Synthesis of aromatic and indole alpha-glucosinolates

Article abstract of DOI:10.1016/j.carres.2017.11.004

Aromatic and indole glucosinolates are important members of the glucosinolate family of compounds du to their potential medicinal properties. They are known to exert antioxidant and anti-carcinogenic activity either by the natural products themselves, or their metabolic products including indole-3-carbinol and isothiocyanates. Natural glucosinolates are all β-glucosinolates; however, α-glucosinolates are also promising compounds for medicinal applications and hence have to be produced synthetically for any bio-activity studies. Here we report on the successful synthesis of a series of α-glucosinolates: α-neoglucobrassicin, α-4-methoxyglucobrassicin, 2,3-dichlorophenyl-α-glucosinolate for the first time. Testing for anti-inflammatory properties of these synthetic GLs, however, did not yield the expected activity.

Full text of DOI:10.1016/j.carres.2017.11.004

Carbohydrate Research 455 (2018) 45e53
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of aromatic and indole alpha-glucosinolates
, b,
Quan V. Vo a *, Simone Rochfort c, Pham C. Nam d, Tuan L. Nguyen e, Trung T. Nguyen e,
Adam Mechler f
a Institute of Research and Development, Duy Tan University, Da Nang, Viet Nam
b Department of Natural Sciences, Quang Tri Teachers Training College, Quang Tri Province, Viet Nam
c Department of Primary Industries, Victorian AgriBiosciences Centre, La Trobe University Research and Development Park, 1 Park Drive, Bundoora 3083,
Victoria, Australia
d Department of Chemical Engineering, The University of Da Nang e University of Science and Technology, Viet Nam
e Department of Chemistry, Quy Nhon University, Quy Nhon, Binh Dinh Province, Viet Nam
f Department of Chemistry and Physics, 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:
Aromatic and indole glucosinolates are important members of the glucosinolate family of compounds du
to their potential medicinal properties. They are known to exert antioxidant and anti-carcinogenic ac-
tivity either by the natural products themselves, or their metabolic products including indole-3-carbinol
Received 20 September 2017
Received in revised form
10 November 2017
and isothiocyanates. Natural glucosinolates are all
promising compounds for medicinal applications and hence have to be produced synthetically for any
bio-activity studies. Here we report on the successful synthesis of a series of -glucosinolates: -neo-
glucobrassicin, -4-methoxyglucobrassicin, 2,3-dichlorophenyl- -glucosinolate for the first time. Testing
b-glucosinolates; however, a-glucosinolates are also
Accepted 10 November 2017
a
a
a
a
Keywords:
for anti-inflammatory properties of these synthetic GLs, however, did not yield the expected activity.
© 2017 Elsevier Ltd. All rights reserved.
a-Glucosinolates
Neoglucobrassicin
4-Methoxyglucobrassicin
Tetraacetyl glucopyranosyl thiol
Synthesis
1. Introduction
been reported in several studies [7e18], while
much less attention [14,19]. The first synthesis of
a
-GLs attracted
a-glucobrassicin
Glucosinolates (GLs) are natural compounds found in a large
number of Brassica species such as cabbage, broccoli and canola.
Increasing interest in GLS and their metabolic products is due to
their potential as human cancer-prevention agents [1,2], as well as
crop-protection compounds [3,4] and biofumigants [5,6] in agri-
(
a
-GB) and some other
Muesser et al. [19]. Here we report the successful synthesis of ar-
omatic and indole -GLs, as well as a study of the stability and anti-
a-thiohydroxymates was reported by Blanc-
a
inflammatory activity of these compounds.
culture. Natural GLs display exclusively a
the anomeric carbon. Their counterparts,
N-hydroxysulfates with a side chain (R) and a sulfur-linked
glucopyranose moiety as shown in Fig. 1. -GLs are considered
unnatural. However, the modification is small in terms of molecular
morphology, raising the possibility that the configuration retains,
or improves on, bioactivity; hence, there is an impetus to study the
bioactivity of -GLs and this requires the establishment of suitable
b
-gluco configuration at
-GLs are -thioglucoside
-D-
2. Results and discussion
a
a
a
2.1. Synthesis of 2,3,4,6-tetra-O-acetyl-
a-D-glucopyranosyl thiol
a
From previous studies, it is known that 2,3,4,6-tetra-O-acetyl-
D-glucopyranosyl thiol 1 could be synthesized by three pathways
[19e23]. Dere's group reported the acid-catalyzed reaction of
2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl-trichloroacetimidate
with bis-trimethylsilyl sulfide in the presence of trimethylsilyl tri-
a-
a
a
synthetic pathways.
Synthesis and biological activity of aromatic and indole GLs have
fluoromethanesulfonate to yield a mixture of
a/
b-isomers in 65%
yield [22]. However, this approach lead to low
a-isomer yield and
difficulties in purification. Therefore, it was decided to synthesize
the thiol 1 by Floyd's pathway [23], and Fujihira's pathway
[21,24,25].
* Corresponding author. Institute of Research and Development, Duy Tan Uni-
versity, Da Nang, Viet Nam.
E-mail addresses: quan_vv@qtttc.edu.vn, vovanquan1980@gmail.com (Q.V. Vo).
https://doi.org/10.1016/j.carres.2017.11.004
0008-6215/© 2017 Elsevier Ltd. All rights reserved.

46
Q.V. Vo et al. / Carbohydrate Research 455 (2018) 45e53
Fig. 1. The structure of a/b-GLs.
Initially, the thiol 1 was synthesized by Fujihira's pathway
(Scheme 1) [21,24]. The acetylation of D-glucose was achieved with
acetic anhydride in pyridine and catalyzed by N,N-dimethylami-
nopyridine (DMAP) to form 3 in 90% yield. Compound 3 was then
treated with HBr/HOAc in DCM to yield the a-bromide 4, which was
used directly for the next steps. From the previous study [21], the
reaction of 4 with N,N-dimethylthioformamide was carried out in
the presence of 0.2% water at 100 ꢀC for 5 min, and then cooled to rt
and methanolysed to yield a mixture of (1)a/b(5)-isomers (1:1, by
NMR).
Scheme 2. Synthesis of 1. Reagents and conditions: a) Ac2O, NaOAc, reflux, 20 min; b)
PCl5, BF3.Et2O, DCM, 10 min; c) KSAc, DMPU, 0e5 ꢀC, 3 d; d) AcOHgPh, EtOH, reflux,
2 h; e) H2S, EtOH, 50e60 ꢀC, 30 min.
However, attempts to purify the
a-isomer from the mixture of a/
b
-isomers by column chromatography either as described in the
literature [21] or by HPLC were unsuccessful. The results suggest
that the thiol 1 should be synthesized by more selective pathways.
Therefore, the 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl thiol
(Scheme 2) was created following Floyd's pathway (Scheme 2)
[19,23].
the reaction temperature was lowered to 0e5 ꢀC to reduce side
products and improve the yield. After work-up and purification by
silica gel column chromatography eluting with hexane/ethyl ace-
tate, the compound 8 was obtained in 63% yield. Compound 8 was
then reacted with phenylmercuric (II) acetate in absolute EtOH for
2 h at reflux temperature to yield the phenylmercury (II) thio de-
rivative 9 in 51% yield. The thiol 1 was obtained in 92% yield, at the
last step, by bubbling hydrogen sulfide into a solution of 9 in EtOH
at 60 ꢀC. As a result, the compound 1 was formed stereospecifically
in 26% overall yield over 5 steps.
The 2,3,4,6-tetra-O-acetyl-b-D-glucopyranose 6 was easily
synthesized in 98% yield [26]. The pentaacetate was then used
directly in the next step that involved treatment with phosphorus
pentachloride in the presence of BF3.Et2O in DCM in an argon at-
mosphere at rt to yield b-glucopyranosyl chloride 7 in 90% yield. It
was found that the chloride was not stable in the presence of silica
gel, thus purification by re-crystallization from diethyl ether/hex-
ane was used instead. The S-acetylation by treatment with potas-
sium thioacetate in 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-
pyrimidinone (DMPU) in an atmosphere of argon for around 3 days
was not easy because of the low yield and side products [19]. Thus,
The NMR, optical rotation and melting point data confirmed the
difference between the
and SH signals in the -thiol 5 are
2.29 (d, J1,SH ¼ 9.9 Hz) [27], respectively, while those of the
b- and a
-isomers. The 1H-NMR data of H1
b
d
4.51 (t, J1,2 ¼ J1,SH ¼ 9.9 Hz) and
-thiol 1
¼ 5.7 Hz) and 1.90 (d, J1, ¼ 5.7 Hz),
a
are
d
5.82 (t, J1,2 ¼ J1,
SH
SH
respectively. Furthermore, the ½a D20
ꢁ
of
a
-thiol 1 is þ 169.5 (c ¼ 1.0,
CHCl3), whereas that of the
b
-thiol is þ10.5 (c ¼ 1.0, CHCl3). The
melting point of the
for the -thiol [27].
a
-thiol is 94e95 ꢀC, compared with 114e115 ꢀC
b
2.2. Synthesis of
a-thiohydroxymates
2.2.1. Synthesis of aromatic
a-thiohydroxymates
In a previous study [16], 3,4-dimethoxyphenyl glucosinolate
was found to be the most active anti-inflammatory of the glucosi-
nolates assayed. Thus 3,4-dimethoxyphenyl glucosinolate and two
other aromatic GLs were chosen for synthesis and to benchmark for
anti-inflammatory activity. The aromatic hydroxymoyl chlorides
12a-c were synthesized following the aldoxime pathway in excel-
lent yields (80e97% over two steps from the aldehydes 10a-c) [16].
The coupling of aromatic hydroxymoyl chlorides with the thiol 1
was possible via a general method (Scheme 3) [10,11,19]. The aro-
matic hydroxymoyl chlorides 12a-c were reacted with the thiol 1 in
the presence of triethylamine catalyst in DCM:Et2O (1:2) to yield
Scheme 1. Synthesis of the thiols 1 and 5 following Fujihira's pathway. Reagents and
conditions: a) Ac2O, Pyridine, DMAP, 6 h; b) HBr/HOAc, DCM, 1 h; c) N,N-dimethylth-
ioformamide, MeOH.
the a-thiohydroxymates 13a-c in 53e86% yield.

Q.V. Vo et al. / Carbohydrate Research 455 (2018) 45e53
47
Scheme 3. Synthesis of aromatic
a
-thiohydroxymates 13a-c. Reagents and conditions: a) NH2OH.HCl, MeOH, pyridine, 2.5 h; b) NCS, DMF; c) 1, Et3N, DCM:Et2O (1:2), 2 h.
2.2.2. Synthesis of indolyl
a
-thiohydroxymates
It was found that the a-thiohydroxymates were not very stable,
It was shown that neo-glucobrassicin (NGB) and 4-
thus the compounds should be used directly in the next step after
purification by column chromatography with silica gel eluting with
hexane/EtOAc or DCM/MeOH. This is likely the main reason for the
methoxyglucobrassicin
inflammatory activity at low concentrations [17]. Thus,
and
inflammatory activity. It was found that the nitronate pathway is
the most convenient method to synthesize the indole -thiohy-
(MGB)
exerted
significant
anti-
a
-NGB
a-MGB were chosen for synthesis and for testing their anti-
low yields of the
-thiohydroxymates. Comparison of the optical rotation of
hydroxymates and -thiohydroxymates indicated that the values of
the -isomers are generally higher than those of -isomers, while
the values of melting points of -isomers are lower than those of
isomers. The values of -isomers are
(1H NMR data of H1) of the
higher than that of the corresponding -isomers (Table 1). The
NMR, optical rotation and melting point results confirmed that the
synthetic compounds 13a-e were -isomers. It was shown before
that this coupling pathway is stereospecific in that only the (Z)-
a-thiohydroxymates compared with those of the
b
a-thio-
a
b
droxymates [17]. The conventional Henry reaction of the aldehydes
14 and 15 with nitromethane in the presence of ammonium acetate
at reflux for 2 h yielded the nitroalkenes 16 and 17 in 78% and 99%
yields, respectively (Scheme 4). The nitrovinyl group was then
reduced with NaBH4 in THF/MeOH to form the nitroalkanes 18 and
19 in 77e79% yield [28]. The nitroalkanes 18 and 19 were then
reacted with sodium methoxide in MeOH to make sodium nitro-
nate derivatives which were treated with sulfonyl chloride in DME
at ꢂ40 ꢀC to convert them to (indol-3-yl)acetohydroxymoyl chlo-
rides. The coupling of the indole hydroximoyl chlorides and thiol 1
was performed by a general method in DCM in the presence of Et3N
a
b
a
b-
d
a
b
a
isomers form [8]. X-ray analysis of S-(2,3,4,6-tetra-O-acetyl-
b
-D-
glucopyranosyl)-2,3-dichlorophenylacetothiohydroxymate
also
determined that the thiohydroxymate can be obtained as a (Z)-
thiohydroxymate [29].
to make indole
a-thiohydroxymates 13d and 13e in 40% and 36%
yield, respectively from the nitroalkanes. Consequently, the
ohydroxymates 13d and 13e were obtained in 24% and 28% overall
yield, respectively (Scheme 4).
a
-thi-
2.3. Synthesis of
a
-glucosinolates
-thiohydroxymates 13a-e by the typical
The sulfation of
a
method used pyridine-sulfur trioxide complex in DCM or pyridine
as the solvent and then treatment with an aqueous solution of
potassium hydrogen carbonate (Scheme 5) [16,17,27]. The products
were purified by flash column chromatography with silica gel
eluting with DCM/MeOH and were obtained in good yield (69e87%
yield).
The de-O-acetylation of 20a-e was carried out in MeOH with
MeOK catalyst. The final
flash chromatography on silica gel eluting with EtOAc/MeOH/H2O
in excellent yields (80e95% yield) [29]. Three -GLs, which include
2,3-dichlorophenyl- -glucosinolate 21b, -NGB 21d, and -MGB
21e were synthesized for the first time in 15%, 7% and 6% overall
yield (over 8 steps from glucose), respectively. Two other -GLs
(21a, 21c) could not been obtained because of the decomposition in
the last step. It was shown that the overall yields of synthetic -GLs
were lower than those of -GLs [16,17]. The reasons for this may be
the instability of the intermediates in the synthetic process and of
the final -GLs. The final products were recrystallised to ensure
a-GLs 21b, 21d and 21e were obtained by
a
a
a
a
a
a
b
a
compound purity before conducting bioassays [29].
2.4. Biological studies
Following the methodology used in previous works on aromatic
and indole GLs [16,17], the anti-inflammatory properties of the
synthetic a-GLs were tested using a bioassay based on the THP-
1 cell line [30]. Lipopolysaccharides (LPS) were used to trigger an
inflammative response in a tissue culture. LPS originate from Gram
negative bacteria where they cover the outer membrane of the
Scheme 4. Synthesis of indole a-thiohydroxymates 13d-e. Reagents and conditions: a)
MeNO2, AcONH4, reflux, 2 h; b) NaBH4, THF, MeOH, rt, 6 h; c) (i) MeONa, MeOH; (ii)
SOCl2, DME, ꢂ40 ꢀC; (iii) 1, DCM/Et2O, Et3N.

48
Q.V. Vo et al. / Carbohydrate Research 455 (2018) 45e53
Table 1
Comparison of the yield, melting point and optical rotation and 1H-NMR data of H1 of
b
- and
a-thiohydroxymates.
Group (R)
Thiohydroxymates
) [16]
Yield (%)
Mp (ꢀC)
½
a D
ꢁ
H1 d
3,4-dimethoxyphenyl
(b
96
57
90
86
83
53
46
40
45
36
60e61
45e46
172e173
72e74
147e148
56e57
65e67
59e60
93e95
69e71
þ26.6
þ132.8
þ22.0
þ144.5
þ31.4
þ147.9
þ10.0
þ115.7
ꢂ3.0
4.58e4.55 (m)
5.95 (d, J ¼ 6.0)
4.14e4.03 (m)
5.46 (d, J ¼ 5.7)
5.53e4.48 (m)
5.90 (d, J ¼ 5.7)
5.55(d,J ¼ 10.2)
6.22 (d, J ¼ 5.7)
5.13 (d, J ¼ 9.6)
6.12 (d, J ¼ 6.0)
13a (
) [16]
13b (
) [16]
13c (
) [17]
13d (
) [17]
13e (
a)
2,3-dichlorophenyl
(b
a
)
4-bromophenyl
(b
a
)
(1-methoxyindol-3-yl) methyl
(4-methoxyindol-3-yl) methyl
(b
a
)
(b
a
)
þ122.2
Scheme 5. Sulfation and de-O-acetylation of the thiohydroxymates 13a-e. Reagents and conditions: a) (i) Pyr.SO3, DCM or pyridine, 18 h, (ii) KHCO3/H2O; b) MeOK/MeOH, 3 h.
bacteria cell. When the monocytes are treated with this bacterial
product an innate immune response is activated which leads to the
tetra-O-acetyl-3,4-dimethoxyphenyl-
cessfully synthesized for the first time. The study found that the
synthetic -GLs exert very weak anti-inflammatory effect in a
bioassay, in contrast to the high inhibition of the corresponding
synthetic -GLs [16,17]. Thus, the results suggest that future efforts
should focus on -GLs as well as natural GLs.
a-glucosinolate) were suc-
release of cytokines including tumor necrosis factor-
In the THP-1 cell assay LPS were used to stimulate this immune
response and the resultant TNF- was measured by ELISA. Anti-
inflammatory activity was measured as the inhibition of the
release of TNF- . To account for any variations in the plate condi-
a
(TNF-
a) [31].
a
a
b
b
a
tions (e.g. cell density) individual references were recorded on each
plate.
4. Experimental
Wells containing THP-1 cells were individually treated in
replicate with compounds 21b-e at different concentrations for
4.1. General methods
4 h at an LPS concentration of 50 mg/L. For each plate LPS controls
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
were measured. The results are summarized in Fig. 2. As a positive
control catechin, an anti-inflammatory agent was used that is
known for a wide range of cardio and chemoprotective effects
[30,32,33]. The catechin control had a strong anti-inflammatory
response (37% inhibition) at a concentration of 0.5 mM (P < 0.05).
The anti-inflammatory effect of the GLs was compared to both LPS
d-line (589 nm); [a]
D values are given in 10ꢂ1 degcm2gꢂ1. Infrared
spectra (nmax) were recorded on a 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.
and catechin controls.
It was shown that in the presence of
slightly inhibited while catechin had a much stronger effect (ꢃ42%
at concentration 15 M of catechin). Hence, the results of anti-
inflammatory activity of -GLs appear to suggest that these un-
a-GLs, TNF-a secretion was
Chemical shifts were recorded as
d values in parts per million
m
(ppm). Spectra were acquired in deuterated chloroform (CDCl3) at
300 K unless otherwise stated. For 1H NMR spectra recorded in
a
natural compounds have lesser biological activity than natural
isomers.
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/second 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
3. Conclusion
2,3,4,6-Tetra-O-acetyl-
synthesized at reasonable yield. The compound was used for the
synthesis of -GLs. A series of -GLs (2,3-dichlorophenyl- -gluco-
sinolate, -MGB, -NGB) and intermediates (potassium 2,3,4,6-
tetra-O-acetyl-4-bromophenyl- -glucosinolate, potassium 2,3,4,6-
a-D-glucopyranosyl thiol was re-
a
a
a
a
a
a

Q.V. Vo et al. / Carbohydrate Research 455 (2018) 45e53
49
Fig. 2. Effects of synthetic GLs on TNF-a secretion in LPS-stimulated THP-1 cells.
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 5 l/min.
products were 2,3,4,6-tetra-O-acetyl-
(650 mg, 72%), ratio (1):
b
/
a
-D-glucopyranosyl thiols
m
a
b
(5) ¼ 1:1 (by NMR) as a colourless syrup.
The heated capillary was maintained at 320 ꢀC with a source heater
temperature of 350 ꢀC and the sheath, auxiliary and sweep gases
were at 40, 15 and 8 units, respectively. The 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-
4.2.2. Floyd's pathway [23]
NaOAc (1.58 g, 19.27 mmol) was suspended in Ac2O (21 mL,
222 mol) and the resulting mixture was heated to reflux for 20 min.
The heating bath was removed and D-glucose 2 (3.17 g, 17.55 mmol)
was added in small portions, keeping the reaction mixture just at
reflux temperature. Subsequently, the mixture was stirred for an
additional 15 min under heating and, after cooling to rt, it was
poured onto crushed ice (80 mL). After keeping mixture in the
refrigerator at 4 ꢀC for 3 h, the resulting precipitate was filtered and
washed with ice-water (125 mL) and cold EtOH (10 mL). After
drying under reduced pressure, the crude product was recrystal-
raphy was performed on silica gel 60 particle size 0.040e0.063
(230e400 mesh).
mm
4.2. 2,3,4,6-Tetra-O-acetyl-
a-D-glucopyranosyl thiol (1)
lised from EtOH (20 mL) to yield 1,2,3,4,6-penta-O-acetyl-
b-D-
glucopyranoside 6 (6.71 g, 98%). To pentaacetyl- -D-glucopyranose
b
4.2.1. Fujihira's pathway [21]
6 (6.71 g, 17.2 mmol) and phosphorus pentachloride (3.89 g,
To a solution of D-glucose 2 (3.00 g, 16.6 mmol) in dry pyridine
(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 became 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 (3 ꢄ 45 mL), evapora-
tion of the solvent and co-evaporations with dry toluene
(3 ꢄ 20 mL), peracetylated glucose was obtained as a yellow solid
(5.84 g, 90%). A solution of pentaacetyl-D-glucopyranose 3 (1.12 g,
2.9 mmol) in DCM (20 mL) was stirred in an ice bath while HBr/
HOAc (6 mL, 45 wt%) was added drop-wise. After an hour, the so-
lution was washed with ice-water and cold saturated NaHCO3 so-
lution, dried over MgSO4 and concentrated to leave the glucosyl
bromide 4 as a pale yellow oil (1.03 g). The freshly prepared 4
(1.03 g, 2.5 mmol) and N,N-dimethylthioformamide (230 mg,
2.6 mmol) in the presence of 0.2 wt% H2O were stirred under an
argon stream at 100 ꢀC for 5 min. Dry MeOH (20 mL) was added
after cooling to rt, and the mixture was stirred for about 10 min
(until the solid dissolved). The solvent was evaporated under
reduced pressure, and the precipitated product was separated and
purified by flash column chromatography on silica gel eluting with
66% hexane/EtOAc and then hexane:DCM:MeOH (25:24:1). The
18.92 mmol) in DCM (50 mL) in an atmosphere of argon was added
BF3.Et2O (25 ml). The reaction was stirring for 30 min at rt in an
atmosphere of argon (monitoring by TLC), and then was partitioned
between DCM (50 mL) and H2O (100 mL). The aqueous phase was
re-extracted with DCM (2 ꢄ 40 mL). The combined organics were
washed with sodium hydrogen carbonate (100 mL), brine (100 mL),
dried over MgSO4, filtered and concentrated in vacuo. The resulting
solid was co-evaporated with toluene, triturated under cyclo-
hexane and crystallized from diethyl ether/petrol to afford 2,3,4,6-
tetra-O-acetyl-
white crystalline solid. To a solution of 2,3,4,6-tetra-O-acetyl-
glucopyranosyl chloride 7 (5.67 g, 15.48 mmol) in DMPU (15 mL) at
ꢀC under an atmosphere of argon was added potassium thio-
b
-D-glucopyranosyl chloride 7 (5.67 g, 90%) as a
b
-D-
0
acetate (2.32 g, 17.05 mmol). The reaction mixture was stirring for 3
days under an atmosphere of argon at 0 ꢀC (monitoring by TLC) and
then was partitioned between ethyl acetate (50 mL) and H2O
(50 mL). After extraction with EtOAc (3 ꢄ 50 mL), the organic layers
were washed with brine, dried over MgSO4, filtered and concen-
trated in vacuo. The resulting residue was purified by silica gel
chromatography eluting with 70% hexane/EtOAc to afford 1-S-
acetyl-2,3,4,6-tetra-O-acetyl-1-thio-a-D-glucopyranoside 8 (3.96 g,
63%) as a peach-coloured solid. A mixture of 8 (3.96 g, 9.76 mmol)
and phenylmercuric acetate (3.48 g, 10.24 mmol) in absolute
ethanol (40 mL) was boiled under reflux for 2 h. The resulting black

50
Q.V. Vo et al. / Carbohydrate Research 455 (2018) 45e53
0
0
0
precipitate was filtered off (Celite). The filtrate was evaporated to
dryness on a rotary evaporator and the resulting syrup was purified
by column chromatography eluting with 60% hexane/EtOAc to yield
9 (3.19 g, 51%) as a solid foam. Hydrogen sulfide was bubbled for
J ¼ 1.8 Hz, J4 ,5 ¼ 7.8 Hz,1H, H5 -Ph-H), 5.46 (d, J1,2 ¼ 5.7 Hz,1H, H1),
5.34 (t, J2,3 ¼ J3,4 ¼ 9.6 Hz, 1H, H3), 4.98 (t, J3,4 ¼ J4,5 ¼ 9.6 Hz, 1H,
H4), 4.88 (dd, J ¼ 5.7 Hz, 1H, H2), 4.36e4.29 (m, 1H, H5), 4.25 (dd,
J5,6b ¼ 3.6 Hz, J6a,6b ¼ 12.3 Hz, 1H, H6b), 4.01 (dd, J5,6b ¼ 2.1 Hz,
J6a,6b ¼ 12.3 Hz, 1H, H6a), 2.06, 2.04, 1.99, 1.98 (4 ꢄ s, 12H,
20
min
into
a
solution
of
2,3,4,6-tetra-O-acetyl-1-
phenylmercury(II)-thio-a-D-glucopyranose 9 (3.19 g, 4.98 mmol)
CH3COO ꢄ 4); 13C NMR (75 MHz, CDCl3) (300 K)
d 170.2, 169.5,
in EtOH at 55 ꢀC. The resulting precipitate was removed by filtration
and the filtrate was purified by column chromatography eluting
with 60% hexane/EtOAc. The compound 1 was obtained as a white
solid (1.67 g, 92% (26% overall yield)). Rf ¼ 0.45 in 60% hexane/
169.4, 169.1 (4 ꢄ CH3COO), 149.3 (C¼N), 133.5 (C-3 of Ph), 132.2,
132.1 (C-2 and C-1 of Ph), 131.6 (C-4 of Ph), 129.2 (C-6 of Ph), 127.1
(C-5 of Ph); 79.9 (C-1), 69.9 (C-5), 69.6 (C-3), 68.4 (C-2), 67.5 (C-4),
61.1 (C-6), 20.3(2), 20.2(2) (4 ꢄ CH3COO); HRMS (ESI) m/z for
EtOAc; mp ¼ 94e95 ꢀC (Lit. 92e93 ꢀC) [20]; ½a 2D0
ꢁ
¼ þ169.5 (c ¼ 1.0,
C
21H23NaO10Cl2NS [MþNa]þ, calcd 574.0317, found 574.0211.
CHCl3), (Lit. þ168 [20], þ166.3 [34]); 1H NMR (300 MHz, CDCl3)
(300 K)
d
5.82 (t, J1,2 ¼ J1,SH
¼
5.7 Hz, 1H, H1), 5.27 (t,
4.3.3. S-(2,3,4,6-Tetra-O-acetyl-a-D-glucopyranosyl)-4-
bromophenylacetothiohydroximate (13c)
J3,4 ¼ J2,3 ¼ 9.9 Hz, 1H, H3), 4.97 (dd, J3,4 ¼ 9.9 Hz, J4,5 ¼ 10.2 Hz, 1H,
H4), 4.92 (dd, J1,2 ¼ 5.7 Hz, J2,3 ¼ 9.9 Hz, 1H, H2), 4.35e4.29 (m, 1H,
H5), 4.22 (dd, J5,6b ¼ 4.2 Hz, J6a,6b ¼ 12.3 Hz, 1H, H6b), 4.01 (dd,
J5,6a ¼ 2.1 Hz, J6a,6b ¼ 12.3 Hz, 1H, H6a), 1.97e1.93 (4 ꢄ br s, 12H,
4 ꢄ CH3COO), 1.90 (d, J1,SH ¼ 5.7 Hz, 1H, SH); 13C NMR (75 MHz,
Pure 13c was obtained as a white solid (246 mg, 53%). Rf ¼ 0.32
in 60% hexane/EtOAc; mp ¼ 56e57 ꢀC; ½a D20
¼ þ147.9 (c ¼ 3.3,
ꢁ
DCM); IR (NaCl) nmax 3359 (OH), 1750 (C¼O), 1599 (C¼N); 1H NMR
(300 MHz, CDCl3) (300 K)
d
7.54 (d, J ¼ 8.4 Hz, 2H, H20 and H60-Ph-
CDCl3) (300 K)
d
170.2, 169.8, 169.2, 168.9 (4 ꢄ CH3COO), 76.7 (C-1),
H), 7.44 (dd, J ¼ 8.4 Hz, 2H, H30 and H50-Ph-H), 5.90 (d, J1,2 ¼ 5.7 Hz,
1H, H1), 5.45 (t, J2,3 ¼ J3,4 ¼ 9.9 Hz, 1H, H3), 5.01e4.95 (m, 2H, H2
69.8 (C-5), 69.4 (C-2), 67.9 (C-3, C-4), 61.6 (C-6), 20.4, 20.2(2), 20.1
(4 ꢄ CH3COO); HRMS (ESI) m/z for C14H20NaO9S [MþNa]þ, calcd
387.0726, found 387.0721.
and H4), 4.41e4.38 (m, 1H, H5), 4.19 (dd, J5,6b
¼
4.8 Hz,
J6a,6b ¼ 12.3 Hz,1H, H6b), 4.01 (dd, J5,6b ¼ 2.4 Hz, J6a,6b ¼ 12.3 Hz,1H,
H6a), 2.08, 2.02, 2.01, 2.00 (4 ꢄ s, 12H, CH3COO); 13C NMR (75 MHz,
4.3. General procedure for the preparation of aromatic
thiohydroximates (13a-c)
CDCl3) (300 K)
d
170.3, 169.5, 169.4, 169.2 (4 ꢄ CH3COO), 151.2
(C¼N), 131.7 (C-3, C-5 of Ph),131.5 (C-4 of Ph), 129.3 (C-2, C-6 of Ph),
124.3 (C-1 of Ph), 81.7 (C-1), 70.1 (C-5), 69.9 (C-3), 68.7 (C-2), 67.8
(C-4), 61.2 (C-6), 20.3(2), 20.2, 20.1 (4 ꢄ CH3COO); HRMS (ESI) m/z
for C21H25O10BrNS [MþH]þ, calcd 562.0377, found 562.0376.
To stirred a solution of hydroximoyl chloride 12a-c (the com-
pounds 12a-c were synthesized following the literate [16]) (1.5 eq.)
in dry Et2O:DCM (2:1, 45 mL) was added a solution of 2,3,4,6-tetra-
O-acetyl-
a
-D-glucopyranosylthiol 1 (1 eq.) in dry DCM (6 mL). The
4.4. General procedure for the preparation of indole
thiohydroximates (13d,e)
resulting mixture was treated with Et3N (6 eq.) in Et2O (12 mL). The
reaction mixture was stirred for 2 h at rt under N2 then acidified
with 1 M H2SO4 (7 mL/mmol of sugar). The mixture was left to
stand for 10 min and then separated. The aqueous phase was
extracted with DCM (3 ꢄ 30 mL). The combined organic layers were
dried over MgSO4, filtered and the filtrate was concentrated under
reduced pressure. The thiohydroxymate 13a-c was obtained by
flash chromatography eluting with 0e3% MeOH/DCM.
4.4.1. 2,3,4,6-Tetra-O-acetyl-a-D-glucopyranosyl-((1-
methoxyindole-3-yl)methyl) thiohydroximate (13d)
To a stirred solution of the indole nitroalkane 18 (the compound
18 was prepared following the literature [17]) (264 mg,1.2 mmol) in
dry MeOH (15 mL) in a nitrogen atmosphere was added sodium
methoxide (130 mg, 2.4 mmol). After 20 min the reaction was
concentrated at reduced pressure giving the nitronate as a white
solid which was dried under a high vacuum for 15 min. The
nitronate was cooled to ꢂ40 ꢀC in a nitrogen atmosphere. Then it
was treated with dry DME (20 mL) at ꢂ40 ꢀC. A solution of thionyl
chloride (0.23 mL, 3.12 mmol) in dry DME (5 mL) at ꢂ40 ꢀC was
added to the nitronate dropwise to give a burgundy coloured so-
lution. After 30 min at ꢂ40 ꢀC, water (30 mL) was added and the
solution was extracted with DCM (3 ꢄ 50 mL). The organic extracts
were dried (MgSO4) and concentrated under reduced pressure to
give (1-methoxyindol-3-yl) acethydroximoyl chloride which was
left under a high vacuum for 15 min and then it was reacted directly
in the next step. The indole hydroximoyl chloride in dry Et2O:DCM
(2:1, 15 mL) was treated with a solution of 2,3,4,6-tetra-O-acetyl-l-
4.3.1. S-(2,3,4,6-Tetra-O-acetyl-a-D-glucopyranosyl)-3,4-
dimethoxyphenyl thiohydroximate (13a)
Pure 13a was obtained as a white solid (340 mg, 57%). Rf ¼ 0.28
in 40% hexane/EtOAc; mp ¼ 45e46 ꢀC; ½a D20
¼ þ132.8 (c ¼ 2.55,
ꢁ
DCM); IR (NaCl) nmax 3420 (OH), 2959 (CH3OPh), 1750 (C¼O), 1601
(C¼N), 1226, 1041 cmꢂ1
; d 7.14
1H NMR (300 MHz, CDCl3) (300 K)
(d, J5 ,6 ¼ 8.4 Hz, 1H, H60-Ph-H), 7.05 (s, 1H, H20-Ph-H), 6.86 (d,
0
0
J5 ,6 ¼ 8.4 Hz, 1H, H50-Ph-H), 5.95 (d, J1,2 ¼ 6.0 Hz, 1H, H1), 5.44 (t,
J2,3 ¼ J3,4 ¼ 9.6 Hz, 1H, H3), 5.00e4.96 (m, 2H, H2 and H4),
4.41e4.35 (m, 1H, H5), 4.19 (dd, J5,6b ¼ 4.5 Hz, J6a,6b ¼ 12.3 Hz, 1H,
H6b), 4.01 (dd, J5,6b ¼ 2.3 Hz, J6a,6b ¼ 12.3 Hz, 1H, H6a), 3.87, 3.85
0
0
(2 ꢄ s, 6H, CH3OPh), 2.05, 2.00, 1.99, 1.98 (4 ꢄ s, 12H, CH3COO); 13
C
NMR (75 MHz, CDCl3) (300 K)
d
170.3, 169.5, 169.4, 169.2
thio-a-D-glucopyranose 1 (440 mg, 1.2 mmol) and dry triethyl-
(4 ꢄ CH3COO), 150.4 (C¼N and C-4 of Ph), 148.6 (C-3 of Ph), 124.9
(C-1 of Ph), 121.1 (C-2 of Ph), 110.5 (C-5 and C-6 of Ph), 80.4 (C-1),
70.2 (C-5), 70.1 (C-3), 68.6 (C-2), 67.8 (C-4), 61.3 (C-6), 55.5
(2 ꢄ CH3OPh), 20.2(2) 20.1(2) (4 ꢄ CH3COO); HRMS (ESI) m/z for
amine (1.0 mL, 7.2 mmol) in dry DCM (10 mL). The reaction was
stirred for 2 h giving an orange solution. The reaction mixture was
acidified with 1 M H2SO4 (7 mL/mmol sugar) and then it was
extracted using DCM (3 ꢄ 50 mL). The organic extracts were dried
(MgSO4) and concentrated under reduced pressure. The compound
13d was obtained as foam solid (0.27 g, 40%) by running flash
chromatography on silica gel eluting with 50% hexane/EtOAc.
C
23H28O12NS [M-H]-, calcd 542.1332, found 542.1329.
4.3.2. S-(2,3,4,6-Tetra-O-acetyl-a-D-glucopyranosyl)-2,3-
dichlorophenyl thiohydroximate (13b)
Rf ¼ 0.17 in 40% hexane/EtOAc; mp ¼ 59e60 ꢀC; ½a D20
¼ þ115.7
ꢁ
Pure 13b was obtained as a white solid (284 mg, 86%). Rf ¼ 0.45
(c ¼ 2.42, DCM); IR (KBr drift) nmax 3304, 2945, 2836, 1751,
in 40% hexane/EtOAc; mp ¼ 72e74 ꢀC; ½a D20
ꢁ
¼ þ144.5 (c ¼ 2.18,
1600 cmꢂ1; 1H NMR (300 MHz, MeOD) (300 K)
d
7.57 (d, J ¼ 7.5 Hz,
CHCl3); IR (NaCl) nmax 3320 (OH), 1750 (C¼O), 1600 (C¼N) cmꢂ1; 1H
1H, H4i), 7.39 (d, J ¼ 7.5 Hz, 1H, H7i), 7.25 (s, 1H, H2i), 7.20 (t,
J ¼ 7.5 Hz,1H, H6i), 7.05 (t, J ¼ 7.5 Hz,1H, H5i), 6.22 (d, J ¼ 5.7 Hz,1H,
H1), 5.32 (t, J2,3 ¼ J3,4 ¼ 9.6 Hz, 1H, H3), 4.98e4.85 (m, 2H, H2 and
0
0
NMR (300 MHz, CDCl3) (300 K)
d
7.55 (dd, J ¼ 1.8 Hz, J4 ,5 ¼ 7.8 Hz,
1H, H40-Ph-H), 7.25 (t, J5 ,6 ¼ 7.8 Hz, 1H, H60-Ph-H), 7.20 (dd,
0
0

Q.V. Vo et al. / Carbohydrate Research 455 (2018) 45e53
51
H4), 4.22e4.19 (m, 1H, H5), 4.12e4.03 (m, 4H, CH3O and H6b),
3.98e3.74 (m, 3H, CH2C¼N and H6a), 2.01, 1.98, 1.94, 1.78 (4 ꢄ s,
6 of Ph), 81.4 (C-1), 70.1 (C-5), 69.9 (C-3), 68.9 (C-2), 67.6 (C-4), 61.1
(C-6), 54.8, 54.7 (2 ꢄ CH3OPh), 18.9(2), 18.7(2) (4 ꢄ CH3COO); HRMS
(ESI) m/z for C23H28O15NS2 [M-K]-, calcd 622.0906, found 622.0998.
12H, 4 ꢄ CH3COO); 13C NMR (75 MHz, MeOD) (300 K)
d 170.4, 169.7,
169.4, 169.0 (4 ꢄ CH3COO), 147.5 (C¼N), 132.2 (C-8i), 122.9 (C-2i),
121.8 (C-6i), 121.2 (C-9i) 119.0, 118.7 (C-4i and C-5i), 107.5 (C-7i),
106.0 (C-3i), 77.9 (C-1), 69.6 (C-5), 69.5 (C-3), 68.3 (C-2), 67.9 (C-4),
64.6 (CH3O), 61.0 (C-6), 29.0 (CH2C¼N), 18.8, 18.7(2), 20.4
(4 ꢄ CH3COO); HRMS (ESI) m/z for C25H30NaO11N2S [MþNa]þ, calcd
589.1468, found 589.1472.
4.5.2. Potassium 2,3,4,6-tetra-O-acetyl-2,3-dichlorophenyl-a-
glucosinolate (20b)
Pure 20b was obtained as a slightly yellow solid (250 mg, 79%).
Rf ¼ 0.25 in 15% MeOH/DCM; mp ¼ 146e148 ꢀC (dec.); ½a D20
ꢁ
¼ þ146.12 (c ¼ 4.1, MeOH); IR (KBr drift) nmax 2940, 2885, 1750
(C¼O), 1650 cmꢂ1; 1H NMR (300 MHz, CD3OD) (300 K)
d 7.67e7.64
4.4.2. 2,3,4,6-Tetra-O-acetyl-
a-D-glucopyranosyl-((4-
(m, 1H, H40-Ph-H), 7.40e7.38 (m, 2H, H50 and H60-Ph-H), 5.47 (d,
J1,2 ¼ 5.7 Hz, 1H, H1), 5.28 (t, J2,3 ¼ J3,4 ¼ 9.6 Hz, 1H, H3), 4.98 (t,
J3,4 ¼ J4,5 ¼ 9.6 Hz, 1H, H4), 4.91 (dd, J ¼ 5.7 Hz, 1H, H2), 4.35e4.29
(m, 1H, H5), 4.24 (dd, J5,6b ¼ 4.2 Hz, J6a,6b ¼ 12.6 Hz, 1H, H6b), 4.06
(dd, J5,6b ¼ 2.4 Hz, J6a,6b ¼ 12.6 Hz, 1H, H6a), 2.05, 2.01, 1.99, 1.97
methoxyindol-3-yl)methyl) thiohydroximate (13e)
Compound 13e was synthesized from the indole nitroalkane 19
(the compound 19 was prepared following the literature [17]) by
the same procedure as described for compound 13d and was ob-
tained as cream coloured solid (343 mg, 36%). Rf ¼ 0.22 in 40%
(4 ꢄ s, 12H, CH3COO); 13C NMR (75 MHz, CD3OD) (300 K)
d 170.4,
hexane/EtOAc; mp ¼ 69e71 ꢀC; ½a D20
ꢁ
¼ þ122.2 (c ¼ 2.7, DCM); IR
169.6, 169.4, 169.1 (4 ꢄ CH3COO), 154.0 (C¼N), 132.8 (C-3 of Ph),
131.8 (C-4 of Ph), 131.6 (C-1 of Ph), 131.3 (C-2 of Ph), 129.9 (C-6 of
Ph), 127.4 (C-5 of Ph), 80.3 (C-1), 69.6 (C-5), 69.4 (C-3), 68.7 (C-2),
67.4 (C-4), 60.9 (C-6),18.8(2), 18.7(2) (4 ꢄ CH3COO); HRMS (ESI) m/z
for C21H22O13Cl2NS2 [M-K]-, calcd 629.9915 found 629.9896.
(KBr drift) nmax 3336, 2942, 2839, 1750, 1600 cmꢂ1
;
1H NMR
(300 MHz, MeOD) (300 K)
d
6.94 (t, J6i,7i ¼ J5i,6i ¼ 7.5 Hz, 1H, H6i),
6.91 (d, J6i,7i ¼ 7.5 Hz, 1H, H7i), 6.84 (s, 1H, H2i), 6.46 (d,
J5i,6i ¼ 7.5 Hz, 1H, H5i), 6.12 (d, J1,2 ¼ 6.0 Hz, 1H, H1), 5.28 (t,
J2,3 ¼ J3,4 ¼ 10.2 Hz, 1H, H3), 4.97e4.83 (m, 2H, H2 and H4), 4.35 (d,
Jgem ¼ 16.8 Hz, CHHC¼N), 4.29e4.09 (m, 1H, H5), 4.16 (dd,
J5,6b ¼ 4.8 Hz, J6a,6b ¼ 12.3 Hz, 1H, H6b), 4.03 (d, Jgem ¼ 16.8 Hz,
4.5.3. Potassium 2,3,4,6-tetra-O-acetyl-4-bromophenyl-a-
glucosinolate (20c)
CHHC¼N), 3.87 (s, 3H, CH3O), 3.84 (dd, J5,6b
¼
2.1 Hz,
Pure 20c was obtained as a white solid (144 mg, 71%). Rf ¼ 0.42
J6a,6b ¼ 12.3 Hz, 1H, H6a), 2.01, 1.99, 1.98, 1.93 (4 ꢄ s, 12H, CH3COO);
in 15% MeOH/DCM; mp ¼ 142e144 ꢀC (dec.); ½a D20
¼ þ152.3
ꢁ
13C NMR (75 MHz, MeOD) (300 K)
d
170.5, 169.7, 169.5, 169.0
(c ¼ 2.0, MeOH); IR (KBr drift) nmax 2936, 2875, 1750 (C¼O), 1600
(4 ꢄ CH3COO), 154.2 (C-4i), 149.6 (C¼N), 137.8 (C-8i), 121.7 (C-6i),
120.7 (C-2i), 116.2 (C-9i) 109.8(C-3i), 104.1 (C-7i), 98.5 (C-5i), 78.3
(C-1), 69.7 (C-5), 69.5 (C-3), 68.2 (C-2), 68.0 (C-4), 61.1 (C-6), 53.8
(CH3O), 29.9 (CH2C¼N), 18.8, 18.7(2), 18.5 (4 ꢄ CH3COO); HRMS
(ESI) m/z for C25H30NaO11N2S [MþNa]þ, calcd 589.1468, found
589.1448.
(C¼N) cmꢂ1
; d 7.61e7.51 (m,
1H NMR (300 MHz, CD3OD) (300 K)
5H, H20, H30, H40, H50 and H60-Ph-H), 5.68 (d, J1,2 ¼ 5.7 Hz, 1H, H1),
5.45 (t, J2,3 ¼ J3,4 ¼ 10.2 Hz, 1H, H3), 5.09e4.99 (m, 2H, H2 and H4),
4.41e4.36 (m, 1H, H5), 4.22 (dd, J5,6b ¼ 4.5 Hz, J6a,6b ¼ 12.6 Hz, 1H,
H6b), 4.09 (dd, J5,6b ¼ 2.4 Hz, J6a,6b ¼ 12.6 Hz, 1H, H6a), 2.07, 2.01,
2.00, 1.96 (4 ꢄ s, 12H, CH3COO); 13C NMR (75 MHz, CD3OD) (300 K)
d
170.5, 169.6, 169.4(2) (4 ꢄ CH3COO), 153.7 (C¼N), 131.1 (C-3, C-5 of
4.5. General procedure for the preparation of potassium sulfate
salts of thiohydroximates (20a-e)
Ph), 130.9 (C-4 of Ph), 130.0 (C-2, C-6 of Ph), 124.1 (C-1 of Ph), 81.1
(C-1), 69.9, 69.9 (C-5) and (C-3), 68.9 (C-2), 67.5 (C-4), 61.0 (C-6),
81
18.9(2), 18.7(2) (4 ꢄ CH3COO); HRMS (ESI) m/z for C21H23
O BrNS2
13
To a stirred solution of the thiohydroxymate 13a-e (1 eq.) in dry
DCM or pyridine (40 mL) was added pyridine sulfur trioxide com-
plex (2.5 eq.). After stirring under Ar for 18 h at rt, an additional
portion of the complex (0.3 eq.) was added and stirring was
continued for 2 h. After that, a solution of KHCO3 (4 eq.) in water
(40 mL) was added and the mixture was stirred for 30 min and then
it was concentrated under reduced pressure. The residue was dis-
solved in water and extracted with chloroform (2 ꢄ 30 mL) and
then with 80% CHCl3/MeOH (3 ꢄ 30 mL). The organic layers were
dried (MgSO4), filtered and concentrated under reduce pressure. To
remove excess pyridine, the mixture was co-distilled several times
with toluene. The compounds 20a-e were obtained by flash chro-
matography eluting with DCM/MeOH.
[M-K]-, calcd 641.9800 found 641.9830.
4.5.4. Potassium 2,3,4,6-tetra-O-acetylneo-a-glucobrassicin (20d)
Pure 20e was obtained as a slightly yellow solid (226 mg, 79%).
Rf ¼ 0.28 in 15% MeOH/DCM; mp ¼ 121e123 ꢀC (dec.); ½a D20
ꢁ
¼ þ 54.6 (c ¼ 2.47, MeOH); IR (KBr drift) nmax 2940, 1750, 1599,
1442, 1370, 1233, 1059 cmꢂ1 1H NMR (300 MHz, MeOD) (300 K)
;
d
7.67 (d, J ¼ 8.1 Hz, 1H, H4i), 7.38e7.35 (m, 2H, H2i and H7i), 7.18 (t,
J ¼ 8.1 Hz,1H, H6i), 7.07 (t, J ¼ 8.1 Hz,1H, H5i), 6.14 (d, J ¼ 6.0 Hz,1H,
H1), 5.26 (t, J2,3 ¼ J3,4 ¼ 10.2 Hz, 1H, H3), 4.98 (t, J ¼ 10.2 Hz, 1H, H4),
4.89 (dd, J ¼ 6.0 Hz, 1H, H2), 4.32e4.26 (m, 1H, H5), 4.21e4.16 (m,
3H, H6b and CH2C¼N), 4.07 (s, 3H, CH3O), 3.97 (dd, J5,6a ¼ 2.1 Hz,
J6a,6b ¼ 12.6 Hz, H6a), 2.03, 1.98, 1.92, 1.74 (4 ꢄ s, 12H, 4 ꢄ CH3COO);
13C NMR (75 MHz, MeOD) (300 K)
d 170.5, 169.5, 169.4, 168.8
4.5.1. Potassium 2,3,4,6-tetra-O-acetyl-3,4-dimethoxyphenyl-
glucosinolate (20a)
a
-
(4 ꢄ CH3COO), 155.7 (C¼N), 132.1 (C-8i), 122.7 (C-9i), 121.9 (C-6i),
121.4 (C-2i), 119.2, 118.7 (C-4i and C-5i), 107.4 (C-7i), 104.8 (C-3i),
78.2 (C-1), 69.3, 69.3 (C-5 and C-3), 68.7 (C-2), 67.7 (C-4), 64.6
(CH3O), 60.9 (C-6), 28.6 (CH2C¼N), 18.8, 18.7, 18.6, 18.2
(4 ꢄ CH3COO); HRMS (ESI) m/z for C25H29O14N2S2 [M-K]-, calcd
645.1066, found 645.1069.
Pure 20a was obtained as a white solid (320 mg, 87%). Rf ¼ 0.17
in 15% MeOH/DCM; mp ¼ 110e112 ꢀC (dec.); ½a D20
¼ þ110 (c ¼ 3.0,
ꢁ
MeOH); IR (KBr drift) nmax 2945, 1750 (C¼O), 1600 (C¼N) cmꢂ1; 1H
NMR (300 MHz, CD3OD) (300 K)
d
7.20e7.19 (m, 2H, H20 and H60-
Ph-H), 7.02 (d, J5 ,6 ¼ 8.7 Hz, 1H, H50-Ph-H), 5.75 (d, J1,2 ¼ 5.7 Hz, 1H,
H1), 5.40 (t, J2,3 ¼ J3,4 ¼ 9.9 Hz, 1H, H3), 5.02e4.89 (m, 2H, H2 and
H4), 4.23e4.36 (m, 1H, H5), 4.19 (dd, J5,6b ¼ 4.5 Hz, J6a,6b ¼ 12.6 Hz,
1H, H6b), 4.09 (dd, J5,6b ¼ 2.1 Hz, J6a,6b ¼ 12.6 Hz,1H, H6a), 3.86, 3.85
(2 ꢄ s, 6H, 2 ꢄ CH3OPh), 2.07, 2.01, 2.00, 1.98 (4 ꢄ s, 12H,
0
4.5.5. Potassium 2,3,4,6-tetra-O-acetyl-4-methoxy-a-
glucobrassicin (20e)
Pure 20e was obtained as a white solid (221 mg, 69%). Rf ¼ 0.20
in 15% MeOH/DCM; mp ¼ 124e126 ꢀC (dec.); ½a D20
ꢁ
¼ þ151.2 (c ¼ 1.2,
4 ꢄ CH3COO); 13C NMR (75 MHz, CD3OD) (300 K)
d
170.5, 169.7,
MeOH); IR (KBr drift) nmax 3326, 2941, 2840, 1750, 1599 cmꢂ1 1H
;
169.6,169.5 (4 ꢄ CH3COO),155.2 (C¼N),150.8 (C-4 of Ph),148.6 (C-3
NMR (300 MHz, CD3OD) (300 K)
d 6.98e6.89 (m, 3H, H2i, H6i and
of Ph), 123.8 (C-1 of Ph), 121.7 (C-2 of Ph), 111.5 (C-5 of Ph), 110.7 (C-
H7i), 6.46 (d, J5i,6i ¼ 7.5 Hz, 1H, H5i), 6.06 (d, J1,2 ¼ 5.4 Hz, 1H, H1),

52
Q.V. Vo et al. / Carbohydrate Research 455 (2018) 45e53
5.28 (t, J2,3 ¼ J3,4 ¼ 9.6 Hz, 1H, H3), 5.09e4.90 (m, 2H, H2 and H4),
4.55 (d, Jgem ¼ 16.8 Hz, CHHC¼N), 4.30e4.23 (m, 1H, H5), 4.17e3.50
(m, 3H, H6b, H6a and CHHC¼N), 3.88 (s, 3H, CH3O), 2.02, 1.98, 1.92,
1.83 (4 ꢄ s, 12H, 4 ꢄ CH3COO); 13C NMR (75 MHz, MeOD) (300 K)
4.7. Anti-inflammatory assays
The anti-inflammatory assays were conducted following the
literature [17,30]. Human monocytic leukaemia THP-1 cells were
obtained from the American-Type Culture Collection. The cells
were grown in 10% heated-inactivated fetal bovine serum and
Invitrogen RPMI-1640 containing 2 mM L-glutamine. The cytokine
d
170.4169.6, 169.2, 169.0 (4 ꢄ CH3COO), 158.2 (C¼N), 153.7 (C-4i),
137.6 (C-8i), 122.0 (C-6i), 121.9 (C-2i),115.6 (C-9i),108.3 (C-3i), 104.6
(C-7i), 98.7 (C-5i), 79.4 (C-1), 74.8 (C-5), 73.3 (C-3), 69.2 (C-2), 67.1
(C-4), 60.7 (C-6), 53.9 (CH3O), 29.6 (CH2C¼N), 18.7(2), 18.6(2)
(4 ꢄ CH3COO); HRMS (ESI) m/z for C25H29O14N2S2 [M-K]-, calcd
645.1066, found 645.1069.
(TNF-a) Elisa Kit including the reagents was obtained from BD
Bioscience (R&D systems).
All compounds were dissolved in sterile distilled water then
further diluted in Invitrogen DMEM (Dulbecco's Modified Eagle
Medium). The cells were grown in a 75 mL flask and maintained at
37 ꢀC in humidified 5% CO2 atmosphere. The experiments were
carried out once the cells had reached 1 ꢄ 105 cells/ml. The PMA
was dissolved in DMSO to a concentration of 1 mg/mL then further
diluted before use. The cells were plated out to a cell density of
4.6. General procedure for the preparation of glucosinolates (21a-e)
To a solution of O-acetylglucosinolate 20a-e in anhydrous MeOH
(20 mL) under a N2 atmosphere was added dry MeOK (0.4 eq.) until
pH ¼ 8e9 was reached. After stirring for 3 h at rt, the solution was
made neutral by the addition of glacial acetic acid and then the
solution was concentrated under reduced pressure. The GLs 20a-e
were obtained by flash chromatography eluting with EtOAc:-
MeOH:H2O (16:4:1).
10 ꢄ 104 cell/ml, at 100
ml/well in a 96-well plate then treated with
PMA to a final concentration of 50 nM for 24 h at 37% in humidified
5% CO2 atmosphere.
The LPS (LPS from Escherichia coli O111:B4) was dissolved in
sterile water to a concentration of 5 mg/mL then further diluted to
4.6.1. 2,3-Dichlorophenyl-
a-glucosinolate (21b)
working stock of 10
mg/mL. The THP-1 cells were challenged with
Pure 26b was obtained as a white solid (51.0 mg, 85%). Rf ¼ 0.1 in
various compounds ranging from 0.1
m
M to 15 M. They were
m
EtOAc/MeOH/H2O (16:4:1); mp ¼ 125e127 ꢀC (dec.); ½a D20
ꢁ
¼ þ121.4
stimulated with LPS at a final concentration of 50 ng/mL. Super-
natants were collected after 4 h incubation and stored at ꢂ20 ꢀC
until ELISA analysis.
A sandwich ELISA was used to screen supernatants for the
release of cytokine TNF-a. The ELISA plates were coated with a
(c ¼ 1.9, H2O); IR (KBr drift) nmax 3355 (OH), 1566 (C¼N), 1413, 1275,
1262, 1064 cmꢂ1; 1H NMR (300 MHz, D2O) (300 K)
d 7.67e7.63 (m,
1H, H40-Ph-H), 7.42e7.34 (m, 2H, H5'and H60-Ph-H), 5.14 (d,
J1,2 ¼ 5.4 Hz, 1H, H1), 3.84e3.79 (m, 1H, H5), 3.71e3.57 (m, 3H, H6b,
H6a and H2), 3.49 (t, J ¼ 9.6 Hz, 1H, H3), 3.34 (dd, J ¼ 9.6 Hz, 1H,
capture antibody (1:250) which was diluted in coating buffer and
H4); 13C NMR (75 MHz, D2O) (300 K)
d
160.2 (C¼N), 132.7 (C-3 of
left at 4 ꢀC overnight. The ELISA plates were aspirated and washed 3
Ph), 132.3 (C-4 of Ph), 131.0 (C-1 of Ph), 130.6 (C-2 of Ph), 129.4 (C-6
of Ph), 127.8 (C-5 of Ph), 84.4 (C-1), 73.2 (C-5), 72.9 (C-3), 70.1 (C-2),
68.4 (C-4), 59.4 (C-6); HRMS (ESI) m/z for C13H14O9Cl2NS2 [M-K]-,
calcd 461.9493 found 461.9560.
times with 1 x PBST (0.05% Tween-20) before adding 200 ml/well
assay diluent incubated at room temperature for 1 h. Standards
were prepared by 2 fold serial dilutions to range from 500 pg/
mLꢂ7.8 pg/mL in assay buffer diluent. Standards and sample were
added in quadruplicate into appropriate wells and incubated at
room temperature for 2 h. After the 2 h incubation the plates were
aspirated and washed with a total of 5 washes. The detection
antibody and HRP reagent was added (100 ml/well) and incubated
at room temperature for 1 h. The plates were aspirated and washed
again, this time with a total of 7 washes and were soaked for 30 s
4.6.2.
a-Neoglucobrassicin (21d)
Pure 21d was obtained as a red-brown solid (74.0 mg, 80%).
Rf ¼ 0.11 in EtOAc/MeOH/H2O (16:4:1); mp ¼ 115e117 ꢀC (dec.);
½
a 2D0
ꢁ
¼ þ47.8 (c ¼ 1.6, H2O); IR (KBr drift) nmax 3125, 2936, 1714,
1575, 1453, 1242, 1060 cmꢂ1 1H NMR (300 MHz, D2O) (300 K)
;
d
7.66 (d, J ¼ 7.5 Hz,1H, H4i), 7.45e7.40 (m, 2H, H7i and H2i), 7.24 (t,
between each wash. The substrate solutions were added (100
well) and incubated at room temperature for 30 min in the dark.
The reaction was stopped by adding 50 l/well of kit stop solution
then read at 450 nm in a plate reader within 30 min with
ml/
J ¼ 7.5 Hz, 1H, H6i), 7.09 (t, J ¼ 7.5 Hz, 1H, H5i), 5.81 (d, J ¼ 5.1 Hz,
H1), 4.12 (d, Jgem ¼ 16.8 Hz, 1H, CHHCN), 3.99e3.97 (m, 4H,
CHHC¼N and CH3O), 3.69e3.60 (m, 2H, H2 and H5), 3.56e3.41 (m,
2H, H3 and H6b), 3.34e3.25 (m, 2H, H6a and H4); 13C NMR
m
l
correction at 570 nm. All the results are mean (SD) of 3 different
(75 MHz, D2O) (300 K)
d
161.6 (C¼N),131.9 (C-8i),122.7 (C-9i),122.6
experiments run in duplicate (P ꢅ 0.09).
(C-6i), 122.5 (C-2i), 120.0 (C-5i), 118.9 (C-4i), 108.3(C-7i), 105.3 (C-
3i), 82.5 (C-1), 72.9 (C-5), 72.8 (C-3), 70.2 (C-2), 68.7 (C-4), 65.6
(CH3O), 59.4 (C-6), 28.8 (CH2C¼N); HRMS (ESI) m/z for
17H21O10N2S2 [M-K]-, calcd 477.0643, found 477.0667.
Abbreviations
C
DBU, 1,5-diazabicyclo [5.4.0]undecene; DCM, dichloromethane;
DME, 1,2-dimethoxyethane; DMF, N,N-dimethylformamide;
DMF.DMA, dimethylformamide dimethyl acetal; ELISA, enzyme-
linked immunosorbent assay; ESI, electrospray ionization; FTMS,
Fourier transform mass spectrometry; GB, glucobrassicin; GLs,
glucosinolates; HESI, heated electrospray ionization; HRMS, high
resolution mass spectrometry; IR, infra-red; LPS, lipopolysaccha-
rides; MGB, 4-methoxyglucobrassicin; MS, mass spectrometry;
NCS, N-chlorosuccinimide; NGB, neo-glucobrassicin; NMR, nuclear
magnetic resonance; PMA, phorbol-12-myristate-13-acetate; THF,
tetrahydrofuran; TLC, thin layer chromatography; TOMAC, tri(n-
4.6.3.
a-4-Methoxyglucobrassicin (21e)
Pure 21e was obtained as a red-brown solid (14.0 mg, 95%).
Rf ¼ 0.08 in EtOAc/MeOH/H2O (16:4:1); mp ¼ 106e109 ꢀC (dec.);
½
a 2D0
ꢁ
¼ þ138.4 (c ¼ 1.0, H2OIR) (KBr drift); nmax 3454, 3267, 2908,
2842,1260,1060 cmꢂ1; 1H NMR (300 MHz, D2O) (300 K)
d 7.10e7.01
(m, 3H, H2i, H6i and H7i), 6.57 (d, J5i,6i ¼ 6.9 Hz, 1H, H5i), 5.80 (d,
J1,2 ¼ 5.4 Hz, 1H, H1), 4.41 (d, Jgem ¼ 16.8 Hz, 1H, CHHC¼N), 4.17 (d,
Jgem ¼ 16.8 Hz, 1H, CHHC¼N), 3.83 (s, 3H, CH3O), 3.75e3.72 (m, 1H,
H5), 3.66e3.39 (m, 4H, H2, H3, H6b and H6a), 3.33 (t, J ¼ 9.6 Hz, 1H,
H4); 13C NMR (75 MHz, D2O) (300 K)
d
163.1 (C¼N), 153.7 (C-4i),
137.5 (C-8i), 122.6 (C-6i and C-2i), 115.9 (C-9i) 108.3(C-3i), 105.1 (C-
7i),100.0 (C-5i), 82.7 (C-1), 72.9 (C-5 and C-3), 70.3 (C-2), 68.7 (C-4),
59.6 (C-6), 55.2 (CH3O), 29.9 (CH2C¼N); HRMS (ESI) m/z for
octyl)methyl ammonium chloride; TNF-a, tumor necrosis factor-
a; UV, ultra-violet.
C
17H21O10N2S2 [M-K]-, calcd 477.0643, found 477.0686.

Q.V. Vo et al. / Carbohydrate Research 455 (2018) 45e53
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53
Acknowledgments
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[17] Q.V. Vo, C. Trenerry, S. Rochfort, J. Wadeson, C. Leyton, A.B. Hughes, Bioorg.
Med. Chem. 22 (2014) 856e864.
This research was supported by Vietnam National Foundation
for Science and Technology Development (NAFOSTED) [ grant
number 104.06-2016.03 (3/2017/104/HÐTN)].
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