Synthesis of stable isotope-labeled precursors for the biosyntheses of capsaicinoids, capsinoids, and capsiconinoids

Article abstract of DOI:10.1271/bbb.110187

Stable isotope-labeled precursors were synthesized for an analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate the biosynthetic flow of capsaicinoids, capsinoids, and capsiconinoids. [1- ¹³C][5-²H]-Vanil

Full text of DOI:10.1271/bbb.110187

Biosci. Biotechnol. Biochem., 75 (8), 1611–1614, 2011
Note
Synthesis of Stable Isotope-Labeled Precursors for the Biosyntheses
of Capsaicinoids, Capsinoids, and Capsiconinoids
y
1;
Kenji KOBATA,1;2 Makoto MIMURA,1 Mai SUGAWARA,1 and Tatsuo WATANABE
1Graduate School of Nutritional and Environmental Sciences and Global COE Program, University of Shizuoka,
52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
2Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan
Received March 10, 2011; Accepted May 10, 2011; Online Publication, August 7, 2011
[doi:10.1271/bbb.110187]
Stable isotope-labeled precursors were synthesized
for an analysis by liquid chromatography-tandem mass
spectrometry (LC-MS/MS) to elucidate the biosynthetic
flow of capsaicinoids, capsinoids, and capsiconinoids.
[10-13C][5-2H]-Vanillin was prepared by the condensa-
tion of guaiacol with [13C]-chloroform and a D2O
treatment. Labeled vanillylamine, vanillyl alcohol,
ferulic acid, and coniferyl alcohol were prepared from
the labeled vanillin. The labeled vanillylamine was
converted to labeled capsaicinoid in a crude enzyme
solution extracted from pungent Capsicum fruits.
produced via the phenylpropanoid pathway (Fig. 1).5)
Although the factor for the conversion of vanillin to
vanillyl alcohol seems to be crucial for capsinoid
biosynthesis, it has not yet been elucidated. It is also
not clear whether CS participates in the condensation of
the vanillyl alcohol with a fatty acid in the final stage of
capsinoid biosynthesis. A new class of coniferyl esters,
namely capsiconinoids, has recently been found in
several cultivars of pepper.6) One of the possible
biosynthetic precursors of capsiconinoids is coniferyl
alcohol from the viewpoint of its chemical structure.
Clarifying details about the flow of metabolites is a
relatively direct approach to elucidating the biosynthetic
pathway, although a molecular biological approach
would also be helpful in understanding the pathway.
We chemically prepared stable isotopes of the putative
precursors of capsaicinoids, capsinoids, and capsiconi-
noids in the present study as analytical tools to elucidate
details of their biosynthesis (Fig. 2). The natural
abundance of the [M þ 2] molecule to [M] molecule
of these compounds is about 1%, although that of
[M þ 1] to [M] is about 9%. We therefore synthesized
doubly labeled compounds to get higher sensitivity, and
then detected the enzyme activity of CS by liquid
chromatography-tandem mass spectrometry (LC-MS/
MS), using the labeled precursor.
[10-13C]-Vanillin was prepared according to the
method of Ohara and Fukuda7) with a slight modification.
Briefly, a mixture of 4 mL of 5 M guaiacol (20 mmol) in
MeOH, 390 mL of [13C]-chloroform (4.9 mmol; Taiyo
Nippon Sanso Co., Tokyo, Japan), and 11 mL of 2.8 M
NaOH aq. was heated at 65 ꢀC for 24 h. An ethyl acetate
extract from the reaction mixture was purified by silica
gel and subsequently by successive octadecylsilyl silica
gel column chromatography to afford 111.9 mg of [10-
13C]-vanillin (0.73 mmol, 15% yield from [13C]-chloro-
form). The 13C-nuclear magnetic resonance (NMR)
spectrum8) of the compound showed an extremely
intense signal at 191 ppm indicating a carbonyl carbon
containing the 13C isotope. The 1H-NMR spectrum8)
showed a set of doublet signals coupled with 173 Hz at
9.82 ppm, indicating the presence of an aldehyde proton
attached to the 13C carbonyl carbon. The other signals in
both NMR spectra of the compound were completely
coincident with those of standard vanillin. The positive
Key words: stable isotope; capsaicinoid; capsinoid;
capsaicin synthase; liquid chromatography-
tandem mass spectrometry (LC-MS/MS)
The substances responsible for the pungency of
Capsicum peppers are a group of lipophilic alkaloids
knows as capsaicinoid(s). The fundamental structure of
capsaicinoids comprises a fatty acid amide of vanillyl-
amine. Early studies in which classical in vivo tracer
experiments were performed with radioactive precursors
revealed the outline of the biosynthetic pathway for
capsaicinoids (Fig. 1).1) The aromatic moiety of capsai-
cinoids is derived from phenylalanine via the phenyl-
propanoid pathway. However, details of the downstream
events on the pathway, i.e., the conversion of vanillin to
vanillylamine, have not yet been elucidated. Recent
molecular biological approaches suggest that the gene
of a putative aminotransferase (pAMT) encodes an
enzyme that catalyzes the conversion of vanillin to
vanillylamine.2) A putative acyltransferase encoded
in Pun1, namely capsaicin synthase (CS), is also
considered a candidate enzyme that catalyzes the
condensation of vanillylamine with a fatty acid to
produce capsaicinoids.3)
Recent studies have revealed that many kinds of
pungent and non-pungent Capsicum cultivars contained
a novel group of non-pungent capsaicinoid-like sub-
stances named capsinoid(s).4) The fundamental structure
of capsinoids comprises a fatty acid ester of vanillyl
alcohol. We performed in our previous study an in vivo
tracer experiment using radioactive precursors and
found that the aromatic moiety of capsinoids, i.e.,
vanillyl alcohol, was derived from vanillin that was
y
To whom correspondence should be addressed. Fax: +81-54-264-5550; E-mail: watanbt@u-shizuoka-ken.ac.jp
Abbreviations: APCI-MS, atmospheric pressure chemical ionization-mass spectrometry; CS, capsaicin synthase; LC-MS/MS, liquid
chromatography-tandem mass spectrometry; MRM, multiple reaction monitoring; NMR, nuclear magnetic resonance

1612
K. KOBATA et al.
Phenylalanine
Phenylpropanoid pathway
Coniferyl alcohol
Ferulic acid
Vanillin
?
O
CH3O
HO
O
?
pAMT
Capsiconinoids
(e.g., capsiconiate)
Vanillylamine
Vanillyl alcohol
Branched-chain fatty acid-CoA
?
CS
O
O
CH3O
HO
CH3O
HO
N
H
O
Capsaicinoids
Capsinoids
(e.g., capsaicin)
(e.g., capsiate)
Fig. 1. Proposed Biosynthetic Pathway for Capsaicinoids, Capsinoids, and Capsiconinoids in Capsicum Plants.
pAMT, putative aminotransferase; CS, capsaicin synthase.
*
CH3O
NH2
HO
D
[1'-13C][5-2H]-vanillylamine
1) CH3ONH2, Pyr, rt, 24 h
2) H2, Pd/C, HCl aq, rt, 3 h
*
CHO
*
*
CH3O
HO
CH3O
HO
*
CH3O
CHO
CH3O
HO
NaBH4
CHCl3
NaOH aq, 65°C, 24 h
D2O
OH
Et3N, 100°C, 24 h
MeOH, rt, 3.5 h
HO
D
D
guaiacol
[1'-13C]-vanillin
[1'-13C][5-2H]-vanillin
[1'-13C][5-2H]-vanillyl alcohol
Malonic acid
Pyr, aniline, 55°C, 14 h
*
*
CH3O
HO
COOH
CH3O
OH
1) EtOH, conc H2SO4, 100°C, 3 h
2) LiAlH4, EtOH, rt, 0.5 h
HO
D
D
[1'-13C][5-2H]-coniferyl alcohol
[1'-13C][5-2H]-ferulic acid
Fig. 2. ꢁ Synthetic Scheme for the Stable Isotope-Labeled Precursors.
13C; D, 2H.
,
atmospheric pressure chemical ionization-mass spectro-
metric (APCI-MS) spectrum9) showed a protonated
molecular ion peak at m=z 154.
13C][5-2H]-vanillin. The 1H-NMR spectrum of the
compound showed signal attenuation at 7.04 ppm, this
being assigned to the 5-position of the benzene ring of
vanillin. The rate of substitution of a proton with
deuterium at the 5-position of vanillin was calculated
as 89% from the area of the attenuated proton signal.
The positive APCI-MS spectrum showed a protonated
molecular ion peak at m=z 155.
[10-13C][5-2H]-Vanillin was prepared according to the
method of Krings et al.10) with a slight modification.
Briefly, a mixture of 53.7 mg of [10-13C]-vanillin
(0.37 mmol), 290 mL of deuterium oxide (14.5 mmol),
and 52 mL of triethylamine was heated at 100 ꢀC for 24 h
under nitrogen gas. The ethyl acetate extracted from the
reaction mixture gave 55.2 mg (0.36 mmol) of [10-
[10-13C][5-2H]-Vanillylamine was prepared from [10-
13C][5-2H]-vanillin via an oxime according to the

Stable Isotope-Labeled Capsaicinoid Precursors
1613
method of Gannett et al.11) with a slight modification.
Briefly, a mixture of 100.8 mg of [10-13C][5-2H]-vanillin
(0.65 mmol), 60.0 mg of methoxyamine hydrochloride
(0.78 mmol), and 18 mL of dry pyridine was agitated for
24 h at room temperature. The reaction mixture was then
subjected to rotary evaporation in vacuo to eliminate
pyridine. CHCl3 extracted from the residue gave
109.5 mg of [10-13C][5-2H]-4-hydroxy-3-methoxyben-
zaldehyde O-methyloxime (0.60 mmol, 92% yield). A
mixture of 20.2 mg of the oxime (0.11 mmol), 42 mg of
palladium carbon, and 79 mL of 12 M HCl was agitated
at room temperature for 3 h in 150 cm3 of hydrogen gas.
The reaction mixture was passed through Celite to
remove the palladium carbon, and was then subjected to
rotary evaporation in vacuo to afford 21.7 mg of [10-
13C][5-2H]-vanillylamine (51% purity by HPLC, 60%
olefinic proton attached to the 13C isotope with trans
configuration. The other signals in these spectra were
similar to those of standard ferulic acid, except for the
signal corresponding to a deficiency at the 5-position
in the labeled compound in its 1H-NMR spectrum.
The negative APCI-MS data showed a deprotonated
molecular ion peak at m=z 195.
[10-13C][5-2H]-Coniferyl alcohol was prepared from
[10-13C][5-2H]-ferulic acid via its ethyl ester according
to the method of Quideau and Ralph12) with a slight
modification. A mixture of 81.0 mg of [10-13C][5-2H]-
ferulic acid (0.41 mmol), 800 mL of conc. HCl, and
10 mL of ethyl alcohol was heated for 3 h at 100 ꢀC.
After evaporating the reacted mixture, the resulting
residue gave a corresponding amount of an ethyl ester.
This ethyl ester was dissolved in 5 mL of dehydrated
diethyl ether, and 137 mg of LiAlH4 was then added to
the solution. The mixture was allowed to remain at room
temperature for 30 min, and then the catalyst was
removed by filtration. The ethyl acetate extract from
the residue was purified by ODS column chromatog-
raphy to afford 22 mg of [10-13C][5-2H]-coniferyl alco-
hol (0.12 mmol, 29% yield). The 1H-NMR spectrum
showed a set of double-doublet signals coupled with 150
and 16 Hz at 6.53 ppm, indicating a shift of the olefinic
proton at the 10-position because of the reduction from
carboxylic acid to an alcohol. The other signals in the
spectrum were similar to those of standard coniferyl
alcohol, except for the signal indicating a deficiency at
the 5-position in the labeled compound. The positive
APCI-MS data showed a dehydroxy molecular ion peak
at m=z 165, this being 2 masses larger than that of
standard coniferyl alcohol.
1
total yield from [10-13C][5-2H]-vanillin). The H-NMR
spectrum showed a set of doublet signals of 2 protons
coupled with 142 Hz at 4.01 ppm, indicating the pres-
ence of a labeled methylene group together with amine
and phenyl groups. Labeled methylene was observed as
an extremely intense signal at 44 ppm in the 13C-NMR
spectrum. The other signals in these spectra were similar
to those of standard vanillylamine, except for the signal
indicating deficiency at the 5-position in the labeled
compound in its 1H-NMR spectrum. The positive APCI-
MS data for standard vanillylamine shows a typical
fragment ion peak predominantly at m=z 137 caused by
the elimination of an amine group. Labeled vanillyl-
amine showed a fragment ion peak at m=z 139,
indicating that 2 labels remained in the fragment.
[10-13C][5-2H]-Vanillyl alcohol was prepared by
NaBH4 reduction of [10-13C][5-2H]-vanillin. We added
12 mg of NaBH4 (0.32 mmol) to 2.4 mL of an MeOH
solution containing 23.9 mg of [10-13C][5-2H]-vanillin
(0.16 mmol), and placed the mixture at room temper-
ature for 3.5 h. Excess water was added to the reaction
mixture, and subsequent ethyl acetate extraction gave
24.8 mg of [10-13C][5-2H]-vanillyl alcohol (0.16 mmol).
A preliminary experiment was carried out for meas-
uring the capsaicin synthase (CS) activity in a crude
enzyme solution extracted from the Capsicum pepper
fruits according to the method of Fujiwake et al.13) with
a slight modification. Conversion of the labeled vanillyl-
amine into a labeled vanillyl octanamide, a model
analog of capsaicin, was measured by an LC-MS/MS
system (LC: Nanospace SI-1, Shiseido, Tokyo, Japan;
MS/MS: API 2000, Applied Biosystems, Carlsbad, CA,
USA). The placenta and dissepiment (1.4 g) were
dissociated from 3 pods of fresh fruits of Habanero
(Capsicum chinense) and were ground at 4 ꢀC with 3 mL
of 50 mM Tris–HCl (pH 8.0) containing 1% Triton
X-100. After centrifuging at 2000 g and 4 ꢀC for 30 min,
the supernatant was used as the crude enzyme solution
(8.6 mg/mL in protein content). We incubated 100 mL of
a 50 mM Tris–HCl (pH 8.0) solution containing 2 mM
1
The H-NMR spectrum showed a set of doublet signals
of 2 protons coupled with 144 Hz at 4.59 ppm, indicating
the labeled methylene group together with hydroxy and
phenyl groups. The other signals in these spectra were
similar to those of standard vanillyl alcohol, except for
the signal indicating a deficiency at the 5-position in the
1
labeled compound in its H-NMR spectrum. The posi-
tive APCI-MS data for the labeled compound showed a
typical fragment ion peak predominantly at m=z 138
caused by elimination of the aliphatic hydroxy group of
vanillyl alcohol.
[10-13C][5-2H]-Ferulic acid was prepared from [10-
13C][5-2H]-vanillin according to the method of Krings
et al.10) with a slight modification. A mixture of 23.0 mg
of [10-13C][5-2H]-vanillin (0.15 mmol), 31.0 mg of
malonic acid (0.30 mmol), 46 mL of pyridine, and 5 mL
of aniline was heated at 55 ꢀC for 14 h. We then added
200 mL of 5 M HCl and 30 mL of water to the reaction
mixture. Subsequent ethyl acetate extraction gave
24.0 mg of [10-13C][5-2H]-ferulic acid (0.12 mmol,
80.0% yield). The 13C-NMR spectrum of the compound
showed an extremely intense signal at 148 ppm indicat-
[10-13C][5-2H]-vanillylamine, 1 m
M octanoyl-CoA (Sigma),
and 10 mL of the enzyme solution at 37 ꢀC for 1 h. The
reaction was terminated by adding 6 M HCl to the
mixture. The ethyl acetate extract from the mixture was
dried and then dissolved in MeOH containing 0.1%
acetic acid for an LC-MS/MS analysis. The LC-MS/MS
conditions for measuring vanillyl octanamide were as
follows: LC column, a reversed-phase Unison UK-C18
silica gel column (2 mm i.d. ꢂ 150 mm (Imtakt Co.,
Kyoto, Japan)); column temperature, 40 ꢀC; solvent, 70–
100% MeOH containing 0.1% acetic acid (0–10 min);
flow rate, 0.2 mL/min; injection volume, 5 mL; MS/MS
ion source, ESI; polarity, positive; detection mode,
multiple reaction monitoring (MRM); detected ions,
1
ing a sp2 13C isotope of carbon. The H-NMR spectrum
showed a set of double-doublet signals coupled with
155 and 16 Hz at 7.59 ppm, indicating a conjugated

1614
K. KOBATA et al.
precursor/product, 280/137 for the natural predominant
isotope of vanillyl octanamide [M] and 282/139 for the
labeled one [M þ 2]. The optimum parameters for
detecting vanillyl octanamide were automatically tuned
by using an authentic sample with Analyst software
(Applied Biosystems). The ions of vanillyl octanamide
were observed in the mass chromatogram at 4.38 min.
The enzyme solution from native Habanero contained
a trace of vanillyl octanamide (the naturally predom-
inant isotope [M], 3.9 nmol/mg of protein). A negligible
amount of the native [M þ 2] isotope (50 pmol/mg of
protein) was observed as 1.2% of the abundance ratio
that coincides with the calculated abundance ratio of the
isotope (1.3%). Incubating the enzyme solution with
labeled vanillylamine and octanoyl-CoA substantially
increased the amount of the [M þ 2] isotope of vanillyl
octanamide to 370 pmol/mg of protein, and its abun-
dance ratio was 8.8%. No marked increase in the
[M þ 2] isotope was apparent in the absence of the
enzyme or when using its boiled solution.
References and Notes
1) Suzuki T and Iwai K, ‘‘Alkaloids’’ Vol. 23, ed. Brossi A,
Academic Press, New York, pp. 227–299 (1984).
´
2) del Rosario Abraham-Juarez M, del Carmen Rocha-Granados
´
M, Lopez MG, Rivera-Bustamante FR, and Ochoa-Alejo N,
Planta, 227, 681–695 (2008).
3) Stewart CJr, Kang BC, Liu K, Mazourek M, Moore SL, Yoo
EY, Kim BD, Paran I, and Jahn MM, Plant J., 42, 675–688
(2005).
4) Singh S, Jarret R, Russo V, Majetich G, Shimkus J, Bushway R,
and Perkins B, J. Agric. Food Chem., 57, 3452–3457 (2009).
5) Sutoh K, Kobata K, Yazawa S, and Watanabe T, Biosci.
Biotechnol. Biochem., 70, 1513–1516 (2006).
6) Tanaka Y, Hosokawa M, Otsu K, Watanabe T, and Yazawa S,
J. Agric. Food Chem., 57, 5407–5412 (2009).
7) Ohara M and Fukuda J, Pharmazie, 33, 467 (1978).
8) 1H- and 13C-NMR spectra (tetramethylsilane was used as the
internal standard) of all the compounds were recorded on a
JEOL ꢀ-400 instrument (JEOL, Tokyo, Japan) at 399.65 and
100.40 MHz, respectively.
9) APCI-MS analyses of all the compounds were performed with
the API2000 system (Applied Biosystems, Carlsbad, CA, USA).
Thirty mL/min of 10 mM compound was infused into the system
with 200 mL/min of MeOH containing 0.1% acetic acid. The
ion source was APCI and detection polarity was positive.
The optimum parameters for the detection of each compound
were tuned automatically by the Analyst software (Applied
Biosystems).
We synthesized in this study stable isotope-labeled
precursors for capsaicinoid, capsinoid, and capsiconi-
noid biosyntheses and showed that the precursors could
be useful tools for biosynthetic studies. Further inves-
tigations are in progress using the labeled precursors for
information on the flow and properties of enzymes
involved in the biosyntheses.
10) Krings U, Pilawa S, Theobald C, and Berger RG, J. Biotechnol.,
85, 305–314 (2001).
11) Gannett PM, Nagel DL, Reilly PJ, Lawson T, Sharpe J, and
Toth B, J. Org. Chem., 53, 1064–1071 (1988).
Acknowledgments
12) Quideau S and Ralph J, J. Agric. Food Chem., 40, 1108–1110
(1992).
This work was supported by Grant-Aid for Scientific
Research (C, 19580125) from JPSP (Japan) and by the
Global Center of Excellence (COE) program from
MEXT (Japan).
13) Fujiwake H, Suzuki T, Oka S, and Iwai K, Agric. Biol. Chem.,
44, 2907–2912 (1980).