-labeled compound (0.2 μCi), then 10 μL of unlabeled
ferulic acid (0.04 μmol) in feeding experiments of 14C-labeled Phe and Met,
and incubated at 26 °C under an illumination of ca. 5300 lx in a growth
cabinet. After 1, 3, 6, 12, and 24 h, each set of 10 disks was frozen and
stored at -80 °C until the following extraction. Feeding experiments of
14C-labeled 4-coumaric acid and ferulic acid into disks of 6-month-old
vanilla pods were carried out in duplicate.
(d) [O-methyl-14C]-Ferulic Acid. 14C-Labeled ferulic acid was
synthesized by the condensation of malonic acid and vanillin that was
prepared by methylation of 3,4-dihydroxybenzaldehyde with 14CH3I via
the modification of the 4-position with a benzyl group (13) as follows.
Liquid 14CH3I (250 μCi) was added to a mixture of 4-benzyl-3-hydrox-
ybenzaldehyde (37.5 mg), methyl ethyl ketone (3 mL), and K2CO3
(60 mg), and the solution was kept for 1 h at room temperature, followed
by further incubation for 16 h at 60 °C. After concentration of the reaction
mixture, the reaction mixture was incubated with 20 mL of 6 N HCl for 2 h
at 120 °C under a stream of nitrogen gas to eliminate the benzyl group.
Extraction with diethyl ether and successive chromatography on a silica
gel column (1 ꢀ 15 cm) with a solvent of benzene/ethyl acetate (3:1)
produced 267 μCi of [O-14CH3]-vanillin. In the next step, 267 μCi of
[O-14CH3]-vanillin, 100 μL of pyridine, 4 μL of aniline, and 20 mg of
malonic acid were incubated at 65 °C for 12 h, and 134.7 μCi of [O-14CH3]-
ferulic acid (specific radioactivity of 7.6 mCi/mmol; radiochemical purity
of 82.1%) was obtained by chromatography with a silica gel column
(1ꢀ15 cm) and a solvent of CHCl3/MeOH/AcOH (95:5:1).
Extractions and Separations of 14C-Labeled Metabolites. Ten
disks of vanilla pods were homogenized in 60 mL of MeOH for 1 min with
a Polytron (Kinematica AG, Switzerland) and held for 1 h at room
temperature. After filtration of the mixture with a filter paper under
suction, the residue was re-extracted with 60 mL of 80% MeOH for 1 h.
Filtrates were combined, concentrated with a rotary evaporator, and
lyophilized. The filtrate was dissolved in 30 mL of water and chlorophyll
removed by extraction twice with 5 mL of n-pentane. 14C-Labeled
aglycones were extracted 10 times with 5 mL of diethyl ether, and the
combined diethyl ether solution was concentrated and made up to 25 mL
with MeOH, and the radioactivity of the solution was measured. The
remaining water solution was concentrated to remove diethyl ether and
made up to 50 mL. This solution was passed through an Amberlite XAD-2
column (2ꢀ25 cm) equilibrated with water after thorough washing with
MeOH. The column was washed with 200 mL of water, followed by the
elution of 14C-labeled glucosides with 250 mL of MeOH. The MeOH
eluate was concentrated and made up to 25 mL with MeOH, and the
radioactivity of the solution was measured. The solutions containing
aglycones or glucosides were concentrated and lyophilized. The lyophi-
lized powders were dissolved in 50% MeOH, which were used to analyze
the metabolites by HPLC.
Syntheses of 4-Coumaroyl Glucose and Feruloyl Glucose.
4-Coumaroyl-β-D-glucose (14) was prepared by the following four steps:
(a) Preparation of Acetyl-4-coumaric Acid Chloride. 4-Cou-
maric acid (1.0 g) and pyridine (1.2 mL) were dissolved in 4 mL of acetic
acid and held overnight at room temperature. The reaction mixture was
slowly dropped into 200 mL of water with ice, and the resulting white
crystals were filtered with a glass filter. The crystals (1.1 g) were dried
thoroughly in a desiccator under vacuum and then refluxed with thionyl
chloride (3.35 g) at 100 °C for 3 h. After the solvent of the reaction mixture
had been evaporatedunder vacuum, the reactionmixture was further dried
in a desiccator under vacuum. Crude crystals were recrystallized from
benzene to obtain 0.36 g of acetyl-4-coumaric acid chloride.
Large-Scale Purification and Identification of Metabolites from
14C-Labeled 4-Coumaric Acid and Ferulic Acid. After 4-coumaric
acid (1 μCi, 0.05 mCi/mmol) had been fed to 170 disks of 3-month-old
green vanilla pods (74.8 g) for 3 h, the extraction and separation
procedures described above were carried out. The MeOH eluate
(b) Preparation of 2,3,4,6-Tetraacetyl-β-D-glucose (15).
Acetobromoglucose (10 g) was dissolved in dry acetone (15 mL) and
5.63 g of AgCO3 in 0.28 mL of water at 0 °C. The reagents were stirred for
30 min, and the reaction mixture was heated to 50 °C and filtered. The
filtrate was concentrated at room temperature to obtain crystals. After the

9958 J. Agric. Food Chem., Vol. 57, No. 21, 2009
Negishi et al.
Figure 1. Feeding of 14C-4-coumaric acid (A, B) and 14C-ferulic acid
(C, D) to 3-month-oldpods(A, C) and 6-month-oldpods(B, D):O, vanillin;
b, glucovanillin; ., vanillyl alcohol glucoside; 0, 4-coumaric acid; 9,
4-coumaroyl glucose (trans); black box with cross, 4-coumaroyl glucose
(cis); ], ferulic acid; [, feruloyl glucose (trans); black diamond with cross,
feruloyl glucose (cis); 4, 4-hydroxybenzaldehyde.
Figure 2. Feeding of 14C-4-hydroxybenzaldehyde (A, B) and 14C-4-
hydroxybenzyl alcohol (C, D) to 3-month-old pods (A, C) and 6-month-
old pods (B, D): 4, 4-hydroxybenzaldehyde; 2, 4-hydroxybenzaldehyde
glucoside; 3, 4-hydroxybezyl alcohol; 1, 4-hydroxybezyl alcohol gluco-
side; outlined cross, glucosides A and B (bis[(β-D-glucopyranosyl-
oxy)benzyl]-2-isopropyltartrate and bis[(β- -glucopyranosyloxy)benzyl]-
D
2-(2-butyl)tartrate, respectively); f, unknown 1.
(0.48 μCi) from Amberlite XAD-2 column chromatography was further
purified with a Toyopearl HW-40S column (2.5ꢀ35 cm) (Tosoh Corp.)
equilibrated with MeOH/1% AcOH (5:95). Metabolites were eluted by a
linear gradient of MeOH/1% AcOH from 5 to 30% MeOH. Eluates were
monitored at 280 nm and their radioactivities measured. The fractions
were purified by HPLC with an Inertsil prep ODS column (250 mmꢀ6.0 mm
i.d., 10 μm) (GL Science, Japan). The unknown fraction was hydrolyzed
separately with 1 N NaOH and β-glucosidase to identify the metabolites by
HPLC. These treatments revealed whether the metabolites were glucosides
or glucose esters (19). Unknown metabolites from 14C-ferulic acid were
also purified and identified as well as 14C-4-coumaric acid.
by both treatments. The retention times of unknown compounds
were the same as their synthesized glucose esters. These results
indicate that the unknown metabolites are glucose esters (19). In
addition, the cis and trans configurations of the two esters were
observed by HPLC analysis (19).
Conversion of 14C-Labeled 4-Coumaric Acid and Ferulic Acid to
Glucovanillin. In 6-month-old vanilla pods (mature pods) 15% of
14C-4-coumaric acid was incorporated into glucovanillin within
24 h (Figure 1B). Early in the feeding experiments higher
incorporation of 14C into 4-coumaroyl glucoses (cis and trans)
was observed. Furthermore, 4-coumaric acid was also converted
into vanillin and 4-hydroxybenzaldehyde. In 3-month-old pods
(immature pods), 14C was incorporated into 4-coumaroyl glu-
coses with only small amounts converted into glucovanillin
(Figure 1A). A small amount of 14C activity in ferulic acid was
also detected when 14C-4-coumaric acid was fed to disks of both
mature and immature pods (data not shown). On the other hand,
14C-ferulic acid labeled with a methyl group decreased rapidly
within 6 h after feeding, but glucovanillin increased immediately
to ca. 25% within 3 h in mature beans (Figure 1D). Incorporation
of 14C-ferulic acid into glucovanillin was 29% in 24 h. The O-Me
group of ferulic acid appears not to be eliminated in this
metabolism. Two glucose esters of ferulic acid similar to
4-coumaric acid metabolites were detected in both immature
and mature pods (Figures 1C,D). Glucovanillin was also detected
in immature pods, but not accumulated. These feeding experiments
RESULTS AND DISCUSSION
Identification of Metabolites from 14C-Labeled 4-Coumaric Acid
and Ferulic Acid. In the feeding experiments with 14C-labeled
4-coumaric acid and ferulic acid, several unknown metabolites
were detected. We tried to identify the major unknown com-
pounds from 4-coumaric acid and ferulic acid. After the applica-
tion of 14C-labeled 4-coumaric acid or ferulic acid to disks of
3-month-old vanilla pods (immature pods) for 3 h, the metabo-
lites were extracted. The crude extracts were purified by Toyo-
pearl HW-40S column chromatography. Peaks of unknown
compounds were separated from others by a gradient of MeOH
from 15 to 20%. Unknown compounds were further purified by
HPLC and treated with base and β-glucosidase, and the products
were analyzed by HPLC. Compounds prepared from the feeding
experiments with 14C-labeled 4-coumaric acid and ferulic acid
were hydrolyzed to 4-coumaric acid and ferulic acid, respectively,

Article
J. Agric. Food Chem., Vol. 57, No. 21, 2009 9959
Figure 3. Feeding of 14C-methionine to 6-month-old pods: (A) 14C-Met
(control); (B) 14C-Met þ unlabeled ferulic acid;O, vanillin; b, glucovanillin;
], ferulic acid; [, feruloyl glucoses (cis and trans); white box with cross,
unknown 2. Decreases in fed 14C-Met were not determined.
Figure 4. Feeding of 14C-phenylalanine to 6-month-old pods:(A) 14C-Phe
(control); (B) 14C-Phe þ unlabeled ferulic acid; O, vanillin; b, gluco-
vanillin; 0, 4-coumaric acid; 9, 4-coumaroyl glucoses (cis and trans); ],
ferulic acid; [, feruloyl glucoses (cis and trans). Decreases in fed 14C-Phe
were not determined.
with two compounds suggest that ferulic acid is the precursor
closer to glucovanillin than 4-coumaric acid in the biosynthetic
pathway of vanillin.
Conversion of 14C-Labeled Phenylalanine to Glucovanillin with
Addition of Unlabeled Ferulic Acid. The conversion of phenylala-
nine, a precursor of phenylpropanoids, to glucovanillin was
examined. 14C from phenylalanine was incorporated into
4-coumaric acid, ferulic acid, their glucose esters, vanillin, and
glucovanillin (Figure 4). In the control experiment (Figure 4A),
incorporation into glucovanillin was ca. 11% during 6-24 h,
whereas by the addition of unlabeled ferulic acid (0.04 μmol/
disk), the incorporation into glucovanillin was 7.6% within 6 h
with an increase to 13.4% within 24 h (Figure 4B). More 14C was
found as ferulic acid in the early stages of incubation, and
incorporation into 4-coumaroyl glucose and feruloyl glucose
increased compared to the control (Figure 4B). The incorporation
into glucovanillin reached a maximum in a short time (Figure 4A),
whereas the amount continued to increase at 24 h (Figure 4B),
which was similar to the incorporation of 14C-methionine with
added unlabeled ferulic acid. In the addition of ferulic acid, the
14C-labeled glucose esters were greater than the control, but their
decrease was small. The glucose esters do not appear to influence
the 14C incorporation into glucovanillin. The high incorporation
of these amino acids into phenylpropanoids and glucovanillin
indicates that the biosynthesis of vanillin in V. planifolia is very
active.
Biosynthetic Pathway for Vanillin. It is known that vanillin is
synthesized in vanilla pods (4, 6, 8, 11), but it is difficult for a
whole pod to take up 14C-labeled compounds. Although Zenk (6)
carried out feeding experiments by incubating the disks of pods in
a solution of 14C-labeled compound, we fed concentrated solu-
tions to the disks so that the reactions proceeded securely.
Zenk (6) determined the amount of vanillin by TLC after the
hydrolysis of the incubated solutions. In addition, he proposed
the pathway that vanillin is synthesized via vanilloyl-CoA from
ferulic acid. On the other hand, we determined glucovanillin and
other intermediates by HPLC to investigate the time course of the
metabolite conversions in detail. The results of our feeding
experiments are summarized as follows: phenylpropanoids from
phenylalanine are precursors of vanillin, methionine is a donor of
a methyl group to phenylpropanoids via S-adenosylmethionine,
and ferulic acid is a good precursor of vanillin. These results are
similar to those from Zenk (6); however, the rate of radioactivity
incorporated into glucovanillin toward total radioactivity applied
to disks by our method (Figures 1-4) was much higher than that
by his method. Glucosylated metabolites were detected in feeding
Conversion of 14C-Labeled 4-Hydroxybenzaldehyde and 4-Hy-
droxybenzyl Alcohol to Glucovanillin. Panels A and B and panels
C and D of Figure 2 indicate conversions of 4-hydroxybenzalde-
hyde and 4-hydroxybenzyl alcohol, respectively. In immature
pods, 4-hydroxybenzaldehyde was converted rapidly to 4-hydro-
xybenzyl alcohol and further to its glucoside. Rapid glucosylation
was recognized from the feeding experiments with 4-hydroxy-
benzyl alcohol (Figure 2C). On the other hand, similar amounts of
14C labels from 4-hydroxybenzaldehyde were distributed to
4-hydroxybenzyl alcohol and their glucosides in mature pods
(Figure 2B). In addition, conversion of 4-hydroxybenzyl alcohol
to its glucoside appears to be easy (Figures 2C,D). Very small
amounts of 14C labels from 4-hydroxybenzyl alcohol were also
incorporated into 4-hydroxybenzaldehyde and its glucoside. This
conversion is in the opposite direction from immature pods. The
conversion rate (about 10%) from 4-hydroxybenzyl alcohol to
glucosides A and B, esters of tartaric acid derivatives and two
molecules of 4-hydroxybenzyl alcohol glucoside (bis[(β-
D-
glucopyranosyloxy)benzyl]-2-isopropyltartrate and bis[(β- -glu-
D
copyranosyloxy)benzyl]-2-(2-butyl)tartrate, respectively) (7, 8),
in immature pods was much higher than the others. However, the
radioactivities of 4-hydroxybenzaldehyde and 4-hydroxybenzyl
alcohol were not incorporated into glucovanillin.
Trapping of 14C-Methionine by Unlabeled Ferulic Acid. To
examine methylation in the pathway to vanillin, we investigated
the effect of the addition of unlabeled ferulic acid (0.04 μmol/disk)
to vanilla pod disks fed 14C-methionine at the same time. As shown
in Figure 3B, the rate of conversion from methionine to gluco-
vanillin was slow (8-15%/6-24 h) compared to the control
(22-24%/6-24 h) (Figure 3A), but glucovanillin gradually
increased. We observed incorporation of much 14C into ferulic
acid at 3 h (ca. 3%) after feeding and into ferulic acid glucose ester
during the incubation times from 3 to 24 h (3-4%). These results
suggest that ferulic acid was directly converted to vanillin and
further to glucovanillin, not via ferulic acid glucose esters because
their decrease was slow. The slow conversion rates of glucose esters
are shown in Figures 1 and 4. Furthermore, 14C of methionine was
observed in S-adenosylmethionine (SAM), a donor of the methyl
group in methylation, and an unknown compound eluted earlier
than SAM in HPLC. In immature pods, 14C of methionine was not
incorporated into glucovanillin (data not shown).

9960 J. Agric. Food Chem., Vol. 57, No. 21, 2009
Negishi et al.
Figure 5. Proposed biosynthetic pathway for vanillin and related compounds from phenylpropanoids and formation of their glucosides and glucose esters in
Vanilla planiforia: (a) 4-coumaricacid; (b) 4-hydroxybenzaldehyde;(c) 4-hydroxybenzylalcohol; (d) ferulic acid;(e) vanillin; (a)0, (b)0, (c)0, (d)0, and (e)0 show
therespectiveglucoseestersorglucosides. Glc AandBareestersoftartaricacid derivativesandtwomoleculesof(c)0 (bis[(β-
D-glucopyranosyloxy)benzyl]-2-
isopropyltartrate and bis[(β-D-glucopyranosyloxy)benzyl]-2-(2-butyl)tartrate, respectively). Bold and dotted arrows indicate identified and unidentified routes,
respectively, in this study.
experiments with 14C-labeled compounds (Figure 1) and in the
analysis of vanilla pods by Tokoro et al. (7), suggesting that
glucovanillin is synthesized via glucose esters of 4-coumaric acid
and ferulic acid or the glucoside of 4-hydroxybenzaldehyde.
Tokoro et al. (7) and Kanisawa (8) have presented a pathway
via the glucosides to glucovanillin. However, the 4-hydroxyben-
zaldehyde glucoside was not metabolized to glucovanillin in this
study (Figure 2). Furthermore, the formation of glucovanillin was
very fast, with a maximum increase in 6 h after feeding 14C-
labeled compounds, and remained high. However, the glucose
esters of ferulic acid and 4-coumaric acid slowly decreased
(Figure 1). From these results the formation of vanillin via the
glucose ester of ferulic acid may be excluded. In the feeding
experiments withMet and Phe by the addition ofunlabeled ferulic
acid (Figures 3 and 4), the formations of glucovanillin slowed, but
glucovanillin increased over time. In addition, 14C was accumu-
lated as ferulic acid, suggesting that vanillin is directly synthesized
via ferulic acid and then immediately glucosylated to form
glucovanillin. High levels of 4-coumaric acid, ferulic acid, 4-
hydroxybenzaldehyde, 4-hydroxybenzyl alcohol, and vanillin are
immediately glucosylated to become the respective glucose esters
of C6-C3 compounds and glucosides of C6-C1 compounds
(Figure 5), because they appear to be poisonous to the cells of
vanilla plants (11). Large pools of 4-hydroxybenzaldehyde gluco-
side and glucovanillin accumulate in vanilla pods. The contents of
4-hydroxybenzaldehyde glucoside and glucovanillin in 6-month-
old pods (mature pods) were 0.23 and 1.23 mmol/100 g of fresh
weight, respectively. 4-Hydroxybenzaldehyde glucoside may be
the end metabolite from 4-hydroxybenzaldehyde as well as
glucovanillin or may be an intermediate of glucosides A and B
(Figures 2 and 5). Immature and mature pods contain 1.19 and
1.02 mmol of glucosides A and B in 100 g of fresh weight,
respectively. In conclusion, we propose the biosynthetic pathway
for vanillin in Figure 5, which shows that vanillin is synthesized
via ferulic acid from 4-coumaric acid and glucosylated to form
glucovanillin in mature vanilla pods. The pathway from ferulic
acid to vanillin may be supported by demonstrating the existence
of an enzyme catalyzing the reaction for shortening of the
phenylpropanoid side chain. As reported by Podstolski et
al. (10), we also detected enzyme activity in cell-free extracts of
vanilla pods. Further investigation to purify, isolate, and identify
the key enzyme is in progress.
ACKNOWLEDGMENT
We thank Takasago International Corp. for supplying authen-
tic glucosides and green vanilla pods and for their helpful advice
on the synthesis of authentic compounds.
LITERATURE CITED
(1) Odoux, E.; Escoute, J.; Verdeil, J. L.; Brillouet, J. M. Localization of
β-D-glucosidase activity and glucovanillin in vanilla bean (Vanilla
planifolia Andrews). Ann. Bot. 2003, 92, 437–444.
(2) Gobley, T. W. Recherches sur la Principe odorant de la Vanille. J.
Pharm. Chem 1858, 3, 401–405.
(3) Tiemann, F.; Haarmann, W. Ueber das Coniferin und seine Un-
wandlung in das aromatische Princip der Vanille. Ber. Dtsch. Chem.
Ges. 1874, 7, 608–623.
(4) Walton, N. J.; Mayer, M. J.; Narbad, A. Vanillin. Phytochemistry
2003, 63, 505–515.
(5) Anwar, M. H. Paper chromatography of monohydroxyphenols in
vanilla extract. Anal. Chem. 1963, 35, 1974–1976.
(6) Zenk, M. H. Biosynthese von vanillin in Vanilla planifolia Andr. Z.
Pflanzenphysiol. 1965, 53, 404–414.
(7) Tokoro, K.; Kawahara, S.; Amano, A.; Kanisawa, T.; Indo, M.
Glucosides in vanilla beans and changes of their contents during
maturation. In Flavour Science and Technology; Bessiere, Y., Thomas,
A. F., Eds.; Wiley: Chichester, U.K., 1990; Vol. 73, pp 73-76.
(8) Kanisawa, T. Flavor development in vanilla beans. Koryo 1993, 180,
113–123.
(9) Yazaki, K.; Heide, L.; Tabata, M. Formation of p-hydroxybenzoic
acid from p-coumaric acid by cell free extract of Lithospermum
erythrorhizon cell cultures. Phytochemistry 1991, 30, 2233–2236.
(10) Podstolski, A.; Havkin-Frenkel, D.; Malinowski, J.; Blount, J. W.;
Kourteva, G.; Dixon, R. A. Unusual 4-hydroxybenzaldehyde
synthase activity from tissue cultures of the vanilla orchid Vanilla
planifolia. Phytochemistry 2002, 61, 611–620.
(11) Dignum, M. J. W.; Kerler, J.; Verpoorte, R. Vanilla production:
technological, chemical, and biosynthetic aspects. Food Rev. Int.
2001, 17, 199–219.
(12) Negishi, O.; Ozawa, T. Determination of hydroxycinnamic acids,
hydroxybenzoic acids, hydroxybenzaldehydes, hydroxybenzyl alco-
hols and their glucosides by high-performance liquid chromatogra-
phy. J. Chromatogr., A 1996, 756, 129–136.
(13) Funk, C.; Brodelius, P. E. Phenylpropanoid metabolism in
suspension cultures of Vanilla planifolia Andr. II. Effects of
precursor feeding and metabolic inhibitors. Plant Physiol. 1990,
94, 95–101.

Article
J. Agric. Food Chem., Vol. 57, No. 21, 2009 9961
(14) Shimizu, T.; Kojima, M. Partial purification and characterization of
UDPG:t-cinnamate glucosyltransferase in the root of sweet potato,
Ipomoea batatas Lam. J. Biochem. 1984, 95, 205–212.
(18) Birkofer, L.; Kaiser, C.; Hillges, B.; Becker, F. NMR-specktrosko-
pische Untersuchungen von Acyl-glykosen. Liebigs Ann. Chem.
1969, 725, 196–202.
(15) McCliskey, C. M.; Coleman, G. H. β-
D
-Glucose-2,3,4,6-tetraacetate.
(19) Funk, C.; Brodelius, P. E. Phenylpropanoid metabolism in suspen-
sion cultures of Vanilla planifolia Andr. III. Conversion of 4-
methoxycinnamic acids into 4-hydroxybenzoic acids. Plant Physiol.
1990, 94, 102–108.
In Organic Synthesis; Horning, E. C., Ed.; Wiley: New York, 1955;
Collect. Vol. 73, pp 434-436.
::
(16) Birkofer,L.;Kaiser,C.;Nouvertne
´
,W.;Thomas,U.Naturlich vorkommende
::
Zuckerester von Phenolcarbonsauren. Z. Naturforsch. 1961, 16, 249–251.
(17) Birkofer, L.; Kaiser, C.; Kosmol, H.; Romussi, G.; Donike, M.;
Received for review April 9, 2009. Revised manuscript received June 19,
2009. Accepted September 8, 2009.
Michaelis, G. D-Glucose- und L-Rhamnoseester der p-Cumar- und
::
Ferulasaure. Liebigs Ann. Chem. 1966, 699, 223–231.