Synthesis and elucidation of deuterated vanillylamine hydrochloride and capsaicin

Article abstract of DOI:10.1002/jlcr.1678

Capsaicin is the major pungent component of hot peppers, which belong to the plant genus Capsicum. Although the biosynthesis of capsaicin is known to involve the condensation of vanillylamine and 8-methylnonenoic acid by capsaicin synthase, the mechanism of biosynthesis is still not fully understood. In this study, deuterium labelled versions of capsaicin and the precursor vanillylamine were synthesized in order to investigate the biosynthesis of capsaicin in hot peppers. Copyright

Full text of DOI:10.1002/jlcr.1678

Research Article
Received 27 March 2009,
Revised 29 July 2009,
Accepted 19 August 2009
Published online 18 September 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1678
Synthesis and elucidation of deuterated
vanillylamine hydrochloride and capsaicin
Sang Wook Kim,a Jeong Hoon Park,a Soon Jae Jung,a,b Tae Bum Choi,b
Ã
Min Goo Hur,a Seung Dae Yang,a and Kook Hyun Yub
Capsaicin is the major pungent component of hot peppers, which belong to the plant genus Capsicum. Although the
biosynthesis of capsaicin is known to involve the condensation of vanillylamine and 8-methylnonenoic acid by capsaicin
synthase, the mechanism of biosynthesis is still not fully understood. In this study, deuterium labelled versions of capsaicin
and the precursor vanillylamine were synthesized in order to investigate the biosynthesis of capsaicin in hot peppers.
Keywords: [2H3]Capsaicin; synthesis; biosynthetic pathway; hot pepper
from 4-hydroxybenzaldehyde (1) using a reported procedure.7 It
was found that the optimum reaction time for this bromination
Introduction
Capsaicin, trans-8-methyl-N-vanillyl-6-nonenamide, is the major
pungent component in hot peppers. It is an irritant for mammals,
including humans, and produces a burning sensation in tissue with
which it comes into contact. Capsaicin and several related
compounds are called capsaicinoids and are produced as
secondary metabolites by the plant genus Capsicum. Hot peppers
are widely used as a food additive in many East Asian and Latin-
American countries and are known to be rich in vitamin C.1 Besides
its use as a food additive, the medicinal and pharmacological
properties of capsaicin have been the focus of recent attention. It
has been reported that capsaicin has anticancer effects on several
cancer cell lines and can provide relief in arthritis and respiratory
ailments.1,2 Capsaicin is currently used as an ingredient of a muscle
pain relieving drug and is also used for the treatment of gastric
ulcers and rheumatism.2 As interest in the biological activity of
capsaicin has increased, the need for an understanding of its
biosynthesis has become evident. It has been reported that
capsaicin synthase (CS) is responsible for the condensation reaction
between vanillylamine and 8-methyl-nonenoic acid, leading to
capsaicin production.2–4 However, the biosynthetic pathway is not
fully understood. To investigate this pathway, methods for the
synthesis of isotopically labelled precursors are required. The total
synthesis of a carbon-14 labelled version of capsaicin, labelled in
the benzylic position, was published by Bodine and Chien5. An
improved version of this synthesis has subsequently been
reported.6 In this paper we report the synthesis of deuterium
labelled versions of vanillylamine and capsaicin, which can be used
to probe the biosynthetic pathway through the use of techniques
was 30 s to 1 min. Longer reaction times resulted in the formation
of 3,5-dibromo-4-hydroxybenzaldehyde or conversion back to
starting material.7 Conveniently, the crude reaction mixture could
be used immediately in the next step. The isotopic label was
introduced by displacement of bromine from (2) with sodium
[2H3]methoxide. This reaction was sensitive to moisture so
precautions were taken to ensure that the sodium [2H3]methoxide
solution was kept as dry as possible. Sodium [2H3]methoxide was
freshly prepared from [2H4]methanol.8 Vanillyloxime (4) was found
to be a 1:1 mixture of E- and Z-isomers as expected. The benzylic
protons of the E- and Z-isomers had chemical shifts at d 8.04 and d
7.91 respectively in the NMR spectrum. The presence of E- and Z-
isomers of (4) had no consequence in subsequent steps. The
crude oxime mixture was hydrogenated over palladium on carbon
in the presence of hydrochloric acid. Thorough flushing of the
hydrogenation vessel with inert gas before introduction of
hydrogen was essential to optimize the yield. Vigorous stirring
during hydrogenation was also essential. [2H3]Vanillylamine
hydrochloride was obtained as its hydrochloride salt in 31.4%
yield from (3). (E)-8-Methyl-6-nonenoyl chloride (6) was prepared
according to a published method.9 Coupling of [2H3]vanillylamine
free base with (6) gave [2H3]capsaicin (7) in 47.5% yield.
Conclusion
In conclusion, [2H3]vanillylamine hydrochloride (5) and [2H3]cap-
saicin (7) have been effectively prepared. These compounds can
be used to investigate the biosynthesis of capsaicin in the plant
genus Capsicum and probe the function of CS.
1
such as H, 13C NMR and mass spectrometry.
Results and discussion
aRadiation Research Division for Industry and Environment, Korea Atomic
Energy Research Institute, Jeongeup 580-185, Korea
Synthesis of the target compounds [2H3]vanillylamine hydro-
chloride (5) and [2H3]capsaicin (7) is shown in Scheme 1.
Overall yield of [2H3]vanillylamine hydrochloride (5) from (2) was
about 9.6%. 3-Bromo-4-hydroxybenzaldehyde (2) was prepared
bDepartment of Chemistry, Dongguk University, Seoul 100-715, Korea
*Correspondence to: Kook Hyun Yu, 3 Pil-dong Chung-gu, Seoul 100-715, Korea.
E-mail: yukook@dongguk.edu
J. Label Compd. Radiopharm 2009, 52 563–565
Copyright r 2009 John Wiley & Sons, Ltd.

S. W. Kim et al.
H
O
H
O
H
O
Br2
NaOCD3, CuBr
EtOAc
CD3OD
CD3
O
Br
OH
OH
OH
3
1
2
CH
NHOH
CH2NH2·HCl
H2, Pt
HCl
NaOAc/NH2OH
Acetic acid
CD3
CD3
O
O
OH
OH
4
5
O
6
CH2NH
O
Cl
Ether
CD3
O
OH
7
Scheme 1. Synthetic scheme of deuterated capsaicin.
added 1.1 g (9 mmol) of 4-hydroxybenzaldehyde (1) and the mixture
was stirred at 151C for 30s to give 3-bromo-4-hydroxybenzalde-
hyde (2). The contents of this flask were immediately poured into
flask A and the resulting mixture was heated at 1201C for 1.5h.
The mixture was cooled to room temperature and 60mL
(180mmol) of 3.0 M hydrochloric acid was added. The mixture
was transferred to a separating funnel and extracted with
3 Â 20mL of dichloromethane. The combined extracts were dried
over sodium sulphate and filtered. The filtrate was concentrated
to dryness and the residue was purified by column chromato-
graphy eluting with dichloromethane–hexane (9:1 v/v) to give (3)
as a light yellow solid (410mg, 2.64mmol, 26%).
Rf: 0.55(DCM:Hexane = 9:1). mp: 81–831C. 1H NMR (CDCl3) d:
9.83 (s, 1H, –CHO), 7.42 (m, 2H, H-5,6), 7.05 (d, 1H, H-2), 6.24
(s,1H, –OH). 13C NMR (CDCl3) d: 191.05, 151.80, 147.17, 129.99,
127.48, 114.42, 109.07, 55.19 (septet; D3). FAB-MS (m/z): 156.07
[M1H]1.
Experimental
General
All commercial reagents and solvents were purchased from Aldrich
and Merck in analytical grade and were used without further
purification. Melting point was measured on a Mel-Temp capillary
tube apparatus and was uncorrected. H, 13C NMR spectra were
1
recorded on a JEOL ECA-500 NMR spectrometer, chemical shift
data for the proton resonances were reported on ppm scale with
TMS as an internal standard. Mass spectra were recorded on JEOL
JMS-AX505WA mass spectrometer, compounds were measured by
FAB method using m-nitrobenzyl alcohol as a matrix at National
Center for Inter-University Research Facilities (NCIRF). Gravity
column chromatography was performed on Merck silica gel 60
(70-230 mesh ASTM). Thin layer chromatography was performed
on Merck silica gel 60F-254 glass plates and visualized by UV light.
The hydrogenation was carried out using our own device at 4 bar.
Synthesis of [2H3]vanillylamine hydrochloride (5)
Synthesis of [2H3]vanillin (3)
Flask A: A mixture of 45 mL (180 mmol) of 4.0 M NaOCD3/CD3OD
solution and 2.58 g (18 mmol) of copper(I) bromide was heated
at 1001C for 5–7 min under anhydrous conditions.
Flask B: 15 mL (7.5 mmol) of a 0.5 M solution of bromine in CD3OD
was cooled in an ice-water bath for 5 min. To this solution was
Synthesis of [2H3]vanillyloxime
2 g (12.8 mmol) of [2H3]vanillin was suspended in 10 mL of
glacial acetic acid and combined with 1.1 g (13.4 mmol)-
of anhydrous sodium acetate. Then, 0.9 g (12.9 mmol) of
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J. Label Compd. Radiopharm 2009, 52 563–565

S. W. Kim et al.
hydroxylamine hydrochloride was added, the reaction mixture The excess oxalyl chloride was evaporated and the residue was
was heated to 301C with stirring and stirred for 30 h.
added to the suspension of 500 mg (3.20 mmol) of base free
vanillylamine in 25 mL of tetrahydrofuran. The mixture was
stirred at room temperature for 2 h and then refluxed for an
additional 3 h. The reaction mixture was filtered, and the filtrate
was evaporated. The residue was purified by column chromato-
graphy (hexane:ethyl acetate = 2:1) on silica gel to give a white
solid (473 mg, 1.52 mmol, 47.5%).
Rf: 0.68 (DCM:MeOH = 9:1). mp: 64–661C. 1H NMR (DMSO-d6)d:
8.77 (s, 1H, –OH), 8.12 (t, 1H, –NH–), 6.76 (s, 1H, H-2), 6.65 (d, 1H,
H-6), 6.59 (d, 1H, H-5), 5.30 (m, 2H, HCCH), 4.10 (d, 2H, –CH2NH),
2.17 (m, 1H, CH), 2.07 (t, 2H, CH2CO), 1.90 (q, 2H, CH2CC), 1.47
(p, 2H, –CH2), 1.26 (p, 2H, –CH2), 0.89 (d, 6H, –CH3). 13C NMR
(DMSO-d6)d: 172.40, 147.93, 145.87, 137.89, 131.05, 127.15,
120.16, 115.67, 112.12, 56.02 (septet; D3), 42.31, 35.75, 32.21,
30.90, 29.22, 25.43, 23.08. FAB-MS (m/z): 309.22 [M1H]1.
Synthesis of [2H3]vanillylamine hydrochloride
The reaction mixture obtained in the earlier step (‘Synthesis of
[2H3]vanillyloxime’) was combined with 2.6 mL of hydrochloric
acid and 0.4 g of Pd/C. Hydrogen was piped into the reaction
mixture, with stirring, at 101C over a period of 4 h under a
pressure of 4 bar. After the addition of 5 mL of water the mixture
was heated to 601C and stirred for 1 h. The catalyst was filtered
off and the acetic acid was eliminated from the filtrate by
evaporation at 601C. Then the reaction mixture was combined
with 10 mL of water in order to dissolve the [2H3]vanillylamine
hydrochloride, and the salts were stirred at 601C for 0.5 h. After
the addition of 3 mL of hydrochloric acid was stirred for 1 h, the
suspension formed was cooled to 31C and after 3 h the
precipitate was filtered off, washed with acetone and dried at
601C. The [2H3]vanillylamine hydrochloride was obtained in
31.4% yield (780 mg, 4.02 mmol).
Rf: 0.10 (MeOH:DW = 9:1). mp: 215–2161C. 1H NMR (CDCl31
DMSO-d6)d: 9.16 (s, 1H, –OH), 8.24 (s, 2H, –NH2), 7.12 (s, 1H, H-2),
6.81 (d, 1H, H-6), 6.75 (d, 1H, H-5), 3.84 (s, 2H, -CH2NH2). 13C NMR
(CDCl31DMSO-d6)d: 147.50, 146.82, 124.65, 121.75, 115.26,
113.58, 55.74 (septet; D3), 42.17. FAB-MS (m/z): 157.10 [M1H]1.
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Synthesis of [2H3]Capsaicin (7)
To a aqueous solution of The [2H3]vanillylamine hydrochloride
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J. Label Compd. Radiopharm 2009, 52 563–565
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