A total synthesis of (R,S)S-glucoraphanin

Article abstract of DOI:10.1016/j.tet.2013.07.097

A total synthesis of (R,S)_S-glucoraphanin (GRP) has been completed by a novel, simple and convenient method in high overall yield (17% over seven steps). The study describes a method for the synthesis of natural and unnatural (methylsulfinyl)alkyl glucosinolates (GLs) and also opens useful pathways to synthesize GRP as well as other sulfinyl GLs.

Full text of DOI:10.1016/j.tet.2013.07.097

Tetrahedron 69 (2013) 8731e8737
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
A total synthesis of (R,S)S-glucoraphanin
,
,
*
Quan V. Vo a, Craige Trenerry b, Simone Rochfort c d, Andrew B. Hughes a
a Department of Chemistry, La Trobe University, Victoria 3086, Australia
b Department of Primary Industries, Knoxfield Centre, 621 Burwood Highway, Knoxfield, Victoria 3180, Australia
c Department of Primary Industries, Victorian AgriBiosciences Centre, La Trobe University Research and Development Park, 1 Park Drive, Bundoora, 3083
Victoria, Australia
d La Trobe University, Victoria 3086, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 April 2013
Received in revised form 15 July 2013
Accepted 30 July 2013
Available online 11 August 2013
A total synthesis of (R,S)S-glucoraphanin (GRP) has been completed by a novel, simple and convenient
method in high overall yield (17% over seven steps). The study describes a method for the synthesis of
natural and unnatural (methylsulfinyl)alkyl glucosinolates (GLs) and also opens useful pathways to
synthesize GRP as well as other sulfinyl GLs.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Glucoraphanin
Sulforaphane
Sulfinylalkyl glucosinolates
Glucosinolates
Brassica
1. Introduction
2. Results and discussion
GLs are
b-thioglucoside N-hydroxysulfates with a side chain (R)
2.1. Synthesis of 5-(methylthio)pentanal
and a sulfur-linked
b-D-glucopyranose moiety. These are natural
compounds, which are found in a large number of Brassica species
such as cabbage, broccoli, and canola. 4-Methylsulfinylbutyl glucosi-
nolate (GRP) is the most abundant glucosinolate in broccoli1 or Car-
daria draba2 and is hydrolyzed by the enzyme myrosinase to
sulforaphane that imparts numerous health benefits. Studies have
shownthatsulforaphaneeliminatescarcinogensinlivingtissue3,4 and
it is an inducer of phase 2 enzymes (glutathione S-transferase and
quinone reductase),5 whichisthoughttoresultincancerprotection.6,7
A series of synthetic analogs of sulforaphane were developed both as
a mixture of (R,S)S-isomers and as RS-sulforaphane.8e11 Although
sulforaphane haspotential applications, it isvolatilecompared to GRP,
which is a stable non-volatile compound. It is practical therefore to
create a GRP based nutraceutical, which retains the health advantages
of sulforaphane. Some studies in extraction of GRP from plants have
been developed.2,12e15 However, up to now, there have been only
a few studies in synthesis of labeled GRP,16,17 or oxidation of natural
glucoercin.5 The total chemical synthesis of GRP and the applications
of the synthetic compound have not yet been studied. Here, we report
a versatile synthetic approach for the synthesis of GRP.
From previous studies, 5-(methylthio)pentanal 1 could be syn-
thesized by either reduction of the corresponding ester or carbox-
ylic acid.18e20 Dawson’s group used 5-bromopentanenitrile as
starting material to synthesize aldehyde 1 in two steps in 18%
overall yield.18 However, using this pathway to synthesize the al-
dehyde 1 has some disadvantages such as low overall yield and the
toxicity of methanethiol.
To avoid the disadvantages and improve the yield, the aldehyde 1
was synthesized byan oxidationpathway (Scheme 1). The chloride 2
was treated with sodium methanethiolate in MeOH/H2O (3:1) for
4 h at rt to form 3 in 85% yield. The alcohol 3 was then oxidized by
Swern reaction21 to create the aldehyde 1 in 80% yield. Thus, the
overall yield was improved to 68% yield over two steps, compared
with 18% in Dawson’s method. Furthermore, the final aldehyde 1
could be used directly in the next step without purification.
2.2. Synthesis of 5-(methylsulfinyl)pentanal oxime
Firstly, the 5-(methylsulfinyl)pentanal oxime 8 was synthesized
following Morrison’s method (Scheme 1).16,17 To avoid oxidation of
the aldehyde, in the first step, the aldehyde 1 was protected
by reaction with ethane-1,2-diol in toluene in the presence of
* Corresponding author. Tel.: þ613 9479 000 0000; fax: þ613 9479 1266;
e-mail addresses: tahughes@optusnet.com.au, andrew.hughes2@optusnet.co-
m.au (A.B. Hughes).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.07.097

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Q.V. Vo et al. / Tetrahedron 69 (2013) 8731e8737
the products of reaction contained both the sulfoxide and the sul-
fone. Thus, it was difficult to control the amount of oxidant and
reaction conditions to get only compound 5. Moreover, the purifi-
cation of the products was not an easy task.
From the studies of Afzali-Ardakani’s group and Leonard’s
group,28,29 sodium metaperiodate was used as oxidant for the re-
action in three different sets of conditions (Table 1, entries 9e11). As
can be seen from Table 1, the best result is with NaIO4 (Table 1, entry
11). The reaction occurred within a 1 h reaction time in an excellent
isolated yield (82%). Compound 5 was obtained as a mixture of
(R,S)S-isomers and could be used directly in the next step without
purification. Sodium metaperiodate was the most efficient and
convenient reagent for the oxidation of the sulfide to the sulfoxide
and though the yield was similar to that in entry 1, it was used in
the following reactions.
The deprotection of the dioxolane 5 could not be carried out
under normal conditions involving aqueous acid, because of the
formation of condensation products.17 This problem was solved
by carrying out the protection in the presence of excess hy-
droxylamine in order to trap the aldehyde in situ, before com-
peting condensation reactions could occur, and so form the
desired oxime 8 in one step. Following the research on the oxi-
mation,17 the reaction was carried out with excess hydroxylamine
hydrochloride in H2O, but the yield was only about 18% (Table 2,
entry 1). To study the effect of concentration of hydroxylamine
hydrochloride on the reaction, the amount of the reagent was
increased from 2 to 10 equiv. It was found when the concentra-
tion of hydroxylamine hydrochloride was more than 5 equiv, the
yield of the reaction reached a maximum value. Thus 5 equiv of
hydroxylamine hydrochloride was chosen for the next phase of
the study.
Scheme 1. Synthesis of 5-(methylsulfinyl)pentanal oxime 8. Reagents and conditions:
(a) CH3SNa, MeOH/H2O (3:1),
4
h; (b) (COCl)2, DMSO, Et3N, DCM, ꢁ78 ꢀC; (c)
HOCH2CH2OH, p-MePhSO3H, toluene, reflux, 3 h; (d) NaIO4, MeOH/H2O (2:1), 3 h; (e)
H2NOH$HCl 5.0 equiv, 1 M H2SO4, CH3CN/H2O (3:1); (f) H2NOH$HCl, NaOAc, MeOH/
H2O (1:3).
Table 2
Deprotection of the dioxolane and oximation of 5 in a variety of conditions
p-toluenesulfonic acid as a catalyst for 3 h at reflux temperature to
yield the 1,3-dioxolane 4 in 88% yield.22
Entrya
Acid
Solvent
Tempb (ꢀC)
Yield of 8c (%)
1
2
3
4
5
6
7
8
d
H2O
H2O
rt
100
rt
60
rt
60
rt
100
rt
60
rt
18
17
50
46
48
45
37
32
53
41
54
34
From previous studies,23e27 the oxidation reaction of sulfides to
sulfoxides can be performed using a wide range of oxidation re-
agents. So, to find the best conditions for oxidation of 4 to 5,
combinations of reagents and solvents were investigated at differ-
ent temperatures; the results are summarized in Table 1.
d
d
MeOH/H2O
MeOH/H2O
CH3CN/H2O
CH3CN/H2O
H2O
d
d
d
1 M H2SO4
1 M H2SO4
1 M H2SO4
1 M H2SO4
1 M H2SO4
1 M H2SO4
H2O
9
MeOH/H2O
MeOH/H2O
CH3CN/H2O
CH3CN/H2O
Table 1
10
11
12
Oxidation of 4 in a variety of conditions
Temp (ꢀC) Reaction
time (min)
Yield (%)
60
Entry Oxidation
reagents
Solvent(s)
a
Amount of H2NOH$HCl 5 equiv, H3Oþ 1.2 equiv.
Reaction was conducted for 24 h and monitored by TLC.
Isolated yield of 8.
b
c
1
2
3
4
5
6
7
H2O2
H2O2
m-CPBA
NCS
NCS
H2O
MeOH/H2O
DCM
MeOH
Dioxane/H2O rt
60
rt
rt
0
60
180
120
60
60
30
78
47
85a
73a
64
To find the best conditions for the reaction, different reaction
HNO3/wet SiO2
SiO2eHNO3/KBr DCM
DCM
rt
rt
84a
60
conditions were applied. The results are shown in Table 2. It is
shown that the reaction should be conducted at rt rather than with
warming. However, the low yield of reaction was still a limitation
(around 54% yield). This is because the acid conditions reduce the
nucleophilicity of the hydroxylamine. Consequently, after finishing
the reaction, the reaction mixture always had the aldehyde 6
present, although hydroxylamine was added in an excess amount.
It is difficult to purify the oxime from reaction residue because of
the presence of the aldehyde 6 and ethylene glycol. The oxime 8
could be lost in the aqueous phases because of its high polarity and
hence water solubility. An attempt was made to improve the re-
action yield by using MeOH for acetalation of the aldehyde, but the
results were not satisfactory. As a result, the overall yield of 8 was
only 39% for the 1,3-dioxolane (34% for the dimethyl acetal) over
three steps.
30
8
9
10
11
PVPeHNO3/KBr
NaIO4
NaIO4
DCM
MeCN
MeOH/H2O
MeOH/H2O
rt
30
60
120
60
76
80
65
82
ꢁ10
rt
NaIO4
0
a
The converted yield of sulfide; note the product was a mixture of sulfoxide and
sulfone.
Initially, the oxidation reaction employed hydrogen peroxide in
H2O at 60 ꢀC (Table 1, entry 1) and hydrogen peroxide in MeOH/H2O
at rt (Table 1, entry 2). The yield of these reactions was 78 and 47%,
respectively. The reaction was also carried out using m-CPBA/DCM,
N-chlorosuccinimide (NCS)/MeOH, NCS/dioxaneeH2O, HNO3/wet-
SiO2, SiO2eHNO3/KBr, and polyvinyl pyrrolidone (PVP) PVPeHNO3/
KBr (Table 1, entries 3e8) but the yield was not much improved and

Q.V. Vo et al. / Tetrahedron 69 (2013) 8731e8737
8733
Fortunately, from these studies, it was found that the aldehyde 6
could be isolated, so a process to synthesize 8 without carbonyl
protection was planned (Scheme 1). The aldehyde 1 was oxidized
with sodium metaperiodate in MeOH/H2O (2:1) for 3 h at rt. After
work-up and purification by flash column chromatography eluting
with DCM/MeOH (9:1), compound 6 was obtained as a colorless
oil (75% yield). The oximation of aldehydes normally employs hy-
droxylamine hydrochloride in MeOH or EtOH in the presence of
pyridine,9,30 but it was shown that the reaction of 6 with hydrox-
ylamine hydrochloride in MeOH formed the acetal 1,1-dimethoxy-
5-(methylsulfinyl)pentane as a side product. Consequently, the
isolated yield of 8 was low (49%). To improve the yield of the re-
action, the oximation of 6 was carried out following Wang’s
method.31 The aldehyde was treated with hydroxylamine hydro-
chloride (3 equiv) and CH3COONa (3 equiv) in MeOH/H2O (1:3).
After work-up, the oxime 8, as a mixture isomers, was obtained as
a colorless oil (76% yield) by flash column chromatography.
By this method, the overall yield of 8 was enhanced to 57%.
However, it was also found that the aldehyde 6 was not very stable,
which could be a reason for the low yield of the oximation reaction.
This problem was solved by doing the oximation reaction first and
then oxidation of the sulfur atom (Scheme 1). It was pleasing that
the yields of the oximation and oxidation reactions were success-
fully improved to 92 and 91%, respectively, by this strategy. As
a result, compound 8 was obtained in 84% overall yield over two
steps. The study has shown that the pathway to synthesize oxime 8
following the oximation of aldehyde 3 and then oxidation of the
resulting oxime 7 was the simplest and most convenient method.
The results can be widely applied to synthesize other alkylsulfinyl
alkyl oximes.
It was found that when the amount of NCS was larger than
1.1 equiv, side products appeared, which were related to the oxi-
dation of the sulfur atom of the thiol 9 or the oxime 8. This may be
because the excess NCS caused the oxidation reaction over and
above the chlorination reaction. However, when the amount of NCS
was 1 equiv or less, there was insufficient for the chlorination to
reach completion (monitored by TLC). Consequently, there was
a limitation in the yield of 10. It was found that the amount of NCS
should be controlled in the range 1.05e1.10 equiv. This study also
found that the amount of pyridine should be the same as the
amount of NCS to avoid the adverse effect of pyridine in the second
step, which may reduce the yield of the coupling reaction.
Investigation of the solvent effects on the reaction (Table 3) has
found that DCM is the best solvent for the reaction because of the
highest yield and ease of work-up and purification.
Table 3
The coupling of 8 and 9 in a variety of solvents
Entry
Solvent(s)
Yield (%) of 10
1
2
3
4
5
6
Et2O
DCM
DCM/Et2O
THF
DCM/THF
CHCl3
37
47
40
43
38
22
2.4. Synthesis of GRP
Sulfation of 10 was accomplished with pyridine sulfur trioxide
complex in pyridine (Scheme 3).33 The resulting potassium salt 11
(73% yield) was isolated by flash column chromatography on silica
gel. The sulfating reaction was first conducted in DCM but the yield
was not high because of the limited dissolution of 10 in the reaction
mixture. Thus, pyridine was used as a convenient solvent for the
reaction. However, it was found in the work-up that removing the
pyridine solvent in the presence of aqueous potassium bicarbonate
resulted in deterioration of the product by deacetylation and/or
desulfation.34 The problem was solved by quick extraction of or-
ganic phases by chloroform and then CHCl3/MeOH (20%) before
work-up and purification.
2.3. Coupling of thiol glucose 9 with 5-(methylsulfinyl)pen-
tanal oxime
In most cases of glucosinolate synthesis, the oxime 8 is then
converted to the chlorooxime using NCS, before coupling to 2,3,4,6-
tetra-O-acetyl-b-D-glucopyranosyl thiol 9 (compound 9 can be
synthesized according to Floyd’s method32). However, in the liter-
ature,16,17 the chlorooxime was noted as an unstable compound and
in our hands could not be isolated. Therefore, the chlorination and
subsequent coupling reactions were carried out in one pot (Scheme
2). The oxime 8 was reacted with NCS in the presence of pyridine to
Scheme 2. The synthesis of S-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-(5-(meth-
ylsulfinyl)pentyl)thiohydroximate 10. Reagents and conditions: (a) NCS (1.05 equiv),
DCM, pyridine; (b) 9, Et3N, DCM.
form the chlorooxime and then it was immediately treated with
a mixture of thioglucose 9 and triethylamine in DCM. Consequently,
the thiohydroximate 10 was obtained in 47% yield over two steps.
The NMR, MS, IR data confirm that compound 10 is S-(2,3,4,6-tetra-
O-acetyl-
droximate.
b
-
D-glucopyranosyl)-(5-(methylsulfinyl)pentyl)thiohy-
Scheme 3. Synthesis of GRP 12. Reagents and conditions: (a) (1) Py$SO3, pyridine,
24 h, rt, (2) KHCO3, H2O; (b) MeOK, MeOH, 2 h.
De-O-acetylations were performed by dissolving 11 in MeOH in
the presence of MeOK as a catalyst. The final GRP 12 (88% yield) was
successfully purified by flash column chromatography on silica
gel.35 Compound 12 was obtained as a mixture of (R,S)S-isomers in
overall 17% yield over seven steps. This is much higher than the
literature result (9% yield over nine steps).16,17 The optical rotation

8734
Q.V. Vo et al. / Tetrahedron 69 (2013) 8731e8737
data {[
a
]
20 ꢁ14.8 (c 1.0, H2O) (lit. [
a
]
D ꢁ15 (c 1.0, H2O))} compared
1436, 1068, 1049 cmꢁ1; 1H NMR (300 MHz, CDCl3) (300 K)
d 3.59 (t,
D
favorably with the literature.5 However, it was not possible to dif-
ferentiate between the RS-isomer and the SS-isomer using the 1H
and 13C NMR data, which matched the literature data.5,36 An at-
tempt to separate the RS-isomer from the SS-isomer by HPLC was
unsuccessful.
J1,2¼6.3 Hz, 2H, CH2OH), 2.45 (t, J4,5¼7.2 Hz, 2H, CH2S), 2.04 (s, 3H,
CH3S), 1.89 (s, 1H, OH), 1.60e1.46 (m, 6H, CH2CH2CH2CH2OH); 13C
NMR (75 MHz, CDCl3) (300 K)
d 62.2 (C-1), 33.8 (C-5), 31.9 (C-2),
28.5 (C-4), 24.5 (C-3), 15.1 (CH3S); HRMS (ESI) m/z for C6H15OS
[MþH]þ, calcd 135.0838, found 135.0837.
3. Conclusion
4.3. 5-(Methylthio)pentanal (1)
A total synthesis of (R,S)S-GRP has been completed. This com-
pound is now being used for bioassay and other applications. This
study has found a novel method for synthesis of sulfinylalkyl
glucosinolates.
To a stirred solution of oxalyl chloride (1.67 mL, 19.0 mmol) in
DCM (40 mL) at ꢁ78 ꢀC under Ar was added DMSO (2.66 mL,
38.1 mmol). After 15 min, a solution of alcohol 2 (1.70 g, 12.7 mmol)
in DCM (10 mL) was added dropwise and the resulting reaction
mixture was stirred for 15 min. TEA (7.00 mL, 50 mmol) was added
and the reaction mixture was stirred for 15 min and then allowed to
warm to rt. Water (50 mL) was then added and the aqueous layer
was re-extracted with DCM (3ꢂ30 mL). The combined organic
layers were washed with saturated solutions of NH4Cl, NaHCO3, and
NaCl, dried over MgSO4, and concentrated under reduced pressure.
Compound 3 was obtained as a colorless oil (1.34 g, 80%) by flash
column chromatography eluting with 80% hexane/ethyl acetate.
Rf¼0.38 in 70% hexane/EtOAc; IR (NaCl) nmax 2916, 2858, 2719,1722,
4. Experimental
4.1. General methods
Melting points (mp) were recorded on a hot stage apparatus and
are uncorrected. Optical rotations were measured at the stated
temperatures in the stated solvent on a polarimeter at the sodium
D-line (589 nm); [a]
D values are given in 10ꢁ1 deg cm2 gꢁ1. Infrared
1427, 1390, 1355, 1126, 1072 cmꢁ1
(300 K)
9.72 (t, 1H, J¼1.5 Hz, 1H, CHO), 3.49e3.40 (m, 4H, H2 and
H5), 2.05 (s, 3H, CH3S), 1.77e1.56 (m, 4H, H3 and H4); 13C NMR
;
1H NMR (300 MHz, CDCl3)
spectra (nmax) were recorded on an FT-IR spectrometer. Samples
were analyzed as KBr drift (for solids) or as thin films on NaCl plates
(for liquids/oils). Unless otherwise specified, proton (1H) and car-
bon (13C) NMR spectra were recorded on a 300 MHz spectrometer
operating at 300 MHz for proton and 75 MHz for carbon nuclei.
d
(75 MHz, CDCl3) (300 K)
d 200.5 (C-1), 43.0 (C-5), 33.4 (C-2), 28.1
(C-4), 20.7 (C-3), 15.1 (CH3S); HRMS (ESI) m/z for C6H13OS [MþH]þ,
Chemical shifts were recorded as
d values in parts per million
calcd 133.0682, found 133.0681.
(ppm). Spectra were acquired in deuterated chloroform (CDCl3) at
300 K unless otherwise stated. For 1H NMR spectra recorded in
4.4. 2-(4-(Methylthio)butyl)-1,3-dioxolane (4)22
CDCl3, the peak due to residual CHCl3 (dH 7.24) was used as the
internal reference, while the central peak (dC 77.0) of the CDCl3
triplet was used as the reference for proton-decoupled 13C NMR
spectra. Low-resolution mass spectra were measured on a mass
spectrometer at 300 ꢀC and scan rate of 5500 m/z/s using either
water/methanol/acetic acid in a ratio of 0:99:1 or 50:50:1 as
a mobile phase. Accurate mass measurement was by mass spec-
trometry with a heated electrospray ionization (HESI) source. The
mass spectrometer was operated with full scan (50e1000 amu) in
positive or negative FT mode (at a resolution of 100,000). The
analyte was dissolved in water/methanol/acetic acid in a ratio
of 0:99:1 or 50:50:1 and infused via syringe pump at a rate of
A mixture of 5-(methylthio)pentanal 3 (280 mg, 2.1 mmol),
ethylene glycol (0.50 mL, 6.6 mmol), and p-toluenesulfonic acid
(44.0 mg, 0.2 mmol) in toluene (20 mL) was heated at reflux for 3 h
using a DeaneStark trap. The mixture was cooled to rt and K2CO3
(1 g) was added. After stirring for 10 min, the contents were washed
with 10% K2CO3 solution. The aqueous layer was extracted with
DCM (3ꢂ20 mL) and the combined organic layers were dried over
Na2SO4 and concentrated under reduced pressure. Compound 4
was obtained as colorless oil (330 mg, 88%). Rf¼0.44 in 70% hexane/
EtOAc; 1H NMR (300 MHz, CDCl3) (300 K)
d
4.85 (t, 1H, J¼4.5 Hz,
(CH2O)2CH), 3.98e3.86 (m, 4H, (CH2O)2CH), 2.51e2.44 (m, 2H,
5
m
L/min. The heated capillary was maintained at 320 ꢀC with
CH3SCH2), 2.08 (s, 3H, CH3S), 1.71e1.48 (m, 6H, CH3S(CH2)3); 13C
a source heater temperature of 350 ꢀC and the sheath, auxiliary and
sweep gases were at 40, 15 and 8 units, respectively. Source voltage
was set to 4.2 kV. Solvents were dried over standard drying agents
and freshly distilled before use. Ethyl acetate and hexane used for
chromatography were distilled prior to use. All solvents were pu-
rified by distillation. Reactions were monitored by TLC on silica gel
60 F254 plates with detection by UV fluorescence or charring with
a basic potassium permanganate stain. Flash column chromatog-
NMR (75 MHz, CDCl3) (300 K)
d 104.4 (CH(OCH2)2), 64.7
(CH(OCH2)2), 34.1, 33.5, 29.0, 23.2, 15.3 (CH3S); HRMS (ESI) m/z for
C8H17O2S [MþH]þ, calcd 177.0949, found 177.0940.
4.5. 2-(4-(Methylsulfinyl)butyl)-1,3-dioxolane (5)
A solution of sodium metaperiodate (652 mg, 3.0 mmol) in
water (10 mL) was added dropwise to a vigorously stirred ice-cold
solution of 4 (511 mg, 2.9 mmol) in MeOH (20 mL). The reaction
mixture was stirred for 3 h, then filtered at the pump and the filter
cake was washed with MeOH. The combined filtrates were con-
centrated to ca. 10 mL and then extracted with CHCl3 (3ꢂ20 mL).
The combined CHCl3 extracts were washed with water, dried, and
concentrated under reduced pressure. Compound 5 was obtained
as a yellow oil (460 mg, 82%) by flash column chromatography
eluting with 90% DCM/MeOH. Rf¼0.28 in 10% MeOH/DCM; IR (NaCl)
nmax 3408, 2949, 2916, 2887, 1720, 1409, 1130, 1018 cmꢁ1; 1H NMR
raphy was performed on silica gel 60 particle size 0.040e0.063
(230e400 mesh).
mm
4.2. 5-(Methylthio)pentan-1-ol (3)
5-Chloropentanol (2.50 g, 20.5 mmol) was added to a stirred
solution of sodium methylmercaptide (1.83 g, 23.5 mmol) in MeOH/
H2O (3:1 v/v) (20 mL). The solution was further stirred at rt over-
night and it was then extracted with chloroform (3ꢂ25 mL). The
organic phase was collected and dried over anhydrous Na2SO4.
Following filtration, the filtrate was evaporated under reduced
pressure and the crude product was purified by flash column
chromatography on silica gel eluting with 95% DCM/MeOH. The
pure alcohol 3 was isolated as a colorless oil (2.33 g, 85%). Rf¼0.64
in 90% DCM/MeOH; IR (NaCl) nmax 3369 (OH), 2933, 2858, 1456,
(300 MHz, CDCl3) (300 K)
d
4.84 (t, 1H, J¼4.5 Hz, (CH2O)2CH),
3.95e3.78 (m, 4H, (CH2O)2CH), 2.51e2.44 (m, 2H, CH3SOCH2), 2.08
(s, 3H, CH3SO), 1.83e1.54 (m, 6H, CH3SO(CH2)3); 13C NMR (75 MHz,
CDCl3) (300 K)
d 103.6 (CH(OCH2)2), 64.5 (CH(OCH2)2), 54.1
(CH3SOCH2), 38.1 (CH3SO), 32.9 (CH2CH2CH), 22.8 (CH2CH2CH2CH),

Q.V. Vo et al. / Tetrahedron 69 (2013) 8731e8737
8735
22.1 (CH2CH2CH2CH); HRMS (ESI) m/z for C8H17O3S [MþH]þ, calcd
The oxime 8, as a mixture (R,S)-sulfoxide isomers, was obtained as
a colorless oil (270 mg, 76%) by flash column chromatography
eluting with 90% DCM/MeOH.
193.0898, found 193.0892.
4.6. 5-(Methylsulfinyl)pentanal (6)
4.8.3. Synthesis of 8 from 7. A solution of sodium metaperiodate
(370 mg, 1.7 mmol) in water (5 mL) was added dropwise to a vig-
orously stirred ice-cold solution of 7 (240 mg, 1.6 mmol) in MeOH
(15 mL). The reaction mixture was stirred for 3 h, then filtered at
the pump and the filter cake was washed with MeOH. The com-
bined filtrates were concentrated to ca. 10 mL and then extracted
with CHCl3 (3ꢂ20 mL). The combined CHCl3 extracts were washed
with water, dried, and concentrated under reduced pressure.
Compound 8, as a mixture (R,S)S-sulfoxide isomers, was obtained
as a colorless oil (240 mg, 91%) by flash column chromatography
eluting with 90% DCM/MeOH. Rf¼0.17 in 10% MeOH/DCM; IR
(NaCl) nmax 3419, 2943, 2866, 2833, 1716, 1647, 1124, 1045,
Compound 6 was prepared by the method of Leonard et al.29 A
solution of sodium metaperiodate (692 mg, 3.2 mmol) in water
(10 mL) was added dropwise to a vigorously stirred ice-cold solu-
tion of 3 (400 mg, 3.0 mmol) in MeOH (20 mL). The reaction mix-
ture was stirred for 3 h, filtered off, and washed with MeOH. The
combined filtrates were concentrated to ca. 10 mL and then
extracted with CHCl3 (3ꢂ20 mL). The combined CHCl3 extracts
were washed with water, dried, and concentrated under reduced
pressure. Flash column chromatography eluting with 90% DCM/
MeOH gave compound 6 as a colorless oil (340 mg, 75%). Rf¼0.33 in
10% MeOH/DCM; IR (NaCl) nmax 3400, 2924, 2852, 2729, 1720, 1664,
1633, 1409, 1024 cmꢁ1; 1H NMR (300 MHz, CDCl3) (300 K)
d
9.68 (t,
1018 cmꢁ1 1H NMR (300 MHz, CDCl3) (300 K)
; d 7.31 (t, 1H,
1H, J¼1.5 Hz, 1H, CHO), 2.66e2.58 (m, 2H, H5), 2.48e2.42 (m, 5H,
J¼6.3 Hz, 1H, CH]N), 6.61 (s, 1H, NOH), 2.71e2.54 (m, 2H, H5),
CH3S and H2), 1.74e1.64 (m, 4H, H3 and H4); 13C NMR (75 MHz,
2.51 (s, 3H, CH3SO), 2.35e2.13 (m, 2H, H2), 1.78e1.49 (m, 4H, H3
CDCl3) (300 K)
d
201.1 (CHO), 53.8 (C-5), 42.9 (C2), 38.2 (CH3SO),
and H4); 13C NMR (75 MHz, CDCl3) (300 K)
d 150.3 (CH]N), 53.4,
21.7, 20.7 (C3 and C4); HRMS (ESI) m/z for C6H13O2S [MþH]þ, calcd
53.3 (C-5 isomers), 38.8 (CH3SO), 28.6 (C-2), 25.2, 24.8 (C-4 iso-
mers), 21.9, 21.6 (C-3 isomers); HRMS (ESI) m/z for C6H14NO2S
[MþH]þ, calcd 164.0740, found 164.0740.
149.0636, found 149.0630.
4.7. 5-(Methylthio)pentanal oxime (7)
4.9. 2,3,4,6-Tetra-O-acetyl-
b-D-glucopyranosyl thiol (9)
A solution of hydroxylamine hydrochloride (360 mg, 5.5 mmol)
and CH3COONa (760 mg, 5.5 mmol) in water (15 mL) was stirred in
an ice-bath. To this solution was added a solution of aldehyde 3
(230 mg, 1.7 mmol) in MeOH (5 mL). The mixture was stirred at 0 ꢀC
for 2 h and then it was stirred at rt for 1 h. The reaction mixture was
concentrated in vacuo; the residue was dissolved in water and
extracted with DCM (3ꢂ20 mL). The combined organic layers were
washed with water, dried over MgSO4, and concentrated under
reduced pressure. The residue was purified by flash column chro-
matography eluting with 90% hexane/ethyl acetate to give oxime 7
as a colorless oil (240 mg, 92%). Rf¼0.2 in 80% hexane/EtOAc; IR
To a solution of -glucose (3.00 g, 16.6 mmol) in dry pyridine
D
(33 mL) at 0 ꢀC under a nitrogen atmosphere was slowly added
acetic anhydride (31.5 mL, 333 mmol). The reaction mixture was
stirred at 0 ꢀC for 1 h before a catalytic amount of DMAP (200 mg,
1.67 mmol) was added. As the reaction mixture was allowed to
reach rt, it becomes slightly exothermic. After 6 h, the clear yellow
mixture was slowly poured into rapidly stirred ice-water (125 mL),
giving a sticky solid. After EtOAc extraction (3ꢂ45 mL), evaporation
of the solvent and co-evaporation with dry toluene (3ꢂ20 mL),
peracetylated glucose was obtained as a yellow solid (5.84 g, 90%). A
(NaCl) nmax 3369 (OH), 2915, 2856, 2720, 1720, 1600 (CH]N) cmꢁ1
;
solution of pentaacetyl-D-glucopyranose (2.00 g, 5.1 mmol) in DCM
1H NMR (300 MHz, CDCl3) (300 K)
d
8.92 (s, 1H, OH), 7.34 (t, 1H,
(20 mL) was stirred in an ice bath while HBr/HOAc (6 mL, 45 wt %)
was added drop-wise. After an hour, the solution was washed with
ice-water and cold saturated NaHCO3 solution, dried over MgSO4,
and concentrated to leave the glucosyl bromide as a pale yellow oil
(1.83 g). The oil was dissolved in dry acetone (20 mL), and the so-
J¼6.3 Hz, CH]N), 2.46e2.09 (m, 4H, H2 and H5), 2.01 (s, 3H, CH3S),
1.58e1.51 (m, 4H, H3 and H4); 13C NMR (75 MHz, CDCl3) (300 K)
d
151.3 (CH]N), 55.4 (C-5), 28.6 (C-2), 28.0 (C-4), 25.2 (C-3), 15.1
(CH3S); HRMS (ESI) m/z for C6H14NOS [MþH]þ, calcd 148.0791,
ꢀ
found 148.0789.
lution was added to freshly activated 4 A molecular sieves (2 g) and
thiourea (500 mg, 6.6 mmol). The mixture was maintained at reflux
temperature (60 ꢀC) under a nitrogen atmosphere for 2.5 h, cooled,
and filtered through Celite. Solvent removal and trituration of the
syrupy residue with hexane (3ꢂ20 mL) gave the isothiouronium
bromide 7 as a colorless amorphous powder. The crude product
was dissolved in DCM (20 mL), a solution of Na2S2O5 (2.00 g) in
water (20 mL) was added, and the mixture was maintained at reflux
under a nitrogen atmosphere for 1 h. After cooling, the organic
layer was separated and washed with water, saturated NaCl solu-
tion, dried over Na2SO4, filtered, and concentrated under reduced
pressure to give a residue. Pure 9 was obtained by flash column
chromatography on silica gel eluting with 0e3% MeOH/DCM as
4.8. 5-(Methylsulfinyl)pentanal oxime (8)
4.8.1. Synthesis of 8 from 5. To a stirred solution of 5 (683 mg,
3.6 mmol) and 2 N H2SO4 (5 mL) in CH3CN/H2O (3:1, 20 mL) was
added hydroxylamine hydrochloride (1.24 g, 17.8 mmol). The mix-
ture was stirred at rt for 48 h. The reaction mixture was concen-
trated in vacuo and then the residue was dissolved in water and
extracted with CHCl3 (3ꢂ20 mL). The combined organic layers were
washed with water, dried over MgSO4, and concentrated under
reduced pressure. The oxime 8, as a mixture (R,S)-sulfoxide iso-
mers, was obtained as a colorless oil (310 mg, 54%) by flash column
chromatography eluting with 90% DCM/MeOH.
a solid (1.60 g, 86%). Rf¼0.3 in 50% hexane/EtOAc; mp¼114e115 ꢀC
20
(lit. 113e114 ꢀC);24
[a
]
þ10.5 (c 1.0, CHCl3) (lit.þ11);24 1H NMR
D
4.8.2. Synthesis of 8 from 6. A solution of hydroxylamine hydro-
chloride (430 mg, 6.5 mmol) and CH3COONa (890 mg, 6.5 mmol) in
water (15 mL) was stirred in an ice-bath. To this mixture was added
a solution of aldehyde 6 (320 mg, 2.2 mmol) in MeOH (5 mL). The
mixture was stirred at 0 ꢀC for 2 h and then it was stirred at rt for
1 h. The reaction mixture was concentrated at reduced pressure
and the residue was dissolved in water and extracted with CHCl3
(3ꢂ20 mL). The combined organic layers were washed with water,
dried over MgSO4, and concentrated under reduced pressure.
(300 MHz, CDCl3) (300 K) d 5.18e5.02 (m, 2H, H3 and H4), 4.93 (dd,
J1,2¼9.6 Hz, J2,3¼9.3 Hz, 1H, H2), 4.51 (dd, J1,2¼9.6 Hz, J1,SH¼9.9 Hz,
1H, H1), 4.23 (dd, J5,6b¼4.8 Hz, J6a,6b¼12.3 Hz, 1H, H6b), 4.11 (dd,
J5,6a¼2.4 Hz, J6a,6b¼12.3 Hz,1H, H6b), 3.66e3.17 (m,1H, H5), 2.29 (d,
J1,SH¼9.9 Hz, 1H, SH), 2.08e1.96 (m, 12H, CH3COO); 13C NMR
(75 MHz, CDCl3) (300 K)
d
170.2, 169.7, 169.2, 168.9 (4ꢂCH3COO),
87.3 (C-1), 75.9 (C-3), 73.2 (C-2, C-3), 67.7 (C-4), 61.6 (C-6), 20.6,
20.3(2), 20.2 (4ꢂCH3COO); HRMS (ESI) m/z for C14H19O9S [MꢁH]ꢁ,
calcd 363.0755, found 363.0746.

8736
Q.V. Vo et al. / Tetrahedron 69 (2013) 8731e8737
4.10. S-(2,3,4,6-Tetra-O-acetyl-
ylsulfinyl)pentyl)thiohydroximate (10)
b
-
D
-glucopyranosyl)-(5-(meth-
eluting with EtOAc/MeOH/H2O (16:4:1). Rf¼0.08 in EtOAc/MeOH/
20
H2O (16:4:1); mp>115 ꢀC (dec); [
a]
ꢁ14.8 (c 1.0, H2O) (lit.5
D
[
a
]
ꢁ15 (c 1.0, H2O)); IR (KBr drift) nmax 3316 (OH), 2976, 2868,
D
To a suspension of 8 (100 mg, 0.6 mmol) in DCM (5 mL) was
added pyridine (0.06 mL, 0.63 mmol), then N-chlorosuccinimide
(80.0 mg, 0.63 mmol). The mixture was stirred for 2.5 h at rt
under N2, then thiol 9 (230 mg, 0.6 mmol) in DCM (5 mL) was
added. The resulting mixture was treated with TEA (0.35 mL,
2.5 mmol). The reaction mixture was stirred for 2 h at rt under N2
then acidified with aqueous 1 M H2SO4 (7 mL/mmol of sugar). The
mixture was left to stand for about 10 min and then separated.
The aqueous phase was extracted with DCM (3ꢂ20 mL). The
combined organic layers were dried over MgSO4, filtered, and the
filtrate was concentrated under reduced pressure. Compound 10,
as a foam (150 mg, 47%), was obtained by flash column chroma-
tography eluting with 90% DCM/MeOH. Rf¼0.43 in 90% DCM/
MeOH; mp¼108e109 ꢀC; IR (NaCl) nmax 3288 (OH), 2922, 2850,
1651 (C]N), 1495, 1265, 1063 cmꢁ1
(300 K)
;
1H NMR (300 MHz, D2O)
d
4.85 (d, J1,2¼9.9 Hz, 1H, H1), 3.75e3.18 (m, 6H, H2, H3, H4,
H5, H6a, and H6b); 2.77e2.75 (m, 2H, SOCH2), 2.55e2.53 (m, 5H,
CH3SO and CH2C]N), 1.66e1.62 (m, 4H, SOCH2CH2CH2); 13C NMR
(75 MHz, D2O) (300 K) d 155.4 (C]N), 81.1 (C-1), 79.7 (C-5), 76.7 (C-
3), 71.8 (C-2), 68.8 (C-4), 60.3 (C-6), 51.8 (C-50), 36.0 (CH3SO), 30.9
(C-20), 25.4 (C-40), 20.8 (C-30); HRMS (ESI) m/z for C12H22O10NS3
[MꢁK]ꢁ, calcd 436.0411, found 436.0433.
Acknowledgements
One of us (Q.V.V.) would like to thank the Vietnamese Govern-
ment for the grant of a Postgraduate Scholarship and the Victorian
Department of Primary Industries for the award of an Aurora
Scholarship.
1751 (C]O), 1600 (C]N), 1375, 1226, 1039 cmꢁ1
;
1H NMR
(300 MHz, CDCl3) (300 K) 5.26e4.99 (m, 4H, H1, H2, H3, H4),
d
4.12e4.07 (m, 2H, H6a and H6b), 3.76e3.71 (m, 1H, H5),
2.81e2.67 (m, 2H, SOCH2), 2.58e2.53 (m, 5H, CH3SO and CH2C]
N), 2.04, 2.01, 1.99, 1.97 (4ꢂs, 12H, CH3COO), 1.82e1.80 (m, 4H,
Supplementary data
SOCH2CH2CH2); 13C NMR (75 MHz, CDCl3) (300 K)
d 170.2, 169.8,
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2013.07.097.
169.0, 168.8 (4ꢂCH3COO), 149.4 (C]N), 79.6 (C-1), 75.4 (C-5), 73.4
(C-3), 69.9 (C-2), 67.8 (C-4), 61.8 (C-6), 53.4 (C-20), 37.8 (CH3SO),
31.8 (C-50), 25.5 (C-40), 21.6 (C-30), 20.4, 20.3, 20.2, 20.17
(4ꢂCH3COO); HRMS (ESI) m/z for C20H31O11NNaS2 [MþNa]þ, calcd
548.1236, found 548.1219.
References and notes
1. West, L. G.; Meyer, K. A.; Balch, B. A.; RossiI, F. J.; Schultz, M. R.; Haas, G. W. J.
Agric. Food Chem. 2004, 52, 916e926.
2. Powell, E. E.; Hill, G. A.; Juurlink, B. H.; Carrier, D. J. J. Chem. Technol. Biotechnol.
2005, 80, 985e991.
4.11. Potassium 2,3,4,6-tetra-O-acetylglucoraphanin (11)
3. Fahey, J. W.; Zalcmann, A. T.; Talalay, P. Phytochemistry 2001, 56, 5e51.
4. Zhang, Y.; Talalay, P.; Cho, C. G.; Posner, G. H. Proc. Natl. Acad. Sci. U.S.A. 1992, 89,
2399e2403.
5. Iori, R.; Bernardi, R.; Gueyrard, D.; Rollin, P.; Palmieri, S. Bioorg. Med. Chem. Lett.
1999, 9, 1047e1048.
6. Kiddle, G.; Bennett, R. N.; Botting, N. P.; Davidson, N. E.; Robertson, A. A. B.;
Wallsgrove, R. M. Phytochem. Anal. 2001, 12, 226e242.
7. Pereira, F. M. V.; Rosa, E.; Fahey, J. W.; Stephenson, K. K.; Carvalho, R.; Aires, A. J.
Agric. Food Chem. 2002, 50, 6239e6244.
8. Vermeulen, M.; Zwanenburg, B.; Chittenden, G. J. F.; Verhagen, H. Eur. J. Med.
Chem. 2003, 38, 729e737.
To a stirred solution of the thiohydroximate 10 (120 mg,
0.2 mmol) in dry pyridine (5 mL) was added pyridineesulfur tri-
oxide complex (95.0 mg, 0.6 mmol). After stirring at rt under N2 for
24 h, an additional portion of the pyridineesulfur trioxide complex
(19.0 mg, 0.1 mmol) was added and stirring was continued for 2 h.
After that, a solution of KHCO3 (850 mg, 8.4 mmol) in water (10 ml)
was added and the mixture was stirred for 30 min and then con-
centrated under reduced pressure. The residue was dissolved in
water and extracted with chloroform (3ꢂ40 mL) and then 20%
MeOH/CHCl3 (2ꢂ30 mL). The organic layers were dried (MgSO4),
filtered, and concentrated under reduced pressure. To remove ex-
cess pyridine the mixture was co-distilled several times with tol-
uene. Compound 11 was obtained by flash chromatography eluting
with 80% DCM/MeOH as a white solid (103 mg, 73%). Rf¼0.16 in 80%
DCM/MeOH; mp¼155e156 ꢀC (dec); IR (KBr drift) nmax 2971, 2873,
9. Mays, J. R.; Roska, R. W.; Sarfaraz, S.; Mukhtar, H.; Rajski, S. R. ChemBioChem
2008, 9, 729e747.
10. Posner, G. H.; Cho, C. G.; Green, J. V.; Zhang, Y.; Talalay, P. J. Med. Chem. 1994, 37,
170e176.
11. Khiar, N.; Werner, S.; Mallouk, S.; Lieder, F.; Alcudia, A.; Ferna’ndez, I. J. Org.
Chem. 2009, 74, 6002e6009.
12. Rochfort, S.; Caridi, D.; Stinton, M.; Trenerry, V. C.; Jones, R. J. Chromatogr., A
2006, 1120, 205e210.
13. Rangkadilok, N.; Nicolas, M. E.; Bennett, R. N.; Premier, R. R.; Eagling, D. R.;
Taylor, P. W. J. Sci. Hortic. 2002, 96, 11e26.
14. Fahey, J. W.; Wade, K. L.; Stephenson, K. K.; Chou, F. E. J. Chromatogr., A 2003,
996, 85e93.
15. Trenerry, V. C.; Caridi, D.; Elkins, A.; Donkor, O.; Jones, R. Food Chem. 2006, 98,
179e187.
16. Botting, P. N.; Robertson, A. A. B.; Morrison, J. J. Labelled Compd. Radiopharm.
2007, 50, 260e263.
17. Morrison, J. J.; Botting, N. P. Tetrahedron Lett. 2007, 48, 1891e1894.
18. Dawson, G. W.; Hick, A.; Bennett, R.; Donald, A.; Pickett, J.; Wallsgrove, R. J. Biol.
Chem. 1993, 268, 27154e27159.
1741 (C]O), 1654 (C]N), 1433, 1368, 1250, 1224, 1061 cmꢁ1
;
1H
NMR (300 MHz, CD3OD) (300 K) 5.38e5.35 (m, 2H, H1 and H3),
d
5.07e4.93 (m, 2H, H2 and H4), 4.19e4.02 (m, 3H, H5, H6a, and H6b),
2.89e2.63 (m, 2H, SOCH2), 2.63e2.61 (m, 5H, CH3SO and CH2C]N),
2.04, 2.01, 1.99, 1.95 (4ꢂs, 12H, CH3COO), 1.91e1.89 (m, 4H,
SOCH2CH2CH2); 13C NMR (75 MHz, CD3OD) (300 K)
d 170.4, 169.7,
169.4, 169.1 (4ꢂCH3COO), 156.6 (C]N), 79.0 (C-1), 75.0 (C-5), 73.3
(C-3), 69.6 (C-2), 67.8 (C-4), 61.5 (C-6), 52.6 (C-50), 36.3 (CH3SO),
31.3 (C-20), 25.0 (C-40), 21.0 (C-30), 18.9(2), 18.7(2) (4ꢂCH3COO);
HRMS (ESI) m/z for C20H30O14NS3 [MꢁK]ꢁ, calcd 604.0834, found
604.0812.
19. Buckingham, D. A.; Rauchfuss, T. B. J. Chem. Soc., Chem. Commun. 1978, 705e707.
ꢁ
20. Smejkal, T.; Breit, B. Angew. Chem. 2008, 120, 4010e4013.
21. Mancuso, A. J.; Huang, S. L.; Swern, D. J. Org. Chem. 1978, 43, 2480e2482.
22. Bates, R. W.; Sridhar, S. Synlett 2009, 1979e1981.
23. Madesclaire, M. Tetrahedron 1986, 42, 5459e5495.
24. Lakouraj, M. M.; Tajbakhsh, M.; Shirini, F.; Asady Tamami, M. V. Synth. Commun.
2005, 35, 775e784.
4.12. (R,S)S-Glucoraphanin (12)
25. Ladziata, U.; Koposov, A. Y.; Lo, K. Y.; Willging, J.; Nemykin, V. N.; Zhdankin, V.
V. Angew. Chem., Int. Ed. 2005, 44, 7127e7131.
26. Zolfigol, M. A.; Amani, K.; Choghamarani, A. G.; Hajjami, M.; Nasrabadi, R. A.;
To a solution of 11 (50.0 mg, 0.08 mmol) in anhydrous MeOH
(10 ml) under N2 atmosphere was added dry MeOK (5.00 mg,
0.1 mmol) until pH¼8e9. After stirring for 2 h at rt, the solution was
made neutral by the addition of glacial acetic acid then it was
concentrated under reduced pressure. GRP 12 as a white solid
(32.4 mg, 88%) was obtained by flash column chromatography
Jafari, S. Catal. Commun. 2008, 9, 1739e1744.
27. Zolfigol, M. A.; Amani, K.; Hajjami, M.; Choghamarani, A. G. Monatsh. Chem.
2008, 139, 895e899.
28. Afzali-Ardakani, A.; Rapoport, H. J. Org. Chem. 1980, 45, 4817e4820.
29. Leonard, N. J.; Johnson, C. R. J. Org. Chem. 1962, 27, 282e284.
30. Davidson, N. E.; Rutherford, T. J.; Botting, N. P. Carbohydr. Res. 2000, 330,
295e307.

Q.V. Vo et al. / Tetrahedron 69 (2013) 8731e8737
8737
31. Wanga, E. C.; Huanga, K. S.; Chena, H. M.; Wua, C. C.; Jiun Lina, G. J. J. Chin. Chem.
Soc. 2004, 51, 619e627.
32. Floyd, N.; Vijayakrishnan, B.; Koeppe, J. R.; Davis, B. G. Angew. Chem., Int. Ed.
34. Zhang, Q.; Lebl, T.; Kulczynska, A.; Botting, N. P. Tetrahedron 2009, 65, 4871e4876.
35. Vo, Q. V.; Trenerry, C.; Rochfort, S.; Wadeson, J.; Leyton, C.; Hughes, A. B. Bioorg
Med. Chem 2013, in press.
2009, 48, 7798e7802.
33. Benn, M. H. Can. J. Chem. 1963, 41, 2836e2838.
36. Vergara, F.; Wenzler, M.; Hansen, B. G.; Kliebenstein, D. J.; Halkier, B. A.; Ger-
shenzon, J.; Schneider, B. Phytochemistry 2008, 69, 2737e2742.