The synthesis of isotopically labelled glucosinolates for analysis and metabolic studies

Article abstract of DOI:10.1002/jlcr.1225

Glucosinolates are dietary natural products with important cancer chemoprevention properties. The syntheses of a number of stable isotopically labelled (²H, ¹³C) glucosinolates, and their desulfo-analogues, are described. These compo

Full text of DOI:10.1002/jlcr.1225

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 260–263.
Published online in Wiley InterScience
JLCR
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1225
Short Research Article
The synthesis of isotopically labelled glucosinolates for
analysis and metabolic studiesy
NIGEL P. BOTTING*, AVRIL A. B. ROBERTSON and JOHN J. MORRISON
School of Chemistry, University of St. Andrews, St. Andrews, Fife KY16 9ST, UK
Received 23 August 2006; Revised 15 December 2006; Accepted 15 December 2006
Abstract: Glucosinolates are dietary natural products with important cancer chemoprevention properties. The
syntheses of a number of stable isotopically labelled (2H, 13C) glucosinolates, and their desulfo-analogues, are
described. These compounds are used as internal standards for analysis and for metabolic studies. Copyright #
2007 John Wiley & Sons, Ltd.
Keywords: glucosinolates; isothiocyanates; glucoraphanin; sulforaphane; Brassicas
Introduction
break down to give nitriles 5 and thiocyanates 6,
depending on the conditions and presence of other
factors such as ESP (epithiospecifier protein) and
ferrous ions.5
Epidemiological and animal studies have provided
convincing evidence that the consumption of broccoli
and other cruciferous vegetables is strongly associated
with a decreased risk of cancer.1 The association is
strongest for cancers of the gastrointestinal and
respiratory tracts. Glucosinolates 1, a class of natu-
rally occurring thioglucosides, are thought to be
responsible for the observed anti-cancer effects, as
they are found in high levels in these vegetables.
Glucosinolates are metabolized by the plant enzyme
myrosinase during food preparation, cooking and
chewing (Scheme 1).2,3 The major product of this
metabolism is the corresponding isothiocyanate
The anti-cancer activity is in fact thought to be due to
the up-regulation of the xenobiotic-detoxifying Phase-II
enzymes and/or the down regulation of the xenobiotic-
activating Phase-I enzymes by the isothiocyanates
derived from the glucosinolates.6 The most active
isothiocyanate appears to be sulforaphane found in
broccoli because of its ability to mono-induce Phase-II
enzymes.7 However, when broccoli is consumed, hu-
mans are not directly exposed to sulforaphane, but
instead to its glucosinolate precursor glucoraphanin.
Enzymatic hydrolysis to release sulforaphane takes
place following tissue disruption by chewing of vege-
tables, although cooking may inactivate myrosinase
and then hydrolysis must occur catalysed by the
bacterial enzymes in the intestinal tract. So the issue
of human exposure is complex and as yet poorly
understood. Stable isotopically labelled derivatives of
glucoraphanin, and other glucosinolates, are thus
required for studies to establish the metabolism and
bioavailability of glucosinolates and isothiocyanates
and to search for possible new biomarkers of exposure.
Another important use for isotopically labelled glu-
cosinolates is as internal standards for analysis by LC-
MS techniques.8 Some methods have been developed
for the measurement of intact glucosinolate, while
others employ a desulfonation step to produce the
uncharged desulfoglucosinolates as analytes, and so
both are required in isotopically labelled form.
¨
Lossen-type rearrangement of
2, formed via
a
the unstable thiohydroximate-O-sulfonate aglycone
which is the initial product. In plant cells the glucosi-
nolates and myrosinase are compartmentalized, so that
they can only interact following tissue damage. In
mammals there also appears to be myrosinase activity
in intestinal bacteria which may contribute to glucosi-
nolate degradation in vivo.4 The aglycone can also
*Correspondence to: Nigel P. Botting, School of Chemistry, University
of St. Andrews, St. Andrews, Fife KY16 9ST, UK.
E-mail: npb@st-andrews.ac.uk
Contract/grant sponsor: Food Standards Agency (FSA); contract/grant
number: T01027
Contract/grant sponsor: BBSRC
yProceedings of the Ninth International Symposium on the Synthesis
and Applications of Isotopically Labelled Compounds, Edinburgh,
16–20 July 2006.
Copyright # 2007 John Wiley & Sons, Ltd.

SYNTHESIS OF ISOTOPICALLY LABELLED GLUCOSINOLATES 261
desulfoglucosinolate 11 in 74% yield. The Z-isomer is
obtained exclusively due to stereoelectronic effects.12
The 1H and 13C NMR data for the final products were
identical to those of the unlabelled compounds except
for the lack of any signal for the aromatic protons, while
electrospray mass spectrometry confirmed the mole-
cular mass. Currently, the [phenyl-2H5]gluconastur-
tiin, and also similarly labelled versions of its
metabolites, are being used to investigate their meta-
bolism in rats.
Results and discussion
Synthesis of isotopically labelled gluconasturtiin
Our initial studies centred on the glucosinolate,
gluconasturtiin, which has a phenethyl side chain. It
was decided to make a derivative with deuterium
substitution in the benzene ring and [2H5]bromoben-
zene 7 was used as the starting material. The full
syntheses of [phenyl-2H5]gluconasturtiin9 8 and [phe-
nyl-2H5]desulfogluconasturtiin10 9 are given in Scheme
2. The required side chain was prepared via conjugate
addition of the Grignard reagent derived from [2H5]bro-
mobenzene with acrolein diethyl acetal. The key step in
constructing the glucosinolate was the coupling of the
oximyl chloride 10 with the thiolate derived from
tetraacetyl thioglucose,11 which gave the protected
Studies using LC-APCI-MS showed that using [phe-
nyl-2H5]desulfogluconasturtiin as an internal standard
combined with single ion monitoring, increased the
levels of detection a hundred fold as compared with
traditional HPLC methods.13 Also the [phenyl-2H5]glu-
conasturtiin has been employed in new LC-MS/MS
method for the analysis of glucosinolates and their
HO
HO
HO
-
-
HO
HO
HO
OSO3
N
OSO3
N
Lossen
O
O
-S
S
Rearrangment
OH
Myrosinase
R
R
N
S
C
4
S
N
OH
1
OH
R
R
2
3
?
ESP
Fe2+
C
6
R
N
C
5
Scheme 1
OH
N
D
D
D
D
D
Br
D
D
D
D
a
b, c, d
OEt
Cl
e
D
D
D
D
D
D
10
7
-
OSO3 K+
AcO
AcO
AcO
AcO
AcO
AcO
OH
O
O
S
N
S
N
D
D
D
D
D
OAc
OAc
f
D
D
11
D
D
D
h
D
g
-
OSO3 K+
HO
HO
HO
HO
OH
O
O
HO
HO
S
N
D
S
N
D
D
D
OH
OH
D
D
8
9
D
D
D
Scheme 2 Reagents and conditions: (a) Mg, THF, then 5% CuBr, THF then acrolein diethyl acetal (100%); (b) acetone/H2O (1:4),
HCl (93%); (c) NH2OH.HCl, NaOAc, EtOH, reflux (88%); (d) N-Chlorosuccinimide, pyridine, CHCl3 (82%); (e) Et3N, THF, tetraacetyl
thioglucose (74%); (f) ClSO3H, pyridine, DCM (20%); (g) KOMe, MeOH (99%); (h) NaOMe, MeOH (99%).
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 260–263
DOI: 10.1002.jlcr

262 N. P. BOTTING ET AL.
metabolites.14 However, a drawback with this strategy
for the design and synthesis of internal standards is
that the deuterium atoms are incorporated into the side
chain of the desulfoglucosinolate and so a different
synthetic procedure will be required for each glucosi-
nolate. Therefore a synthesis has now been developed
whereby three deuterium atoms were incorporated into
the glucose to give [10-2H, 60-2H2]desulfogluconastur-
tiin 12. This means that the same sugar fragment can
be used for the preparation of any desulfoglucosinolate,
regardless of the nature of the side chain, thus making
the procedure much more flexible.
thioglucose fragment. It was decided to use dioxolane
protection for the aldehyde, following some preliminary
studies with other protecting groups. For the isotopic
labelling the end of the side chain was provided by
[2H6]dimethyl sulfoxide, with one mole of [13C]cyanide
ion used for a chain extension reaction, to provide an
overall 6 mass unit increase over the unlabelled
glucoraphanin. The synthesis is shown in Scheme 3.
A key feature of the synthesis was the in situ coupling
of the oximyl chloride with the tetracetylthioglucose 15.
It was found that the oximyl chloride was too unstable
to be isolated, presumably due to competing cyclisation
reactions with the sulfoxide, however when N-chlor-
osuccinimide was used to chlorinate the oxime pre-
cursor 16 in the presence of base and 15, the coupled
product 17 was obtained in a reasonable yield in one
step. The spectroscopic data for the final product,
[10-13C, 11, 12-2H5]glucoraphanin 18, were in accord
with previous literature data.15,16 The presence of the
isotopic labels was confirmed by the increase of 6 mass
units from that of the unlabelled material. The 13C NMR
Synthesis of isotopically labelled glucoraphanin
The next target was an isotopically labelled version of
glucoraphanin 13, the precursor of the isothiocyanate
sulforaphane 14, which was to be employed in meta-
bolic studies. In this case an isotopically labelled
5-carbon chain aldehyde with terminal methyl sulf-
oxide functionality was required for coupling to the
D
D
-
OH
N
OSO3
N
HO
HO
HO
HO
O
O
O
S
S
HO
HO
O
S
S
S
C N
CH3
HO
OH
D
CH3
O
12
13
14
O
O
b
c
a
O
O
13
13 C N
O
13 C
H
O
CH2OH
O
O
N
Br
d
OH
D
D
D
O
f
O
e
CD3
CD3
1C3
16
1C3
S
H
O
S
13
O
CH2Br
D
O
O
AcO
O
g
SH
AcO
AcO
OAc
15
-
OSO3
N
AcO
AcO
AcO
OH
N
AcO
AcO
AcO
O
O
h
S
S
O
O
OAc
13C
OAc
13C
S
S
CD3
CD3
17
D
D
D
D
i
-
OSO3
N
HO
HO
HO
O
S
O
S
OH
13C
CD3
18
D
D
Scheme
3
(a) K13CN, Bu4N+Brꢀ, H2O (75%); (b) DIBAL, Et2O (83%); (c) NaBH4, MeOH (80%); (d) Ph3P, CBr4 (61%);
(e) CD3SOCDꢀ2 Na+, CD3SOCD3, 10 eq. NaH (96%); (f) NH2OH.HCl, H3O+ (100%); (g) N-Chlorosuccinimide, pyridine, then
tetraacetylthioglucose 15 and Et3N (45%); (h) Py.SO3, pyridine (74%); (i) MeOK, MeOH (89%).
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 260–263
DOI: 10.1002.jlcr

SYNTHESIS OF ISOTOPICALLY LABELLED GLUCOSINOLATES 263
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at 21.0 ppm.
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Conclusion
6. Thornallay PJ. Anti-Cancer Drugs 2002; 13:
331–338.
A range of isotopically labelled glucosinolates and their
desulfonated derivatives have been synthesised for use
as internal standards in analysis or for metabolic
studies.
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Acknowledgements
We are grateful to the Food Standards Agency (FSA) for
funding, under contract T01027 (for JJM) and the
BBSRC for a studentship (for AABR).
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Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 260–263
DOI: 10.1002.jlcr