The synthesis of novel hexa-13C-labelled glucosinolates from [13C6]-d-glucose

Article abstract of DOI:10.1016/j.tet.2009.04.043

An isotopically labelled building block, 2,3,4,6-tetra-O-acetyl-1-thio-β-d-[¹³C₆]glucopyranose (4), is obtained from the commercially available [¹³C₆]-d-glucose. This hexa-¹³C-labelled thioglucose can

Full text of DOI:10.1016/j.tet.2009.04.043

Tetrahedron 65 (2009) 4871–4876
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The synthesis of novel hexa-13C-labelled glucosinolates from [13C6]-
-glucose
D
*
Qingzhi Zhang, Tomas Lebl, Agnieszka Kulczynska, Nigel P. Botting
School of Chemistry, The University of St Andrews, Fife KY 17 9ST, UK
a r t i c l e i n f o
a b s t r a c t
An isotopically labelled building block, 2,3,4,6-tetra-O-acetyl-1-thio-b-D
-[13C6]glucopyranose (4), is ob-
Article history:
tained from the commercially available [13C6]- -glucose. This hexa-13C-labelled thioglucose can be em-
D
Received 11 November 2008
Received in revised form 24 March 2009
Accepted 9 April 2009
ployed to make any glucosinolate (8) for use as an internal standard for isotopic dilution LCMS analysis.
Herein three typical glucosinolates in their hexa-13C-labelled form: [glucose-13C6]gluconasturtiin, [glu-
cose-13C6]sinigrin and [glucose-13C6]glucoerucin are synthesised by coupling the isotopically labelled
thioglucose (4) with the corresponding hydroximoyl chlorides followed by sulfation with pyridine sulfur
trioxide and deacetylation with a catalytic amount of potassium methoxide, respectively.
Ó 2009 Elsevier Ltd. All rights reserved.
Available online 17 April 2009
1. Introduction
more accurate quantitative analysis results. To reduce interference
due to the natural isotopic distribution in the mass spectrum, a suit-
Glucosinolates are a family of naturally occurring sulfur-contain-
ing compounds present in all cruciferous plants including broccoli,
cabbage, Brussels sprouts and oilseed rape.1 Glucosinolates share
able internal standard should have at least a three mass unit differ-
ence from the analyte. Most of the existing methods for isotopic
labelling of glucosinolates have incorporated the isotopes into the
side chain, which is more appropriate for metabolism studies.11,14–18
The drawback of this approach is that a new synthesis needs to be
developed for each glucosinolate. Our group has recently demon-
strated a general strategy for glucosinolates deuterated in the glucose
a common structure that comprises a D-glucopyranosyl unit, an
anomeric thiohydroximoyl-O-sulfate function and a variable side
chain. Over 120 different glucosinolates have been isolated and
characterised. In terms of the side chain R, glucosinolates can be di-
vided into several major groups: aliphatic, arylaliphatic,
u-methyl-
fragment, using 2,3,4,6-tetra-O-acetylthio-b-D
-[1-2H, 6-2H2]gluco-
thioalkylandheterocyclic(e.g., indole).1,2 Thereisincreasingevidence
from both epidemiological and animal studies that consumption of
broccoli and other cruciferous vegetables isassociated with a lowered
risk of cancers especially those of the gastrointestinal and respiratory
tracts.3–5 The anti-cancer effects have been attributed to the high
content of glucosinolates in these vegetables. Hydrolysis of glucosi-
nolate during food preparation, cooking, chewing and digestion,
mediated by the compartmentalised plant enzyme myrosinase (thi-
oglucoside glucohydrolase EC 3.2.3.1),1,6 which appears also in
mammalian intestinal bacteria,7 gives an unstable thiohydroximate-
O-sulfate aglycon intermediate. This unstable intermediate reacts
further to give a wide range of degradationproducts (isothiocyanates,
nitriles, epithiocyanates, oxazolidine-2-thiones, thiocyanates) with
diverse biological activities including anti-cancer effects.8,9 This has
generated increasing interest in the accurate analysis of glucosino-
lates using stable isotopic labelled analogues as internal standards for
isotopic dilution methods in LC/MS and/or LC/MS/MS analysis.10–13
Isotopically labelled internal standards allow for correction of losses
of the analytes during sample preparation and consequently provide
pyranose to synthesise 10-2H,60-2H2]desulfogluconasturtiin.19 The
triply deuterated building block itself needs 9–14 synthetic steps,
depending upon the starting material. In this project, a greatly sim-
plified multiply 13C-labelled building block 2,3,4,6-tetra-O-acetyl-1-
thio-
b-D
-[13C6]glucopyranose is prepared in excellent yield in three
steps from the commercially available [13C6]-
D-glucose. Coupling of
this 13C-labelled thioglucopyranose with various hydroximoyl chlo-
rides would afford any glucosinolate in another three-step sequence.
Herein three typical hexa-13C-labelled glucosinolates were syn-
thesised according to our standard methodology for glucosino-
lates,14,19,20 after optimisation using unlabelled materials.
2. Results and discussion
2.1. Synthesis of [glucose-13C6]glucosinolates
Scheme 1 shows the general procedure for the synthesis of the
title compounds. Firstly [13C6]-
2,3,4,6-tetra-O-acetyl-
D
-glucose (1) was converted into
a-D-glucopyranosyl bromide (2) with acetic
anhydride and hydrobromic acid in 88% yield after recrystallisation.
The reaction is governed by the anomeric effect, giving the
a
-anomer as the only product, which is confirmed by the small size
* Corresponding author.
E-mail address: npb@st-andrews.ac.uk (N.P. Botting).
3
of coupling constant, JH1,H2¼3.8 Hz. Heating the bromide (2) and
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.04.043

4872
Q. Zhang et al. / Tetrahedron 65 (2009) 4871–4876
HO
AcO
AcO
*
*
*
*
*
*
O
*
O
*
O
*
*
*
b
a
S
NH2 Br
NH2
HO
AcO
AcO
OH
S
*
*
*
*
HO
AcO
*
AcO
*
*
HO
1
AcO
AcO
Br
3
2
c
*
AcO
AcO
*
AcO
*
OH
N
AcO
O
*
*
OSO3
N
*
K
*
SH
O
*
*
*
O
*
*
S
AcO
d
e
AcO
AcO
*
*
*
AcO
AcO
AcO
*
*
*
AcO
AcO
4
R
R
R
+
N
7a-c
6a-c
HO
f
where
HO
*
*
R
Cl
* = 13C
K
OSO3
N
O
*
5a-c
HO
S
a R = CH2CH2Ph
b R = CH2CH=CH2
*
HO
*
*
8a-c
HO
c R = CH2CH2CH2CH2SCH3
Scheme 1. Hexa-13C-labelled glucosinolates and desulfoglucosinolates from [13C6]-D-glucose. Reagents and conditions: (a) HBr, AcOH, Ac2O (88%); (b) thiourea, acetone (71–80%);
(c) K2S2O5, H2O, CH2Cl2 (67–93%); (d) Et3N, THF (50–93%); (e) (i) Py$SO3 complex, CH2Cl2, (ii) aq 2 M KHCO3 (47–78%); (f) KOMe, MeOH (83–88%).
thiourea in acetone under reflux led to the precipitation of the
isothiouronium bromide (3) in good yield in the -configuration
Forsinigrin, the required allylhydroximoyl chloride(5b, Scheme 2)
was generated immediately before the coupling from the sodium salt
of (E)-4-nitrobut-1-ene using HCl(g) in dioxane and diethyl ether.16
The (E)-4-nitrobut-1-ene was synthesised using (E)-4-bromobut-1-
ene and silver nitrite inwater followed by deprotonationwith sodium
ethoxide.23 For glucoerucin, 4-methylthiobutylhydroximoyl chloride
(5c, Scheme 2) was prepared in the same way as (5b).16 The requisite
nitroalkane was synthesised from 1-bromo-5-chloropentane, react-
ing first with NaNO2 in DMSO and then sodium thiomethoxide in the
presence of sodium iodide in refluxing methanol.24 Coupling of 5b
and 5c with [13C6]thioglucose 4 afforded 6b and 6c in 80% and 50%
yields, respectively (Scheme 1). In all cases the Z-isomer is obtained
exclusively due to stereoelectronic effects.25
The O-sulfation proved to be a tricky step as evidenced by our
previous work,4,14,15 therefore further optimisation with unlabelled
material was carried out using excess Py$SO3 in pyridine. Moni-
toring the reaction closely by TLC has shown that this appears to be
a clean reaction but work-up by removing the solvent pyridine in
the presence of aqueous KHCO3 resulted in deterioration of the
product by deacylation and/or desulfation. This problem was solved
by using DCM as the solvent while keeping the pyridine to as small
amount as possible. The crude product was further purified by
column chromatography, however, recrystallisation from boiling
EtOH (85%)26,18 required extra care as desulfation occurred in our
hands to some extent. Once the O-sulfates (7a–c) were purified, the
final products (8a–c) could be obtained in good yield by straight-
forward deacetylation with a catalytic amount of potassium
methoxide in MeOH.
b
due to the neighbouring group effect of the acetate group adjacent
to the anomeric carbon atom. Simple hydrolysis of the iso-
thiouronium bromide (3) in the presence of potassium meta-
bisulfite in a biphasic system of water and DCM gave thioglucose
(4), the key isotopically labelled building block, in good to excellent
3
yield. The large size of JH1,H2 of 10 Hz in 3 and 4 confirms the
b-configuration.
With the hexa-13C-labelled thioglucose in hand, subsequent
work concentrated on its coupling with the hydroximoyl chloride
(Scheme 2), a protocol initially established by Benn et al.20 Pheneth-
ylhydroximoyl chloride (5a) derived from phenylpropionaldehyde
oxime in DCM in the presence of half an equivalent of pyridine or
triethylamine was known to be unstable and has to be prepared
immediately before the coupling. We reasoned that the instability of
this compound lies in the presence of organic base, which catalyses
the premature removal of HCl and formation of the nitrile oxide.
Therefore the generation of 5a was improved by reacting phenyl-
propionaldehyde oxime with N-chlorosuccinimide in DMF without
base by modifying a literature procedure.21 A more stable, colour-
less oil of 5a, which gradually solidified upon storage at room
temperature, was thus obtained by simple work-up with water and
extraction with diethyl ether.22 To guarantee that no labelled
material was wasted, the coupling reaction was carried out with an
excess of the hydroximoyl chloride to maximise the conversion of
the [13C6]thioglucose. The required 2,3,4,6-tetra-O-acetyl-
conasturtiin (6a) was thus obtained in 88% yield.
b-
D-glu-
2.2. Characterisation of [glucose-13C6]derivatives
OH
H
OH
Cl
N
N
Extensive 1H,13C and 13C,13C coupling leads to severe overlaps and
second order coupling effects in the conventional 1H and 13C NMR
spectra, which substantially hampers characterisation of the iso-
topically labelled glucose derivatives. However, by employing 2D
NMR techniques it was possible to assign all the 1H and 13C reso-
nances, derive significant JHH, JHC and JCC coupling constants and thus
characterise all synthesised 13C-labelled compounds unambiguously.
Alongside standard 1H,1H-COSY spectra, two less common tech-
a
5a
OH
N
O
N
c
d
b
Br
NO2
Cl
O
f
Na
Cl
5b
e
NO2
Br
Cl
NO2
S
niques, 2D 1H J-resolved spectrum with 13C decoupling and 2D 1H,13
C
c
HSQC without 13C decoupling have been used. Examples of the ap-
plication of these techniques on compounds 2 and 3 are shown in
Figures 1 and 2, respectively. Figure 1 shows the 1H J-resolved spec-
trum with 13C decoupling of compound 2, which has only chemical
shift information present on the F2-axis. Homonuclear spin–spin
coupling patterns are displayed along the F1-axis and heteronuclear
coupling was removed by 13C decoupling in order to simplify the
OH
N
O
N
d
S
S
O
Na
Cl
5c
Scheme 2. Synthesis of hydroximoyl chloride. Reagents and conditions: (a) N-chloro-
succinimide, DMF, rt, overnight; (b) AgNO2, CH2Cl2 (45%); (c) NaOEt, EtOH (55% and
88%); (d) HCl(g), Et2O (100%); (e) NaNO2, DMSO, 47%; (f) NaI, CH3SNa, MeOH, reflux,
42%.

Q. Zhang et al. / Tetrahedron 65 (2009) 4871–4876
4873
accurate analysis of individual glucosinolates by LCMS practical.
Further work on other [glucose-13C6]glucosinolates and [glucose-
13C6]desulfoglucosinolates is underway.
4. Experimental
4.1. General
[
13C6]-
D-glucose was purchased from ISOTEC of Sigma–Aldrich.
Other reagents were used as supplied without further purification
unless stated otherwise. Anhydrous tetrahydrofuran, diethyl ether
and dichloromethane were obtained from a solvent purification
system (MBraun, SPS-800). Pyridine, triethylamine and methanol
were distilled over calcium hydride under an argon atmosphere.
Acetone was dried over 4 Å molecular sieve for 48 h. Petrol is defined
as petroleum ether 40–60 ꢀC. All reactions were performed in over-
night oven-dried glassware with magnetic stirring under an argon
atmosphere. Air- and moisture-sensitive liquids and solutions were
transferred by syringe or cannula and introduced into reaction ves-
sels through rubber septa. Thin layer chromatography was performed
on Kieselgel 60 F254 plastic plates pre-coated with a 0.25 mm thick-
ness of silica gel. Visualisation was achieved by inspection under UV
light (254 nm) followed by staining with either potassium perman-
ganate or sulfuric acid/methanol dips. Column chromatography was
performed on Kieselgel 60 (230–400 mesh) silica gel.
Figure 1. Homonuclear J-resolved spectrum of 2.
1H and 13C nuclear magnetic resonance (NMR) spectra were ac-
quiredona Bruker AV-400 spectrometer. Chemicalshifts are reported
as parts per million downfield from tetramethylsilane using residues
of deuterated solvents as references. Connectivities were assigned
using two-dimensional (COSY, HSQC) experiments. Coupling con-
spectrum. Thus, one can get rid of overlaps and easily obtain the 1H
chemical shifts and JHH coupling constants. 2D 1H,13C HSQC without
13C decoupling (Fig. 2) provides standard correlation of 1H and 13C
chemical shifts. In addition, homonuclear 13C,13C coupling patterns
are apparent along the F1-axis and splitting of crosspeaks along the
F2-axis allows derivation of the 1JHC coupling constants.
3
stants JCH, JCC and JHH were denoted 1JCH, 1JCC and JHH, respectively,
and were measured in hertz using 1D or 2D spectra and where nec-
essary, the J-resolving experiments. Mass spectra were acquired by
electrospray ionisation (ESI) on a Micromass LCT spectrometer. In-
frared spectra were recorded as KBr discs on a Perkin Elmer 1420
instrument.
3. Conclusion
In conclusion, we have demonstrated a general synthesis of stable
isotopically labelled derivatives of glucosinolates from the commer-
cially available [13C6]- -glucose. Theoretically, any [glucose-13C6]glu-
D
4.2. Synthetic procedure for [glucose-13C6]derivatives
cosinolate can be achieved by this methodology by varying the
hydroximoyl chloride required for the side chain, which would make
4.2.1. 2,3,4,6-Tetra-O-acetyl-
Hydrogen bromide in acetic acid (45% w/v 40 ml, 222 mmol)
was added dropwise to [13C6]-
-glucose (25.0 g, 134 mmol) in
a-D
-[13C6]glucopyranosyl bromide (2)
D
acetic anhydride (100 ml, 1.06 mol) at ꢁ10 to 5 ꢀC under an argon
atmosphere. After 4 h, further 45% w/v hydrogen bromide in acetic
acid (92 ml, 623 mmol) was added and the solution stirred at room
temperature overnight. The reaction mixture was taken up in
dichloromethane and poured onto ice/water. The organic phase
was carefully neutralised with an ice/saturated sodium hydrogen
carbonate solution. The organic layer was then washed with brine
and dried over magnesium sulfate. The solvent was evaporated at
reduced pressure to give the pale yellow oil, which solidified upon
cooling to 0 ꢀC. The product was recrystallised from diethyl ether to
give 2 as a white crystalline solid (49.3 g, 88%), which is heat labile
and should be stored in the freezer. Mp 89–90 ꢀC (lit.27 (unlabelled)
88–89 ꢀC). [Found: C, 40.47%; H, 4.71. 13C6C8H19BrO9 requires C,
þ194.8 (c 2.42, CHCl3) (lit.28 (unlabelled)
20
40.31; H, 4.59%.] [
a
]
D
þ197. 84 (c 2.42, CHCl3)); nmax (KBr disc) 1744 (C]O), 1246, 1288,
1035 (COO) cmꢁ1
; dH (400 MHz, CDCl3) 6.59 (1H, dd, JCH 185 Hz, JHH
3.8 Hz, H-1), 5.54 (1H, ddd, JCH 149 Hz, 2ꢂJHH 9.6 Hz, H-3), 5.14 (1H,
ddd, JCH 147 Hz, JHH 10.5 and 9.2 Hz, H-4), 4.82 (1H, ddd, JCH 151 Hz,
JHH 10.0 and 4.3 Hz, H-2), 4.31 (1H, ddd, JCH 147 Hz, 2JHH 12.7 Hz, JHH
4.3 Hz, H-6a), 4.21 (1H, m, JCH 146 Hz, JHH not resolved, H-5), 4.12
(1H, ddd, JCH 149 Hz, 2JHH 12.7 Hz, JHH 2.4 Hz, H-6b), 2.23, 2.17, 1.97,
1.91 (12H, 4ꢂs, 4ꢂCH3); dC (100.10 Hz, CDCl3) 170.5, 169.9, 169.8,
169.5 (4ꢂC]O), 86.5 (enhanced d, JCC 38 Hz, C-1), 72.3 (enhanced
dd, 2ꢂJCC 43 Hz, C-5), 71.1 (enhanced m, JCC not resolved, C-3), 70.6
Figure 2. 2D 1H,13C HSQC without 13C decoupling of 3.

4874
Q. Zhang et al. / Tetrahedron 65 (2009) 4871–4876
(enhanced m, JCC not resolved, C-2), 67.1 (enhanced dd, 2ꢂJCC 39 Hz,
C-4), 60.9 (enhanced d, JCC 45 Hz, C-6), 20.7, 20.64, 20.56 (3ꢂs,
4ꢂCH3); m/z (ESþ): 439 and 441 (MþNa)þ.
8.0 mmol).22,23 The mixture was stirred at rt overnight before di-
luting with water and extracting with diethyl ether. The combined
extracts were dried over MgSO4 and the solvent was evaporated
under reduced pressure to give a pale yellow oil of phenethylhy-
droximate chloride 5a (1.5 g, 100%). The oil (1.2 g, 6.5 mmol) was
taken up in anhydrous THF (30 ml) and 4 (1.7 g, 4.6 mmol) was
added. To this solution was added triethylamine (4.5 ml, 32 mmol).
The mixture was stirred overnight before being poured onto ice-
water (200 ml). The precipitate was collected by filtration and
washed with copious amounts of water before being air dried. The
crude product was purified by column chromatography (SiO2,
EtOAc/petrol¼1:2) to give 6a as a white solid (2.21 g, 93%). Mp
4.2.2. 2,3,4,6-Tetra-O-acetyl-b-D
-[13C6]glucopyranosyl
isothiouronium bromide (3)
Thiourea (3.01 g, 39.55 mmol) was added to
2 (16.5 g,
39.55 mmol) in dry acetone (40 ml) under argon. The solution was
heated at reflux for 30 min and then cooled to 0 ꢀC to give a white
precipitate. The precipitate was removed by filtration followed by
washing with acetone to give 3 as a white crystalline solid (13.55 g).
The filtrate was concentrated and triturated with ethyl acetate. The
precipitate was collected by filtration and washed with EtOAc and
ice-cooled acetone successively to give another crop of product
(2.0 g), which was not used for analysis but directly for next step of
reaction. Total yield: 15.55 g, 80%. Mp 203–205 (lit.29 (unlabelled)
205–207 ꢀC (lit.26 (unlabelled) 198 ꢀC). [Found: C, 53.51; H, 5.76; N,
20
2.76. 13C6C17H29NO10S requires C, 53.38; H, 5.65; N, 2.71%.] [
a]
D
ꢁ10.6 (c 1.0, CHCl3) (lit.32 (unlabelled) ꢁ11 (c 2.0, CCl4)); nmax (KBr
disc) 3316 (OH), 1746, 1713 (C]O, C]N), 1253, 1226, 1034
205 ꢀC). [Found: C, 36.68; H, 4.43; N, 5.48. 13C6C9H23BrN2O9S re-
(COO) cmꢁ1
; dH (400 MHz, DMSO-d6) 11.31 (1H, s, OH), 7.30–7.17
20
quires C, 36.51; H, 4.70; N, 5.68%.] [
a]
ꢁ17.0 (c 1.0, MeOH) (lit.30
(5H, m, Ph H), 5.61 (1H, m, JCH 164 Hz, JHH 10.2 Hz, H-1), 5.47 (1H,
dm, JCH 152 Hz, 2ꢂJHH 9.9 Hz, H-2), 4.89 (2ꢂ1H, m, JCH 152 Hz,
4ꢂJHH 9.8 Hz, H-2 and H-4), 4.14 (1H, m, JCH 147 Hz, H-5), 4.01
(2ꢂ1H, m, JCH 147 Hz, H-6), 2.95–2.76 (4H, m, 2ꢂCH2), 2.02, 1.99,
1.96, 1.76 (12H, 4ꢂs, 4ꢂCH3); dC (100.10 Hz, DMSO-d6) 169.9, 169.5,
169.3, 169.1 (4ꢂC]O), 149.1 (C]N), 140.9, 128.3, 128.2, 125.9 (4ꢂs,
Ph-C), 77.8 (enhanced d, JCC 42 Hz, C-1), 74.3 (enhanced dd, 2ꢂJCC
43 Hz, C-5,), 72.7 (enhanced dd, 2ꢂJCC 41 Hz, C-3), 69.6 (enhanced
dd, 2ꢂJCC 41 Hz, C-2), 68.1 (enhanced dd, 2ꢂJCC 41 Hz, C-4), 62.2
(enhanced d, J 45 Hz, C-6), 32.3 (s, 2ꢂCH2), 20.3, 20.2, 20.1 (3ꢂs,
overlapped, 4ꢂCH3); m/z (ESþ) 540 (MþNa)þ, HRMS (ESþ):
(MþNa)þ found 540.1592, 13C6C17H29NO10SNa requires 540.1611.
D
(unlabelled) ꢁ17.3 (c 1.0, MeOH)); nmax (KBr disc) 3312, 3273, 3059,
3021 (NH), 1752 (C]O), 1657 (C]N), 1226, 1039 (COO) cmꢁ1
; dH
(400 MHz, D2O) 5.49 (1H, dd, JCH 167 Hz, JHH 10.0 Hz, H-1), 5.45 (1H,
ddd, JCH 152 Hz, JHH 9.5 Hz, H-3), 5.34 (1H, ddd, JCH 158 Hz, JHH 10.8,
8.9 Hz, H-2), 5.23 (2ꢂ0.5H, ddd, JCH 158 Hz, JHH 10.5 Hz, H-4), 4.41
(1H, ddd, JCH 151 Hz, 2JHH 12.1 Hz, JHH 4.3 Hz, H-6a), 4.29 (1H, m, JCH
152 Hz, 2JHH 12.7 Hz, H-6b), 4.24 (1H, m, JCH 149 Hz, H-5), 2.15, 2.13,
2.11, 2.08 (12H, 4ꢂs, 4ꢂCH3); dC (100.10 Hz, D2O) 173.5, 172.9, 172.5,
172.3 (4ꢂC]O), 167.4 (C]N), 81.25 (enhanced d, JCC 42 Hz, C-1),
75.6 (enhanced dd, 2ꢂJCC 42 Hz, C-5), 73.3 (enhanced dd, 2ꢂJCC
41 Hz, C-3), 69.0 (enhanced ddd, 2ꢂJCC 41 Hz, 2JCC 2.9 Hz, C-2), 67.5
(enhanced ddd, 2ꢂJCC 41 Hz, 2JCC 2.7 Hz, C-4), 61.8 (enhanced d, JCC
44 Hz, C-6), 20.1, 20.0, 19.9 (3ꢂs, 4ꢂAc CH3); m/z (ESþ): 413
(MꢁBr)þ, 337 (MꢁBrꢁNH2C(S)NH2)þ; HRMS (ESþ): (MꢁBr)þ found
413.1331, 13C6C9H23N2O9S requires 413.1326.
4.2.5. 2,3,4,6-Tetra-O-acetyl-b-D
-[glucose-13C6]glucopyranosyl
gluconasturtiin (7a)
To a solution of 6a (2.18 g, 4.21 mmol) in DCM (10 ml) were
added pyridine sulfur trioxide (3.35 g, 21.1 mmol) and pyridine
(3.4 ml, 42.1 mmol). The mixture was heated under reflux for 4 h
and cooled to room temperature. Potassium hydrogen carbonate
(2 M, 35 ml, 70 mmol) was added and the mixture was stirred for
another half an hour and then the two-phase mixture was con-
centrated under reduced pressure. The solid residue was washed
with aqueous KHCO3 (2 M), cold water, cold EtOH and diethyl ether
successively and the residue was purified by chromatography (SiO2,
DCM/MeOH¼100:20) to give the product as a white solid (2.23 g,
83%). Mp 200–202 ꢀC dec (lit.32 (unlabelled) 198–200 ꢀC dec).
4.2.3. 2,3,4,6-Tetra-O-acetyl-1-thio-b-D
-[13C6]glucopyranose (4)
Potassium metabisulfite (3.154 g, 14.19 mmol) and 3 (7.0 g,
14.19 mmol) were suspended in degassed water and dichloro-
methane (1:1, 30 ml). The biphasic solution was heated at reflux
under an argon atmosphere for 30 min and then cooled to room
temperature. The organic phase was washed with water (three
times) and the aqueous layer washed with dichloromethane. The
combined organic layers were dried (MgSO4) and the solvent
evaporated at reduced pressure to give a white gum. The product
was recrystallised from methanol to give 3 as a white crystalline
solid (4.88 g, 93%). Mp 113–115 ꢀC (lit.31 (unlabelled) 112–114 ꢀC).
[Found: C, 42.48; H, 4.41; N, 2.10. 13C6C17H28NO13S2K$H2O requires
20
C, 42.26; H, 4.63; N 2.14%.] [
a
]
D
ꢁ12.0 (c 0.5, MeOH) (lit.32 (unla-
[Found: C, 45.55; H, 5.58. 13C6C8H20O9S requires C, 45.41; H, 5.44%.]
belled) ꢁ11 (c 1.0, 50% aq EtOH)); nmax (KBr disc) 1751 (C]O), 1227,
20
[
a]
þ5.7 (c 1.0, CHCl3) (lit.31 (unlabelled) þ6.3 (c 1.2, CHCl3)); nmax
1063 (ROSOꢁ3 ), 1035 (COO) cmꢁ1
; dH (400 MHz, DMSO-d6) 7.36–7.28
D
(KBr disc) 2584 (S–H), 1744 (C]O), 1239, 1031 (COO) cmꢁ1
;
dH
(5H, m, PhH), 5.68 (1H, dd, JCH 163 Hz, JHH 8.6 Hz, H-1), 5.48 (1H, m,
JCH 152 Hz, JHH 9.7 Hz, H-3), 4.91 (2ꢂ1H, m, JCH 152 Hz, JHH 10.1 Hz,
H-2þH-4), 4.13 (1H, m, JCH 146 Hz, H-5), 4.00 (2ꢂ1H, m, JCH 147 Hz,
H-6), 2.95–2.80 (4H, m, 2ꢂCH2), 2.04, 2.00, 1.96, 1.73 (12H, 4ꢂs,
4ꢂCH3); dC (100.10 Hz, DMSO-d6) 169.6, 169.4 (C]O), (C]N not
observed due to the strong solvent peak), 140.8, 128.4, 128.3, 126.1
(4ꢂPh C), 77.9 (enhanced d, JCC 42 Hz, C-1), 74.4 (enhanced dd,
2ꢂJCC 43 Hz, C-5), 72.7 (enhanced dd, 2ꢂJCC 41 Hz, C-3), 69.5 (en-
hanced dd, 2ꢂJCC 41 Hz, C-2), 68.1 (enhanced dd, 2ꢂJCC 46 Hz, C-4),
62.2 (enhanced d, JCC 44 Hz, C-6), 33.0 (CH2), 32.4 (CH2), 20.4, 20.3,
(300 MHz, CDCl3) 5.17 (1H, m, JCH 148 Hz, JHH 9.0 Hz, H-3), 5.08 (1H,
m, JCH 159 Hz, JHH 9.9 Hz, H-4), 4.96 (1H, m, JCH 150 Hz, JHH 9.6 Hz,
H-2), 4.54 (1H, m, JCH 157 Hz, JHH 9.8 Hz, H-1), 4.23 (1H, m, JCH
149 Hz, 2JHH 12.4 Hz, JHH 4.8 Hz, H-6a), 4.12 (1H, m, JCH 150 Hz, 2JHH
12.4 Hz, JHH 2.4 Hz, H-6b), 3.71 (1H, m, JCH 143 Hz, H-5), 2.31 (1H, dt,
2
JHH 9.9 Hz, JCH¼3JCH 3.9 Hz, SH), 2.10, 2.08, 2.03, 2.01 (12H, 4ꢂs,
4ꢂCH3); dC (100.10 Hz, DMSO-d6) 170.9, 170.3, 169.9, 169.5
(4ꢂC]O), 78.7 (enhanced m, JCC 46 Hz, 2JCC not resolved, C-1), 76.3
(enhanced dd, 2ꢂJCC 46 Hz, C-5), 73.8 (enhanced m, C-3), 73.4
(enhanced m, C-2), 68.0 (enhanced m, JCC 47 Hz, C-4), 62.0 (en-
hanced d, JCC 45 Hz, C-6), 21.0, 20.9 and 20.8 (3ꢂs, partial overlaps,
4ꢂCH3); m/z (ESþ) 393 (MþNa)þ; HRMS (ESþ); (MþNa)þ found
393.0925, 13C6C8H20O9SNa requires 393.0927.
20.1 (3ꢂs, overlapped at
d
20.4, 4ꢂCH3); m/z (ESꢁ) 596 (MꢁK)ꢁ;
HRMS (ESꢁ): (MꢁK)ꢁ, found 596.1214. 13C6C17H28NO13S2 requires
596.1203.
4.2.6. [glucose-13C6]Gluconasturtiin (8a)
4.2.4. 2,3,4,6-Tetra-O-acetyl-
phenethylthiohydroximate (6a)
To solution of 3-phenylpropionaldehyde oxime (1.2 g,
8.0 mmol) in DMF at 0 ꢀC was added N-chlorosuccinimide (1.09 g,
b
-D
-[glucose-13C6]glucopyranosyl
To a suspension of 7a (0.66 g, 1.0 mmol) in methanol (30 ml)
was added a catalytic amount of potassium metal under argon. The
reaction mixture was stirred overnight before Dowex-50 was
added to neutralise the solution. Stirring was continued for
a

Q. Zhang et al. / Tetrahedron 65 (2009) 4871–4876
4875
a further 30 min before the resin was removed by filtration and the
solvent evaporated at reduced pressure to give the product as
a gum. The gum was taken up in water and the solution was freeze
dried to give a white amorphous powder (0.402 g, 83%). Mp 164 ꢀC
dec (lit.32 (unlabelled) 170–172 ꢀC dec). [Found: C, 36.16; H, 3.72; N,
water, cold EtOH and diethyl ether successively to get the crude
product. The residue was purified by chromatography (SiO2,
DCM/MeOH¼100:20) to get the product as a white solid: (0.875 g,
47%). Mp 185–195 ꢀC dec (lit.20 (unlabelled) 193–195 ꢀC). [Found:
C, 37.77; H, 3.93; N, 2.20. 13C6C12H24NO13S2K requires C, 37.83; H,
20
2.78. 13C6C9H20NO9S2K$0.5KHCO3 requires C, 35.97; H, 3.99; N,
4.23; N, 2.45%.] [
a
]
ꢁ14.4 (c 0.14, H2O) (lit.20 (unlabelled) ꢁ16 (c
D
20
2.71%.] [
a
]
ꢁ22 (c 1.0, H2O) (lit.32 (unlabelled) ꢁ23 (c 1.0, H2O));
0.14, H2O)); nmax (KBr disc) 1751 (C]O), 1283, 1244, 1061
D
IR (KBr disc) 3424 (OH), 1237, 1039 (ROSOꢁ3 ) cmꢁ1
;
dH (400 MHz,
(ROSOꢁ3 ) cmꢁ1
; dH (400 MHz, DMSO-d6) 5.96 (1H, ddd, JHH 17.0,
D2O) 7.41–7.28 (5H, m, Ph), 4.99 (1H, dd, JCH 161 Hz, JHH 9.1 Hz,
H-1), 3.94 (1H, m, JCH 147 Hz, 2JHH 12.0 Hz, H-6a), 3.77 (1H, m, JCH
147 Hz, 2JHH 12.0 Hz, H-6b), 3.59 (2ꢂ0. 5H, m, JCH 141 Hz, H-3), 3.56
(1H, m, JCH 144 Hz, H-5), 3.54 (2ꢂ1H, m, JCH 148 Hz, H-2þH-4),
3.12–2.97 (4H, m, 2ꢂCH2); dC (100.10 Hz, D2O) 163.3 (C]N), 140.5,
128.71, 128.68, 126.6 (4ꢂPh C), 81.6 (enhanced d, JCC 40 Hz, C-1),
80.0 (enhanced dd, 2ꢂJCC 42 Hz, C-5), 77.0 (enhanced dd, 2ꢂJCC
39 Hz, C-3), 71.8 (enhanced dd, 2ꢂJCC 39 Hz, C-2), 68.1 (enhanced
dd, 2ꢂJCC 40 Hz, C-4), 62.2 (enhanced d, JCC 43 Hz, C-6), 33.9, 32.5
(2ꢂCH2); m/z (ESꢁ) 428 (MꢁKþ); HRMS (ESꢁ): [MꢁK]ꢁ, found
428.0785. 13C6C9H20NO9S2 requires: 428.0781.
10.4, 6.2 Hz, H-20), 5.47 (1H, dd, JCH 163 Hz, JHH 9.5 Hz, H-1), 5.36
(1H, m, JCH 150 Hz, H-3), 5.23 (1H, dd, JHH 17.2 and 1.5 Hz, H-30),
5.16 (1H, dd, JHH 10.2 and 1.3 Hz, H-30), 4.91 (1H, m, JCH 151 Hz, H-
4), 4.85 (1H, m, JCH 151 Hz, H-2), 4.09 (1H, m, JCH 147 Hz, H-5),
4.05 (2ꢂ1H, m, JCH 147 Hz, H-6), 3.37 (1H, m, H-10), 2.02, 2.01,
2.00, 1.96 (12H, 4s, 4ꢂCH3); dC (100.10 Hz, DMSO-d6) 170.0, 169.6,
169.3, 169.1 (4ꢂC]O), 152.3 (C]N), 133.1 (C-20), 117.52 (C-30),
78.1 (enhanced d, JCC 42 Hz, C-1), 74.3 (enhanced dd, 2ꢂJCC 43 Hz,
C-5), 72.8 (enhanced dd, 2ꢂJCC 41 Hz, C-3), 69.5 (enhanced dd,
2ꢂJCC 41 Hz, C-2), 67.9 (enhanced dd, 2ꢂJCC 40 Hz, C-4), 62.0
(enhanced d, JCC 44 Hz, C-6), 35.7 (C-10), 20.5, 20.4, 20.3 (partially
overlapped, 4ꢂCH3); m/z (ESþ) 594 (MþNa)þ; 610 (MþK)þ; m/z
(ESꢁ) 532 (MꢁK)ꢁ; HRMS (ESꢁ): [MꢁK]ꢁ found 532.0895,
13C6C12H24NO13S2 requires: 532.0890.
4.2.7. 2,3,4,6-Tetra-O-acetyl-b-D
-[glucose-13C6]glucopyranosyl
allylthiohydroximate (6b)
To a suspension of sodium 4-nitrobut-1-ene16 (1.11 g, 9 mmol)
in Et2O (100 ml) cooled to ꢁ78 ꢀC was added dropwise 4 M HCl in
dioxane (16 ml, 72 mmol). The suspension gradually changed from
yellow to bright blue. The suspension was further stirred for 30 min
before the temperature was raised to ꢁ40 ꢀC and triethylamine was
added dropwise. The blue colour of the suspension changed back to
pale brown to which 4 (1.6 g, 4.32 mmol) in THF (30 ml) was added.
The mixture was left stirring at room temperature overnight and
then taken up in water (200 ml). The layers were separated and the
aqueous layer was extracted with EtOAc (two times). The combined
extracts were rinsed with water and brine successively and the
solvent was removed under reduced pressure. The residue was
purified by column chromatography (SiO2, EtOAc/petrol) to give the
product as a white powder (1.56 g, 80%). Mp 168–170 ꢀC (lit.20
4.2.9. [glucose-13C6]Sinigrin (8b)
To a suspension of 7b (0.818 g, 1.43 mmol) in MeOH (30 ml) was
added a catalytic amount of MeOK in MeOH. The mixture was
stirred at room temperature for overnight. The solution was care-
fully neutralised with Dowex-50 to pH 7.0 and then filtered through
a pad of Celite and concentrated under reduced pressure at room
temperature. The residue was dissolved in the smallest amount of
H2O. To this aqueous solution was added ethanol until cloudy. The
mixture was stored at 4 ꢀC overnight. The product precipitated out
as colourless needle crystals (0.475 g, 82%). Mp 128–130 ꢀC (lit.20
(unlabelled) 125–127 ꢀC). [Found, C, 28.91; H, 4.19; N, 3.15.
20
13C6C4H16NO9S2K$H2O requires C, 29.12; H, 4.15; N, 3.39%.] [
a]
D
ꢁ15.9 (c 0.2, H2O) (lit.20 (unlabelled) ꢁ17.0 (c 0.2, H2O)); nmax (KBr
disc) 3492, 3406, 3186 (OH), 1650 (C]C), 1574 (C]N), 1274, 1240,
(unlabelled) 164–165 ꢀC). [Found: C, 47.75; H, 5.23; N, 2.92.
20
13C6C12H25NO10S requires C, 47.68; H, 5.56; N, 3.09%.] [
a
]
ꢁ12.2 (c
1087, 1055, 1010 (ROSOꢁ3 ) cmꢁ1
; dH (400 MHz, D2O) 6.03 (1H, ddd,
D
0.14, CHCl3) (lit.20 (unlabelled) ꢁ13 (c 0.14, CHCl3)); nmax (KBr disc)
JHH 17.2, 10.4 and 6.2 Hz, H-20), 5.32 (1H, ddt, JHH 17.2, 1.7 and 1.2 Hz,
H-30), 5.28 (1H, ddt, JHH 10.4, 1.7 and 1.3 Hz, H-30), 5.05 (1H, dd, JCH
3310 (OH), 1750, 1707 (C]O, C]N), 1605 (C]C), 1253, 1220
(COOꢁ) cmꢁ1
;
dH (400 MHz, DMSO-d6) 11.37 (1H, s, OH), 5.95 (1H,
160.3 Hz, JHH 9.8 Hz, H-1), 4.13 (1H, m, JCH 143 Hz, JHH 12.5 Hz,
2
ddd, JHH 17.1, 10.2, 6.2 Hz, H-20), 5.44 (1H, d, JCH 167 Hz, JHH 8.4 Hz,
H-1), 5.37 (1H, m, 1H, JCH 151 Hz, H-3), 5.18 (1H, ddt, J 17.2, 1.6,
1.2 Hz, H-30), 5.11 (1H, ddt, JHH 10.08, 1.6, 1.2 Hz, H-30), 4.91 (1H, m,
JCH 154 Hz, H-4), 4.85 (1H, m, JCH 155 Hz, H-2), 4.10 (1H, m, JCH
149 Hz, H-5), 4.06 (2ꢂ1H, m, JCH 149 Hz, H-6), 3.30 (2H, m, H-10),
2.01, 2.00, 1.99, 1.96 (12H, 4ꢂs, 4ꢂCH3); dC (100.10 Hz, DMSO-d6)
169.9, 169.6, 169.3, 169.1 (4ꢂC]O), C]N not observed due to the
strong signal of the solvent and the enhanced signal of 13C labelling
atoms, 133.8 (C-20), 116.9 (C-30), 78.0 (enhanced d, JCC 42 Hz, C-1),
74.3 (enhanced dd, 2ꢂJCC 42 Hz, C-5), 72.8 (enhanced dd, 2ꢂJCC
41 Hz, C-3), 69.7 (enhanced dd, 2ꢂJCC 40 Hz, C-2), 68.0 (enhanced
dd, 2ꢂJCC 41 Hz, C-4), 62.1 (enhanced d, JCC 45 Hz, C-6), 35.7 (C-10),
20.47, 20.3 (2ꢂs, overlapped, 4ꢂCH3); m/z (ESþ) 476 (MþNa)þ;
HRESIMSþ (ESþ): 476.1299, 13C6C12H25NO10SNa requires: 476.1298.
H-6a), 3.94 (1H, ddd, JCH 143 Hz, 2JHH 12.5 Hz, JHH 5.6 Hz, H-6b), 3.54
(1H, m, JCH 142 Hz, H-5), 3.53 (1H, m, JCH 143 Hz, H-3), 3.45 (1H, H
m, JCH 148 Hz, H-4), 3.44 (2ꢂ0.5H, m, JCH 146 Hz, H-2), 3.56–3.50
(4H, m, H-3þH-5þ2ꢂ H-10), 3.47–3.42 (2H, m, H-2þH-4); dC
(100.10 Hz, D2O) 163.0 (C]N), 132.0 (C-20), 118.3 (C-30), 81.5 (en-
hanced d, JCC 40 Hz, C-1), 79.9 (enhanced dd, 2ꢂJCC 42 Hz, C-5), 77.0
(enhanced dd, 2ꢂJCC 39 Hz, C-3), 71.8 (enhanced dd, 2ꢂJCC 39 Hz,
C-2), 69.0 (enhanced dd, 2ꢂJCC 40 Hz, C-4), 60.5 (enhanced d, JCC
43 Hz, C-6), 36.4 (C-10); m/z (ES-) 364 (MꢁKþ); HRMS (ESꢁ):
(MꢁK)ꢁ found 364.0470, 13C6C4H16NO9S2 requires: 364.0468.
4.2.10. 2,3,4,6-Tetra-O-acetyl-b-D
-[glucose-13C6]glucopyranosyl-4-
methythiobutylthiohydroximate (6c)
To a suspension of sodium 1-methylthio-5-nitropentane16,25
(0.58 g, 3.13 mmol) in Et2O (100 ml) cooled to ꢁ78 ꢀC was added
dropwise 4 M HCl in dioxane (1.57 ml, 6.3 mmol). The suspension
gradually changed from yellow to bright blue. The suspension was
further stirred for 30 min before the temperature was raised to
ꢁ40 ꢀC and triethylamine (2.78 ml, 12.2 mmol) was added drop-
wise. The blue suspension changed back to pale brown and 4
(0.65 g, 1.76 mmol) was added. The mixture was allowed to warm
up to room temperature and left stirring overnight and then taken
up in water. The layers were separated and the aqueous layer was
further extracted with EtOAc (two times). The combined extracts
were rinsed with water and brine successively, dried over MgSO4
4.2.8. 2,3,4,6-Tetra-O-acetyl-b-D
-[glucose-13C6]glucopyranosyl
sinigrin (7b)
To a solution of 6a (1.427 g, 3.15 mmol) in DCM (60 ml) were
added pyridine sulfur trioxide (2.51 g, 15.77 mmol) and pyridine
(1.5 ml, 18.6 mmol). The mixture was heated under reflux for 6 h
and cooled to room temperature and stirred overnight. Aqueous
potassium hydrogen carbonate solution (2 M, 20 ml, 40 mmol)
was added and the mixture was stirred for 30 min and then the
two-phase mixture was concentrated under reduced pressure.
The solid residue was washed with aqueous KHCO3 (2 M), cold

4876
Q. Zhang et al. / Tetrahedron 65 (2009) 4871–4876
and the solvent was removed under reduced pressure. The residue
was recrystallised from Et2O to give the first crop of product as
a white solid (0.20 g). The mother liquor was concentrated under
reduced pressure and the residue was purified by column chro-
matography [SiO2, EtOAc/petrol¼1:1] to give the second crop of
product total yield: 0.456 g, 50%. Mp 132–134 ꢀC. [Found: C, 46.89;
100%). Mp 158–162 ꢀC dec. [Found: C, 30.65; H, 4.54; N, 2.91.
20
13C6C6H22NO9S3K requires C, 30.95; H, 4.76; N, 3.00%.] [
a
]
ꢁ19.6 (c
D
1.0, H2O) (lit.24 (unlabelled) ꢁ20 (c 1.0, H2O)); nmax (KBr disc) 3423
(br s, OH), 1579 (C]N), 1274, 1240, 1087, 1055, 1010 (ROSOꢁ3 ) cmꢁ1
;
dH (300 MHz, D2O) 5.05 (1H, dd, JCH 160 Hz, JHH 9.5 Hz, H-1), 3.90
(1H, dd, JCH 144 Hz, JHH 12.3 Hz, H-6a), 3.64 (1H, ddd, JCH 142 Hz, JHH
12.5 and 5.6 Hz, H-6b), 3.51 (1H, m, JCH 142.8 Hz, H-5), 3.50 (1H, m,
JCH 144 Hz, H-3), 3.40 (1H, m, JCH 145 Hz, H-4), 3.39 (1H, m, JCH
148 Hz, H-2), 2.74 (2H, t, JHH 7.4 Hz, CH2), 2.59 (2H, t, JHH 7.1 Hz,
CH2), 2.10 (3H, s, SCH3), 1.82 (2H, quintet, JHH 7.4 Hz, CH2), 1.72 (2H,
quintet, JHH 7.0 Hz, CH2); dC (100.10 Hz, D2O) 164.3 (C]N), 81.7
(enhanced d, JCC 40 Hz, C-1), 80.1 (enhanced dd, 2ꢂJCC 41 Hz, C-5),
77.0 (enhanced dd, 2ꢂJCC 39 Hz, C-3), 71.8 (enhanced dd, 2ꢂJCC
39 Hz, C-2), 69.0 (enhanced dd, 2ꢂJCC 39 Hz, C-4), 60.5 (enhanced d,
JCC 43 Hz, C-6), 32.5, 31.6, 27.2, 25.6 (4ꢂCH2), 13.9 (SCH3); m/z (ESꢁ)
426 (MꢁK)ꢁ; HRMS (ESꢁ): [MꢁK]ꢁ found 426.0676, 13C6C6H22NO9S3
requires: 426.0658.
H, 5.87; N, 2.74. 13C6C14H31NO10S2 requires C, 46.59; H, 6.06; N,
20
2.72%.] [
a
]
D
ꢁ16.7 (c 1.0, CHCl3) (lit.24 (unlabelled) ꢁ17 (c 1.0,
CHCl3)); IR (KBr disc) 3320 (OH), 1747, 1712 (C]O), 1612 (C]N),
1253, 1227 (COO) cmꢁ1
; dH (400 MHz, CDCl3) 8.02 (1H, s, OH), 5.27
(2ꢂ0.5H, m, JCH 151 Hz, H-3), 5.02 (1H, m, JCH 156 Hz, H-4), 5.01 (1H,
m, JCH 161 Hz, H-1), 4.99 (1H, m, JCH 155 Hz, H-2), 4.12 (1H, m, JCH
150 Hz, H-6a), 4.09 (1H, m, JCH 149 Hz, H-6b), 3.707 (2ꢂ0.5H, m, JCH
145 Hz, H-5), 2.55 (4H, m, H-10þH-40), 2.11, 2.09, 2.06, 2.04, 2.02
(15H, 5ꢂs, SCH3þ4ꢂCH3), 1.82–1.65 (4H, m, H-20þH-30); dC
(100.10 Hz, DMSO-d6) 170.8, 170.4, 169.6 and 169.4 (4ꢂC]O), 152.2
(C]N), 80.1 (enhanced d, JCC 42 Hz, C-1), 76.2 (enhanced dd, 2ꢂJCC
43 Hz, C-5), 74.0 (enhanced dd, 2ꢂJCC 41 Hz, C-3), 70.3 (enhanced
dd, 2ꢂJCC 42 Hz, C-4), 68.3 (enhanced dd, 2ꢂJCC 42 Hz, C-2), 62.4
(enhanced d, JCC 45 Hz, C-6), 33.9, 32.3, 28.4, 26.1 (4ꢂs, 4ꢂCH2),
20.93, 20.86, 20.78 (3ꢂs, overlapped, 4ꢂCH3), 15.7 (SCH3); m/z
(ESþ) 538 (MþNa)þ; HRMS (ESþ): (MþNa)þ found 538.1473,
13C6C14H31NO10S2Na requires 538.1488.
Acknowledgements
This project is sponsored by Food Standard Agency contract
E01086.
References and notes
4.2.11. 2,3,4,6-Tetra-O-acetyl-b-D
-[glucose-13C6]glucopyranosyl-4-
1. Fenwick, G. R.; Heaney, R. K.; Mullin, W. J. Crit. Rev. Food Sci. Nutr.1983,18,123–201.
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5. Poppel, G.; Verhoeven, D. T. H.; Verhagen, H.; Goldbohm, R. A. Adv. Exp. Med.
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7. Rouzaud, G.; Rabot, S.; Ratcliff, B.; Duncan, A. J. Br. J. Nutr. 2003, 90, 395–404.
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Mitchell-Olds, T. Plant Physiol. 2001, 126, 811–825.
9. Halkier, B. A.; Gershenzon, J. Annu. Rev. Plant Biol. 2006, 57, 303–333.
10. Botting, N. P.; Robertson, A. A. B.; Morrison, J. J. J. Labelled Compd. Radiopharm.
2007, 50, 216–225; Griffiths, W. D.; Bain, H.; Deighton, N.; Botting, N. P.;
Robertson, A. A. B. Phytochem. Anal. 2000, 11, 216–225.
11. Mellon, F. A.; Bennet, R. N.; Holst, B.; Williamson, G. Anal. Biochem. 2001, 306,
83–91.
12. Song, L.; Morrison, J. J.; Botting, N. P.; Thornally, P. J. Anal. Biochem. 2005, 347,
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13. Morrison, J. J.; Botting, N. P. J. Labelled Compd. Radiopharm. 2005, 48, 897–907.
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15. Rossiter, J. T.; Pickett, J. A.; Bennett, M. H.; Bones, A. M.; Powell, G.; Cobb, J.
Phytochemistry 2007, 68, 1384–1390.
methythiobutyl-O-sulfate thiohydroximate (7c)
To a solution of 6c (0.200 g, 0.388 mmol) in DCM (10 ml) were
added pyridine sulfur trioxide (0.3 g, 1.88 mmol) and pyridine
(0.1 ml,1.2 mmol). The mixture was heated under reflux for 4 h and
cooled to room temperature. Potassium hydrogen carbonate (2 M,
10 ml, 20 mmol) was added and the mixture was stirred for an-
other half an hour and then the two-phase mixture was concen-
trated under reduced pressure. The residue was filtered and then
washed with ice cold aqueous KHCO3 (2 M), cold water, cold EtOH
and diethyl ether successively to get the product as a white solid
(0.189 g, 77%). Mp 174–176 ꢀC. [Found: C, 35.90; H, 4.31; N, 2.10.
13C6C14H30NO13S3K$0.5KHCO3 requires C, 36.00; H, 4.49; N, 2.05%.]
20
[
a
]
ꢁ18.8 (c 1.0, H2O); nmax (KBr disc) 1751 (C]O), 1629, 1560
D
(C]N), 1279, 1236, 1064, 1035 (ROSOꢁ3 and COO) cmꢁ1
; dH
(300 MHz, DMSO-d6) 5.51 (2ꢂ0.5H, dd, JCH 165 Hz, JHH 10.0 Hz,
H-1), 5.45 (2ꢂ0.5H, m, JCH 153 Hz, H-3), 4.93 (2ꢂ0.5H, m, JCH
154 Hz, H-4), 4.87 (2ꢂ0.5H, m, JCH 157 Hz, H-2), 4.13 (1H, m, JCH
152 Hz, H-5), 4.05 (2ꢂ1H, m, JCH 148 Hz, H-6), 2.57 (4H, 2ꢂCH2 m,
H-10þH-40), 2.05, 2.03, 2.02, 2.00, 1.96 (15H, 5ꢂs, 4ꢂCH3þSCH3),
1.64 (4H, m, 2ꢂCH2); dC (100.10 Hz, DMSO-d6) 170.0, 169.6, 169.3,
169.1 (4ꢂC]O), 153.5 (C]N), 78.1 (enhanced d, JCC 42 Hz, C-1),
74.3 (enhanced dd, 2ꢂJCC 43 Hz, C-5), 72.7 (enhanced dd, 2ꢂJCC
41 Hz, C-3), 69.5 (enhanced dd, 2ꢂJCC 41 Hz, C-2), 68.0 (enhanced
dd, 2ꢂJCC 41 Hz, C-4), 62.2 (enhanced d, JCC 44.3 Hz, C-6), 32.7, 31.0,
27.9, 25.7 (4ꢂCH2), 20.5, 20.4, 20.3 (acetyl CH3), 14.5 (SCH3); m/z
(ESþ) 656 (MþNa), 672 (MþK); m/z (ES-) 594 [MꢁK]ꢁ; HRMS (ESꢁ):
[MꢁK]ꢁ found 594.1060, 13C6C14H30NO13S3 requires 594.1081.
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To a solution of 7c (0.148 g, 0.2 mmol) in MeOH (30 ml) was
added a catalytic amount of MeOK in MeOH. The mixture was
stirred at room temperature overnight. The solution was neutral-
ised carefully with Dowex-50 ion-exchange resin to pH 7.0 and
filtered through a pad of Celite and concentrated at room temper-
ature. The residue was dissolved in H2O (10 ml) and the solution
was freeze dried to give the product as a fluffy white solid (108 mg,