The synthesis of glucosinolates deuterium labelled in the glucose fragment

Article abstract of DOI:10.1002/jlcr.1141

A facile synthesis is described for 2,3,4,6-tetra-O-acetyl-thio-β-D- [1-²H,6-²H₂]glucopyranose, which has been used to make an isotopically labelled desulphoglucosinolate, [1′- ²H,6′-²H₂]de

Full text of DOI:10.1002/jlcr.1141

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 1201–1211.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1141
Research Article
The synthesis of glucosinolates deuterium labelled in
the glucose fragment
Avril A. B. Robertson and Nigel P. Botting*
School of Chemistry, University of St. Andrews, St. Andrews, Fife KY16 9ST, UK
Summary
A facile synthesis is described for 2,3,4,6-tetra-O-acetyl-thio-b-d-[1-2H,6-2H2]gluco-
pyranose, which has been used to make an isotopically labelled desulphoglucosinolate,
[10-2H,60-2H2]desulfogluconasturtiin. This isotopically labelled building block can be
employed to make any glucosinolate or desulfoglucosinolate for use as internal
standards for MS based analytical methods. Copyright # 2006 John Wiley & Sons,
Ltd.
Received 8 September 2006; Accepted 14 September 2006
Key Words: glucosinolates; isothiocyanates; gluconasturtiin; cancer prevention
Introduction
Glucosinolates (1) are a class of naturally occurring thioglucosides present in
all members of the Cruciferae, including the brassica crops such as cabbage,
Brussels sprouts and oilseed rape.1 Glucosinolates contain a common structure
and over 100 different examples have been isolated and characterized,1 with a
variety of substituents in the side chain R, including allyl, benzyl, indolyl and
4-hydroxybenzyl. They are metabolized by the plant enzyme myrosinase2,3
during food preparation, cooking and chewing (Scheme 1) to give the
-
-
HO
HO
HO
HO
OSO3
N
OSO3
N
Lossen
Rearrangment
O
O
-S
S
OH
Myrosinase
HO
R N C S
HO
OH
1
OH
R
R
2
3
Scheme 1.
*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
Contact/grant sponsor: BBSRC
Copyright # 2006 John Wiley & Sons, Ltd.

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A. A. B. ROBERTSON AND N. P. BOTTING
corresponding isothiocyanate (2) as the major product. The isothiocyanate is
the formed via a Lossen-type rearrangement of the unstable thiohydroximate-
O-sulfonate aglycone (3) initially produced.
There is considerable interest in glucosinolates for two major reasons.
Firstly, they play an important role in plants.4 The glucosinolates and
myrosinase are compartmentalized and only interact to release the noxious
isothiocyanates following tissue damage, usually caused by pests. The
isothiocyanates then deter further attack by non-adapted herbivores.
However, specialist pests have evolved to employ the volatile isothiocyanates
as host–plant recognition cues. Cabbage and turnip root flies (Delia radicum
and Delia floralis), use leaf surface glucosinolates as ovi-position (egg-laying)
stimulants.5 Secondly, there is convincing epidemiological evidence that
consumption of broccoli and other cruciferous vegetables is associated with a
decreased risk of cancer and this association is strongest for cancers of the
gastrointestinal and respiratory tracts.6 The anti-cancer effects have been
attributed to the presence of glucosinolates in these vegetables, although the
anti-cancer activity appears to reside with the isothiocyanate.7 The mechanism
of the anti-cancer activity is still controversial and could be a result of either
inhibition of the Phase 1 detoxification enzymes,8 upregulation of the Phase 2
detoxification enzymes9 or even induction of apoptosis of tumour cells.10
The interest in glucosinolates has generated a need for accurate, and
reproducible, glucosinolate analysis and a number of methods have been
developed using LC-MS11–13 and LC-MS/MS14 techniques. However, there
are currently few isotopically labelled internal standards available to aid
quantitation. Most of the existing methods for isotopic labelling of
glucosinolates have incorporated the isotopes into the side chain.12 The
drawback of this method is that a new synthesis needs to be developed for each
glucosinolate. An answer to this problem is to label the sugar half of the
glucosinolate, and this can then be used whatever the structure of the side
chain. Therefore we herein report the synthesis of [10-2H,60-2H2]
desulfogluconasturtiin, which demonstrates a strategy for the synthesis of
sugar labelled glucosinolates as internal standards for analysis.
Results and discussion
In previous studies in our laboratory a deuterated desulfogluconasturtiin
was prepared as an internal standard for glucosinolate analysis,15 using
[2H5]bromobenzene as the source of the isotopic labels. This synthesis
was then modified to make [ phenyl-2H5]gluconasturtiin which was used in
metabolic studies in rats.16 As a result of this experience gluconasturtiin was
used as our model glucosinolate in these further studies.
For an optimum internal standard a mass increase of three units is required
and it was thought that three deuterium atoms could be incorporated into the
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1201–1211
DOI: 10.1002/jlcr

SYNTHESIS OF GLUCOSINOLATES DEUTERIUM
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OH
AcO
AcO
OSO3- K+
N
HO
HO
AcO
AcO
Cl
OH
N
N
O
O
O
b,c
a
SH
S
S
AcO
HO
AcO
R
OAc
OH
OAc
R
R
4
5
Scheme 2. General glucosinolate synthesis. Reagents and conditions: (a) Et3N,
THF; (b) pyridine.SO3, CH2Cl2; (c) KOMe, MeOH
glucose at C-1 and C-6 via reduction of suitable derivatives with deuterated
reducing agents. As the key step in glucosinolate synthesis, as originally
developed by Benn,17 is the coupling of a protected thioglucose (4) with a
suitable oximyl chloride (5) (Scheme 2), the initial synthetic target was 2,3,4,6-
tetra-O-acetyl-thio-b-d-[1-2H1,6-2H2]glucopyranose (6), which can be then be
incorporated directly into any established glucosinolate synthesis.
Although D-[6-2H2]glucose is commercially available it is also readily made
via reduction of a suitable methyl glucuronide with lithium aluminium
deuteride. The stating material for the labelling was methyl (methyl 2,3,4-tri-
O-benzyl-a-d-glucopyranosid)uronate (7). The method of Bernet and Vasella18
was used to make methyl-2,3,4-tri-O-benzyl-a-d-glucopyranoside which was
oxidized to 7 in two steps using Swern conditions19 followed by pyridinium
dichromate (PDC) in methanol/dichloromethane, to afford the methyl ester (7)
in 58% yield. This oxidation is also reported in the literature in one step using
either chromium trioxide in sulfuric acid/acetone20,21 or PDC in DMF,22
followed by methylation with diazomethane in each case.
The first two deuterium atoms where then introduced at C-6 by reduction
with lithium aluminium deuteride in 91% yield, followed by benzylation to
give the methyl 2,3,4-tribenzyl-a-D-[6-2H2]glucopyranoside (8) (Scheme 3).
Comparison of the spectral data with that of the unlabelled compound clearly
demonstrated the presence of the two deuterium atoms, from the expected
increase in molecular weight and absence of the 6-CH2 signals in the 1H NMR
spectrum. Hydrolysis of the methyl glycoside using glacial acetic acid and
1.0 M sulfuric acid under reflux was followed by oxidation with pyridinium
chlorochromate (PCC) in dry dichloromethane to give the protected
[6-2H2]glucono-g-lactone (9) in 79% yield. Introduction of the third deuterium
atom at C-1 was achieved by reduction with sodium borodeuteride in
quantitative yield. Removal of the three benzyl groups and acetylation were
carried out in a one pot reaction using acetic anhydride and BF3.etherate23 to
give 1,2,3,4,6-pentaacetyl-b-D-[1-2H,6-2H2]glucose (10) as a 5:1 mixture of a
and b anomers.
From this point onwards the synthesis employed our standard glucosinolate
methodology.16,24 The 1,2,3,4,6-pentaacetyl-D-[1-2H,6-2H2]glucose was con-
verted to the glucopyranosyl bromide using HBr in acetic acid, reacted with
Copyright # 2006 John Wiley & Sons, Ltd.
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A. A. B. ROBERTSON AND N. P. BOTTING
D
D
O
D
O
D
O
H3CO
BnO
BnO
BnO
BnO
BnO
a, b
c, d
e, f
O
BnO
BnO
BnO
O
BnO
BnO
BnO
OCH3
OCH3
8
9
7
D
D
D
D
O
D
O
D
O
HO
AcO
AcO
AcO
AcO
OH
N
g. h,i
j, k
HO
HO
S
SH
OAc
AcO
AcO
HO
11
AcO
AcO
D
D
D
10
6
Scheme 3. Synthesis of [10-2H, 60-2H2]desulfogluconasturtiin (11). Reagents
and conditions: (a) LiAlD4, Et2O (91%); (b) NaH, BnBr, DMF (95%);
(c) AcOH, aq. H2SO4 (64%); (d) PCC, CH2Cl2 (79%); (e) NaBD4, THF/H2O
(100%); (f) Ac2O, BF3.Et2O, OoC (82%); (g) HBr, ACOH (83%); (h) thiourea,
acetone (98%); (i) K2S2O5, H2O, CH2Cl2 (71%); (j) phenethyl oximyl chloride,
Et3N, Et2O (100%); (k) KOMe, MeOH (64%)
thiourea in acetone and the resulting thiouronium salt was then hydrolysed
using aqueous potassium metabisulfite to give the target molecule, 2,3,4,6-
tetra-O-acetyl-thio-b-d-[1-2H,6-2H2]glucopyranose (6), in 58% yield over the
three steps. To demonstrate incorporation of the labelled sugar into a gluco-
sinolate framework, as discussed above, [10-2H,60-2H2]desulfogluconasturtiin
was the preferred example. Generally the coupling reaction between the
oximyl chloride and protected thioglucose gives reasonable yields but it was
decided to optimize the reaction further to ensure no waste of labelled
material. Examination of test reactions using unlabelled starting materials
showed that there was some disulfide by-product. Therefore, it was decided to
carry out the reaction with an excess of the oximyl chloride to maximize the
conversion of the labelled thioglucose. Using 2.3 equivalents of the oximyl
chloride the coupled product could thus be obtained in quantitative yield.
Final deprotection using catalytic potassium methoxide gave the
[10-2H,60-2H2]desulfogluconasturtiin (11) in 64% yield. The spectral data
confirmed the labelling pattern. In the 1H NMR spectrum the signals for 10-H
and 60-CH2 were absent and a 3 mass unit increase was observed in the mass
spectrum, compared to the unlabelled material.
A facile synthesis has thus been developed for 2,3,4,6-tetra-O-acetyl-thio-b-
d-[1-2H,6-2H2]glucopyranose and it has shown that this can be used to make
an isotopically labelled desulfoglucosinolate, [10-2H, 60-2H2]desulpho-
gluconasturtiin. In principle this methodology can now be employed to
make any glucosinolate or desulfoglucosinolate that may be required and
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1201–1211
DOI: 10.1002/jlcr

SYNTHESIS OF GLUCOSINOLATES DEUTERIUM
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allow a whole family of compounds to be prepared as internal standards for
MS based analytical methods, using the same isotopically labelled building
block.
Experimental
General
Melting points were determined in open capillary tubes and are uncorrected.
1H and 13C NMR spectra were recorded at 300 and 75.46 MHz, respectively,
using Bruker Avance 500 and Varian Gemini 2000 spectrometers. The
chloroform peaks (7.27 ppm for 1H, 77.00 ppm for 13C) were used as
references. The EI and CI mass spectra were obtained on a VG Autospec
mass spectrometer, and the ESI and APCI mass spectra on a Micromass LCT
mass spectrometer. Diethyl ether and tetrahydrofuran were distilled over
sodium and dichloromethane over calcium hydride. DMSO was freshly
distilled from calcium hydride.
Methyl 2,3,4-tri-O-benzyl-a-d-[6-2H2]glucopyranoside
Methyl (methyl 2,3,4-tri-O-benzyl-a-d-glucopyranosid)uronate (8.0 g, 16.2 mmol)
was dissolved in dry diethyl ether (100 ml) at 08C and treated with lithium
aluminium deuteride (1.0 M solution in diethyl ether) (8.12 ml, 8.12 mmol).
The reaction was stirred at room temperature overnight, quenched using 1.0 M
sulphuric acid and extracted using ethyl acetate (2 ꢀ 100 ml). The organic
extracts were washed with brine (100 ml), dried (MgSO4) and the solvent
evaporated at reduced pressure. The product was purified by column
chromatography on silica gel using ethyl acetate–hexane (1:2) as the eluant
to give methyl 2,3,4-tri-O-benzyl-a-d-[6-2H2]glucopyranoside as a white
crystalline solid (6.87 g, 91%); m.p. 50–518C; [a]D +21.78 (c 1.0 in CHCl3);
(Found: C, 71.58; H, 6.71. Calculated for C28H30 2H2O6: C, 72.08; H, 6.91%);
n
max (nujol)/cmꢁ1 3479 (OH); dH (200 MHz; C2HCl3) 3.37 (3H, s, OCH3), 3.51
(1H, dd, J1,2 3, J2,3 10, H-2), 3.53 (1H, t, J3,4=J4,5 10, H-4), 3.63 (1H, d, J4,5
10, H-5), 4.02 (1H, t, J2,3 = J3,4 10, H-3), 4.57 (1H, d, J 3, H-1), 4.60–5.10 (6H,
m, 3 ꢀ PhCH2), 7.20–7.40 (15H, m, 3 ꢀ PhCH2); dC (50.3 MHz; C2HCl3) 55.7
(OCH3), 71.0 (C-5), 74.0 (PhCH2), 75.6 (PhCH2), 76.3 (PhCH2), 77.8 (C-4),
80.4 (C-2), 82.5 (C-3), 98.7 (C-1), 128.2–129.0 (15CH, 3 ꢀ PhCH2), 138.5,
138.6, 139.2 (3 ꢀ quaternary C, 3 ꢀ Ph); m/z (CI) 467 ([MH]+, 1%), 435 (37),
375 (29), 343 (29), 271 (60), 181 (94) and 91 (100, [PhCH2]+).
Methyl 2,3,4,6-tetra-O-benzyl-a-d-[6-2H2]glucopyranoside (8)
Sodium hydride (60% dispersion in mineral oil) (1.6 g, 61 mmol) was washed
with dry diethyl ether (2 ꢀ 5 ml) then treated with benzyl bromide (4.80 g,
3.4 ml, 27.5 mmol). Methyl 2,3,4-O-benzyl-a-d-[6-2H2]glucopyranoside (6.3 g,
Copyright # 2006 John Wiley & Sons, Ltd.
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13.5 mmol) in dry N,N-dimethylformamide (65 ml) was added to the reaction
and after 2 h at room temperature the reaction was quenched by dropwise
addition of methanol (10 ml). The solvent was evaporated at reduced pressure
then the residue was taken up in diethyl ether (250 ml) and washed with water
(200 ml). The aqueous phase was extracted with diethyl ether (2 ꢀ 100 ml) then
the combined organic extracts were washed with brine (200 ml), dried (MgSO4)
and the solvent evaporated at reduced pressure to give the crude product. The
resulting oil was purified by column chromatography using ethyl acetate-
hexane (1:2) as the eluant to give methyl 2,3,4,6-tetra-O-benzyl-a-d-
[6-2H2]glucopyranoside was given as a clear viscous oil (7.14 g, 95%); [a]D
+37.48 (c 1.0 in CHCl3); (Found: C, 75.53; H, 6.86. Calculated for
C35H362H2O6: C, 75.51; H, 6.88%); dH (200 MHz; CDCl3) 3.40 (3H, s,
OCH3), 3.60 (1H, dd, J1,2 = 3, J2,3 = 10, H-2), 3.70 (1H, t, J2,3 = J3,4 = 10,
H-4), 3.77 (1H, d, J4,5 = 10, H-5), 4.00 (1H, t, J3,4 = J4,5 10, H-3), 4.40–5.10
(9H, m, 4 ꢀ PhCH2, H-1), 7.10–7.40 (20H, m, 4 ꢀ PhCH2); dC (50.3 MHz;
CDCl3) 55.7 (OCH3), 70.4 (C-5), 73.9 (PhCH2), 74.0 (PhCH2), 75.6 (PhCH2),
76.3 (PhCH2), 78.1 (C-4), 80.2 (C-2), 82.6 (C-3), 98.7 (C-1), 128.2–129.0
(20CH, 4 ꢀ PhCH2), 138.4, 138.6, 138.7, 139.2 (4 ꢀ quaternary C, 4 ꢀ Ph);
m/z (CI) 557 ([MH]+, 1%), 271 (60), 219 (33), 181 (94) and 91 (100,
[PhCH2]+).
2,3,4,6-Tetra-O-benzyl-d-[6-2H2]glucopyranoside
Glacial acetic acid (55 ml) and 1.0 M sulfuric acid (14 ml) were heated at
1058C and a solution of methyl 2,3,4,6-tetra-O-benzyl-d-[6-2H2]glucopyrano-
side (6.87 g, 12.3 mmol) in glacial acetic acid (8 ml) was added. The reaction
was kept at 1058C for 1 h then cooled on ice. The crystalline product was
removed by filtration, washed with ice water and dried at reduced pressure.
The white crystals were then washed with hexane and dried in a desiccator to
give 2,3,4,6-tetra-O-benzyl-d-[6-2H2]glucopyranoside as a white crystalline
solid (4.28 g, 64%), which was a mixture of a and b anomers; m.p. 132–1338C;
[a]D +18.48 (c 1.0 in CHCl3); (Found: C, 74.60; H, 6.75. Calculated for
C34H342H2O6: C, 75.25; H, 6.69%); nmax (nujol)/cmꢁ1 3370 (OH); dH
(300 MHz; CDCl3) 2.70 (1H, s, OH), 3.42–3.46 (1H, m, H-2b), 3.55 (1H, d,
J
4,5 =10, H-5 ), 3.60–3.70 (4H, m, H-2a, 3b, 4a and b), 3.98 (1H, t, J2,3 = J3,4
10, H-3a), 4.05 (1H, d, J4,5 = 10, H-5a), 4.40–5.00 (17H, m, 4 ꢀ PhCH2 a and
b, H-1b), 5.20 (1H, d, J1,2 3.6, H-1a), 7.10–7.40 (40H, m, 4 ꢀ PhCH2 a and b);
dC (50.3 MHz; CDCl3) 70.4 (C-5a), 73.8, 74.0 (3 ꢀ PhCH2), 75.0 (3 ꢀ PhCH2,
5b), 76.3 (2 ꢀ PhCH2), 78.2 (C-4a, 4b), 80.4 (C-2a), 82.2 (C-3a), 83.5 (C-2b),
85.0 (C-3b), 91.8 (C-1a), 98.0 (C-1b), 128.1–130.0 (20CH, 4 ꢀ PhCH2), 138.4,
138.6, 138.7, 139.2 (4 ꢀ quaternary C, 4 ꢀ Ph); m/z (FAB) 565 ([M+Na]+,
34%), 543 (2, [MH]+), 417 (15) and 181 (100, [glucoseH]+).
Copyright # 2006 John Wiley & Sons, Ltd.
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SYNTHESIS OF GLUCOSINOLATES DEUTERIUM
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2,3,4,6-Tetra-O-benzyl-d-[6-2H2]glucono-g-lactone (9)
Pyridinium chlorochromate (7.00 g, 40 mmol) was added in one portion to
2,3,4,6-tetra-O-benzyl-a-d-[6-2H2]glucopyranoside (3.62 g, 6.7 mmol) and
powdered 4 A molecular sieves (7.0 g) in dry dichloromethane (90 ml). The
reaction was stirred at room temperature for 1 h then hexane (100 ml) and
diethyl ether (200 ml) were added. The solution was filtered through silica gel
and the solvent evaporated at reduced pressure. The crude product was
purified using column chromatography on silica using hexane-diethyl ether
(7:1) as the eluant. 2,3,4,6-Tetra-O-benzyl-d-[6-2H2]glucono-g-lactone was
obtained as a clear oil (2.83 g, 79%); [a]D +33.28 (c 2.0 in CHCl3); nmax (thin
film)/cmꢁ1 1765 (C=O); dH (200 MHz; CDCl3) 3.95 (1H, t, J 7, H-4), 4.00
(1H, t, J7, H-3), 4.15 (1H, d, J7, H-2), 4.45–5.10 (9H, m, H-5 and 4 ꢀ PhCH2),
7.10–7.50 (20H, m, 4 ꢀ PhCH2); dC (50.3 MHz; CDCl3) 74.0 (PhCH2), 74.3
(2 ꢀ PhCH2), 74.5 (PhCH2), 76.5 (C-5), 77.9, 78.5 (C-2, 4), 81.4 (C-3),
125.5–126.3 (20CH, 4 ꢀ PhCH2), 134.7, 135.3, 135.4 (4 ꢀ quaternary C,
4 ꢀ Ph), 169.9 (C=O); m/z (CI) 541 ([M+H]+, 40%), 540 (5), 271 (64), 181
(64), 107 (65) and 91 (100, [PhCH2]+).
2,3,4,6-Tetra-O-benzyl-d-[1-2H,6-2H2]glucopyranoside
2,3,4,6-Tetra-O-benzyl-d-[6-2H2]glucono-g-lactone (2.7 g, 3.9 mmol) was dis-
solved in tetrahydrofuran (13 ml) and cooled to 08C under nitrogen. Sodium
borohydride (0.84 g, 2.3 mmol) was dissolved in water and added to the
lactone solution dropwise. The reaction was stirred for 24 h, quenched with
1 M sulfuric acid then extracted with ethyl acetate (2 ꢀ 75 ml) and washed with
brine (80 ml). The organic extracts were dried (MgSO4) and the solvent
evaporated at reduced pressure to give 2,3,4,6-tetra-O-benzyl-d-
[1-2H,6-2H2]glucopyranoside as a white crystalline solid (2.7 g, 100%); m.p.
120.5–1228C; [a]D +37.28 (c 1.0 in CHCl3); nmax (nujol)/cmꢁ1 3370 (OH); dH
(200 MHz; CDCl3) 3.1 (1H, s, OH), 3.40–3.70 (6H, m, H-2a, 3b, 4a and b, 5b),
3.98 (1H, t, J2,3 = J3,4 10, H-3a), 4.05 (1H, d, J4,5 10, H-5a), 4.40–5.00 (16H,
m, 4 ꢀ PhCH2 a and b), 7.10–7.40 (40H, m, 4 ꢀ PhCH2 a and b); dC
(50.3 MHz; CDCl3) 70.4 (C-5a), 73.8, 74.0 (3 ꢀ PhCH2), 75.0 (3 ꢀ PhCH2, 5b),
76.3 (2 ꢀ PhCH2), 78.2 (C-4a, 4b), 80.4 (C-2a), 82.2 (C-3a), 83.5 (C-2b), 85.0
(C-3b), 128.1–130.0 (20CH, 4 ꢀ PhCH2), 138.4, 138.6, 138.7, 139.2
(4 ꢀ quaternary C, 4 ꢀ Ph); m/z (FAB) 566 ([M+Na]+), 12%), 544
(2, [MH]+), 543 (5, [M]+), 418 (14) and 181 (100, [glucoseH]+).
1,2,3,4,6-Penta-O-acetyl-d-[1-2H,6-2H2]glucopyranose (10)
2,3,4,6-Tetra-O-benzyl-d-[1-2H,6-2H2]glucopyranose (1.50 g, 0.276 mmol) in
acetic anhydride (200 ml) was treated with boron trifluoride diethyl etherate
(2.88 ml, 22 mmol) at 08C and the reaction stirred overnight. Sodium hydrogen
Copyright # 2006 John Wiley & Sons, Ltd.
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A. A. B. ROBERTSON AND N. P. BOTTING
carbonate was used to neutralize the reaction mixture which was then
extracted using ethyl acetate (2 ꢀ 200 ml) and washed with water (4 ꢀ 200 ml).
The organic extracts were dried (MgSO4) and the solvent evaporated at
reduced pressure. The residue was purified by column chromatography on
silica gel using 40–60 petroleum ether-ethyl acetate (3:2) as the eluant to give
1,2,3,4,6-penta-O-acetyl-d-[1-2H,6-2H2]glucopyranose as a white crystalline
solid (0.89 g, 82%); m.p. 111–1128C; [a]D +98.18 (c 0.6 in CHCl3); (Found: C,
48.34; H, 5.46. Calculated for C16H192H3O11: C, 48.85; H, 5.64%); nmax
(nujol)/cmꢁ1 1735 (CO); dH (200 MHz; CDCl3) 2.00–2.20 (30H, m,
5 ꢀ OC(O)CH3 a and b), 3.80 (1H, d, J4,5 10, H-5b), 4.10 (1H, d, J4,5 10, H-
5a), 5.10 (1H, d, J2,3 10, H-2a), 5.14 (1H, t, J3,4 = J4,5 10, H-4a), 5.46 (1H, t,
J2,3 = J3,4 10, H-3a), 5.00–5.50 (3H, m, H-2b, 3b, 4b); dC (50.3 MHz; CDCl3)
20.9–21.4 (5 ꢀ OC(O)CH3 a and b), 68.1 (C-4b), 68.3 (C-4a), 69.5 (C-2a), 70.1,
70.3 (C-3a, 5a), 70.6 (C-2b), 73.0, 73.2 (C-3b, 5b), 169.3, 169.9, 170.2, 170.7,
171.0 (5 ꢀ OC(O)CH3 a and b); m/z (CI) 394 ([MH]+, 1%), 334 (100, [M-
OAc]+), 274 (28), 214 (6) and 172 (35).
2,3,4,6-Tetra-O-acetyl-a-d-[1-2H,6-2H2]glucopyranosyl bromide
1,2,3,4,6-Penta-O-acetyl-d-[1-2H1,6-2H2]glucopyranose (0.7 g, 1.78 mmol) was
dissolved in acetic anhydride (0.6 ml) and 45% w/v hydrogen bromide in acetic
acid (1 ml) was added. The reaction mixture was stirred for 3 h then further
45% w/v hydrogen bromide in acetic acid (3 ml) was added and stirring
continued overnight. The residue was dissolved in dichloromethane (10 ml)
and poured into ice/water (20 ml) with stirring. The organic layer was then
added carefully to an ice/saturated sodium hydrogen carbonate solution
(20 ml) with stirring. Once the gas evolution became less vigorous the organic
phase was added to saturated sodium hydrogen carbonate solution (20 ml).
The organic layer was dried (MgSO4) and the solvent evaporated at reduced
pressure to give a golden oil which solidified upon cooling to 08C. The product
was recrystallized from 40–60 petroleum ether–diethyl ether to give 2,3,4,6-
tetra-O-acetyl-a-d-[1-2H1,6-2H2]glucopyranosyl bromide as a white crystalline
solid (0.614 g, 83%); m.p. 84–878C; [a]D +1868 (c 2.42 in CHCl3); nmax (nujol)/
cmꢁ1 1730 (CO); dH (200 MHz; CDCl3) 2.00 (3H, s, OC(O)CH3), 2.05 (3H, s,
OC(O)CH3), 2.10 (6H, 2 s, 2 ꢀ OC(O)CH3), 4.20 (1H, d, J4,5 10, H-5), 4.80 (1H,
d, J2,3 10, H-2), 5.15 (1H, t, J3,4 = J4,5 10, H-4), 5.55 (1H, t, J2,3 = J3,4 10, H-3);
dC (50.3 MHz; CDCl3) 21.0–21.1 (4 ꢀ OC(O)CH3), 64.6 (C-4), 70.6 (C-2), 71.0
(C-3), 72.4 (C-5), 169.9, 170.2, 170.3, 171.0 (4ꢀ OC(O)CH3); m/z (CI) 433, 431
([M+NH4]+, 5%), 334 (30, [M-Br]+), 292 (44), 216 (7) and 172 (58).
2,3,4,6-Tetra-O-acetyl-b-d-[1-2H,6-2H2]-glucopyranosylisothiouronium bromide
Thiourea (0.33 g, 4.35 mmol) was added to 2,3,4,6-tetra-O-acetyl-b-d-
[1-2H,6-2H2]glucopyranosyl bromide (0.6 g, 1.45 mmol) in dry acetone (5 ml)
Copyright # 2006 John Wiley & Sons, Ltd.
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SYNTHESIS OF GLUCOSINOLATES DEUTERIUM
1209
under nitrogen. The solution was heated at reflux for 15 min then the reaction
mixture allowed to cool. The solvent was removed at reduced pressure and the
crude product purified by column chromatography on silica gel with 40–60
petroleum ether–ethyl acetate (2:3) followed by methanol as eluant, to give
2,3,4,6-tetra-O-acetyl-b-d-[1-2H1,6-2H2]glucopyranosylisothiouronium
bro-
mide as a white crystalline solid (0.7 g, 98%); m.p. 182.5–183.58C; [a]D
ꢁ15.68 (c 1.0 in CH3OH); nmax (nujol)/cmꢁ1 3360–3310 (NH), 1730 (CO); dH
(200 MHz; D2O) 2.10 (3H, s, COCH3), 2.15 (3H, s, COCH3), 2.20 (3H, s,
COCH3), 2.22 (3H, s, COCH3), 4.20 (1H, d, J4,5 10, H-5), 5.15 (1H, t, J3,4
=
J4,5 10, H-4), 5.25 (1H, d, J2,3 10, H-2), 5.40 (1H, t, J2,3 = J3,4 10, H-3); dC
(50.3 MHz; D2O) 23.3–23.5 (4 ꢀ OC(O)CH3), 68.6 (C-4), 70.1 (C-2), 74.1
(C-3), 76.7 (C-5), 170.5 (C=N), 173.2, 173.3, 173.6, 174.4 (4 ꢀ COCH3); m/z
(CI) 490 ([M]+, 7%), 444 (32), 410 (17), 334 (67), 292 (26), 248 (30), 216 (24)
and 172 (25).
2,3,4,6-Tetra-O-acetyl-thio-b-d-[1-2H,6-2H2]glucopyranose (6)
Potassium metabisulfite (0.24 g, 1.26 mmol) was dissolved in water (4 ml) and
heated to 758C. Dichloromethane (5 ml) was added carefully followed
by 2,3,4,6-tetra-O-acetyl-b-d-[1-2H1,6-2H2]glucopyranosylisothiouronium bro-
mide (0.24 g, 1.22 mmol). The biphasic solution was heated under reflux for
15 min then cooled to room temperature. The organic phase was washed with
water (3 ꢀ 5 ml) then the aqueous layer washed with dichloromethane
(2 ꢀ 5 ml). The combined organic layers were dried (MgSO4) and the solvent
evaporated at reduced pressure to give a white solid. After purification using
column chromatography on silica gel with 40–60 petroleum ether–ethyl acetate
(1:1) as eluant, 2,3,4,6-tetra-O-acetyl-thio-b-d-[1-2H,6-2H2]glucopyranose was
obtained as a white crystalline solid (0.127 g, 71%); m.p. 117–1198C;
[a]D ꢁ8.78 (c 1.0 in CH3OH); (Found: C, 45.66; H, 5.45. Calculated for
C14H172H3O9S: C, 45.77; H, 5.49%); nmax (nujol)/cmꢁ1 3460 (SH), 1735 (CO);
dH (200 MHz; CDCl3) 2.00 (3H, s, OC(O)CH3), 2.02 (3H, s, OC(O)CH3), 2.10
(3H, s, OC(O)CH3), 2.12 (3H, s, OC(O)CH3), 3.70 (1H, d, J4,5 10, H-5), 4.98
(1H, d, J2,3 10, H-2), 5.09 (1H, t, J3,4 = J4,5 10, H-4), 5.18 (1H, t, J2,3 = J3,4
10, H-3); dC (50.3 MHz; CDCl3) 21.1–21.3 (4 ꢀ OC(O)CH3), 68.5 (C-4), 73.9
(C-2), 74.0 (C-3), 76.6 (C-5), 169.9, 170.2, 170.6, 171.2 (4 ꢀ OC(O)CH3); m/z
(CI) 368 ([MH]+, 2%), 334 (100, [M-SH]+), 274 (29) and 172 (62).
2,3,4,6-Tetra-O-acetyl-b-D-[1-2H,6-2H2]desulphogluconasturtiin
Hydrocinnamaldehyde oximyl chloride (0.06 g, 0.32 mmol) was suspended in
dry diethyl ether (3.0 ml) and a solution of 2,3,4,6-tetra-O-acetyl-thio-b-d-
[1-2H1,6-2H2]glucopyranose (0.052 g, 0.14 mmol) in dry diethyl ether (1.5 ml)
was added. The reaction was treated with dry triethylamine (0.3 ml) and stirred
Copyright # 2006 John Wiley & Sons, Ltd.
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1210
A. A. B. ROBERTSON AND N. P. BOTTING
overnight. 1 M sulfuric acid (5 ml) was added to the reaction and the solution
extracted using ethyl acetate (3 ꢀ 15 ml). The organic extracts were dried
(MgSO4) and the solvent evaporated at reduced pressure to give a white solid.
The product was purified by column chromatography on silica gel using 40–60
petroleum ether–ethyl acetate (3:2) as the eluant. 2,3,4,6-Tetra-O-acetyl-b-d-
[1-2H1,6-2H2]desulfogluconasturtiin was obtained as a white solid (0.075 g,
100%); m.p. 152–1548C; [a]D ꢁ19.68 (c 1.0 in CHCl3); (Found: C, 52.66; H,
6.08; N, 2.74. Calculated for C23H262H3NO10S.0.5H2O: C, 52.76; H, 5.78; N,
2.68%); nmax (nujol)/cmꢁ1 3300 (OH), 1750 (C=O); dH (300 MHz; CDCl3)
1.90–2.10 (4 ꢀ 3H, 4 s, 4 ꢀ OC(O)CH3), 2.70–3.00 (4H, m, H-8, 9), 3.65 (1H, d,
J4,5 10, H-5), 4.96–5.00 (2H, 2t, J 10, H-2, 4), 5.20 (1H, t, J 10, H-3), 7.10–7.30
(5H, m, phenyl); dC (75.45 MHz; CDCl3) 20.5–20.6 (4 ꢀ OC(O)CH3), 33.2
(CH2), 34.3 (CH2), 68.1 (C-4), 70.1 (C-2), 73.7 (C-3), 76.0 (C-5), 126.7 (C-40),
128.4 (C-30, 50), 128.8 (C-20, 60), 140.6 (C-10), 152.0 (C-7), 169.4, 169.5, 170.4,
170.8 (4 ꢀ OC(O)CH3); m/z (EI) 515 ([MH]+, 5%), 334 (47), 243 (61), 172
(30), 150 (64), 132 (1 0 0), 105 (15) and 91 (27, [PhCH2]+).
b-D-[10-2H, 60-2H2]desulfogluconasturtiin (11)
To
2,3,4,6-tetra-O-acetyl-b-d-[10-2H,60-2H2]desulfogluconasturtiin
(0.5 g,
0.98 mmol) in methanol was added a catalytic amount of potassium metal
under nitrogen. The reaction was stirred for 18 h then Amberlite IR-120 resin
was added. Stirring was continued for a further 15 min before the Amberlite
was removed by filtration and the solvent removed by evaporation at reduced
pressure to give an extremely hygroscopic white amorphous solid (0.057 g,
64%); m.p. 78–808C; [a]D ꢁ50.38 (c 1.0 in CH3OH); (Found: C, 49.62; H, 6.00;
N, 3.58. Calculated for C15H182H3NO6S.0.8H2O: C, 49.87; H, 6.32; N,
3.88%); nmax (nujol)/cmꢁ1 3300 (OH); dH (300 MHz; CD3OD) 3.00–3.20 (4H,
m, CH2CH2), 3.60–3.80 (4H, m, H-2, 3, 4, 5), 7.25 (5H, s, phenyl); dC
(75.45 MHz; CD3OD) 34.8 (CH2), 35.3 (CH2), 71.3 (C-4), 74.5 (C-2), 79.8 (C-
5), 82.2 (C-3), 127.4 (C-40), 129.6, 129.7 (C-20, 30, 50, 60), 142.7 (C-1’), 154.0
(C=N); m/z (CI) 347 ([MH]+, 1%), 150 (11), 131 (46) and 91 (100,
[PhCH2]+).
Acknowledgements
This work was funded through a BBSRC studentship to AABR.
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DOI: 10.1002/jlcr