Synthetic approaches to C-glucosinolates

Article abstract of DOI:10.1016/S0040-4020(00)00153-8

In these pages, short and efficient synthetic approaches to C-analogs of glucosinolates are described. Starting from D-glucose, C-glucotropaeolin (6) and C-glucocapparin (11) were synthesized in three steps. Preliminary enzymatic assays involving sulfatase and myrosinase have been performed. (C) 2000 Published by Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00153-8

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 2647±2654
Synthetic Approaches to C-Glucosinolates
a
Vincent Aucagne, David Gueyrard, Arnaud Tatibouet, Alain Quinsac and Patrick Rollin
a
a
b
a,
*
È
a
  Â
Institut de Chimie Organique et Analytique, Universite d'Orleans, BP 6759, F-45067 Orleans Cedex 2, France
bCETIOM, avenue de la Pomme de Pin, F-45160 Ardon, France
Received 19 October 1999; accepted 17 February 2000
AbstractÐIn these pages, short and ef®cient synthetic approaches to C-analogs of glucosinolates are described. Starting from d-glucose,
C-glucotropaeolin (6) and C-glucocapparin (11) were synthesized in three steps. Preliminary enzymatic assays involving sulfatase and
myrosinase have been performed. q 2000 Published by Elsevier Science Ltd. All rights reserved.
Introduction
Results and Discussion
Synthesis of C-glucosinolates
Glucosinolates constitute a large family of naturally
occurring thiosugars found mainly in the botanical order
Brassicales. They can be hydrolyzed by the enzyme
myrosinase into d-glucose, hydrogen sulfate and various
co-products depending on the structure of the aglycon.1
Myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1)
isolated from Sinapis alba ripe seeds is a glycoprotein
containing various thiol groups and disul®de bridges
together with ca. 18% carbohydrates, mainly hexoses.
This enzyme consists of two identical subunits with a
molecular weight of 71.7 kDa and it has a pI of 5.1.2 Myro-
sinase is present in all glucosinolate-containing plants.
Different types of glucosinolate analogs have been synthe-
sized with the aim of explaining the mechanism of their
enzymatic hydrolysis.3 Glucotropaeolin (1) is the major
glucosinolate isolated from plants of the genus Nasturtium.
Using synthetic deoxy-glucotropaeolins4,5 or 2-¯uoro-2-
deoxy-glucotropaeolin6,7 and studying the myrosinase
activity in comparison with native glucotropaeolin, the
importance of the hydroxyl group at C-2 for glucosinolates
binding was established and the molecular mechanism for
its hydrolysis by myrosinase could thus be clari®ed. The
design and synthesis of a non-hydrolyzable substrate such
as a C-glycosidic analog would be of great interest in order
to get additional information about the substrate conforma-
tion and binding into the active site of myrosinase by X-ray
analysis.8
A brief retrosynthetic analysis called for a transient b-keto-
C-glucoside which can be obtained following two different
ways: either via an allyl-C-glucoside following a multistep
route or via a Horner±Emmons reaction (Scheme 1).
We have previously reported9 a synthesis of C-gluco-
tropaeolin: following Kishi's procedure,10,11 2,3,4,6-tetra-
O-benzyl-d-glucopyranose was transformed into an allyl
b-C-glycoside which led to the known epoxides 2 by
mCPBA oxidation.12 Regioselective epoxide opening of 2
by phenyllithium13 afforded the diastereomeric mixture of
alcohols 3, which were further oxidized into ketone 4.
Hydrogenolytic deprotection of 4 afforded ketone 5 which
was directly converted into C-glucotropaeolin (6) by
condensation with hydroxylamine-O-sulfonic acid.14
C-glucotropaeolin (6) was obtained as a 3:2 diastereomeric
1
mixture of oxime sulfonates (ratio determined by H NMR
and con®rmed by HPLC) (Scheme 2).
Although this synthetic pathway indeed allowed the
preparation of C-glucotropaeolin (6), quite a number of
steps were required, thus lowering the overall yield to
18% from 2,3,4,6-tetra-O-benzyl-d-glucopyranose. Alter-
natively, a more attractive approach to the key-compound
4 was to be developed as a one step Horner±Emmons type
procedure.15
The synthesis of a dialkyl (2-oxo-3-phenyl)propane-
phosphonate was needed in order to obtain 4. All the
methods proposed in the literature for the synthesis of
such b-keto phosphonates are rather tedious and
inef®cient.16,17 Thus we tried to synthesize dimethyl
(2-oxo-3-phenyl)propanephosphonate (7) following the
method described by Mathey,18 i.e. via the addition of the
Keywords: glucosinolates; myrosinase inhibition; C-glycosides; Horner±
Emmons reaction.
* Corresponding author. Tel.: 133-2-3841-7370; fax: 133-2-3841-7281;
e-mail: patrick.rollin@univ-orleans.fr
0040±4020/00/$ - see front matter q 2000 Published by Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00153-8

2648
V. Aucagne et al. / Tetrahedron 56 (2000) 2647±2654
Scheme 1.
cuprate anion derived from dimethyl methanephosphonate
on phenylacetyl chloride (Method A). The low repro-
ducibility of this reaction led us to try another procedure19
involving the condensation of the lithio derivative of
dimethyl methanephosphonate on ethyl phenylacetate
(Method B), but this latter reaction also proved disappoint-
ing. Finally, replacing the ester by the corresponding
Weinreb amide20,21 (Method C) allowed us to obtain 7
with good yields (Scheme 3).
MgBr2, Et3N/LiCl, MeONa, KOH) or solvent (THF,
MeOH, H2O/CH2Cl2, toluene, dioxane) used: yields inferior
to 30% only were obtained. Application of heat to the
reaction mixture promoted the expected side-reactions
(C-2 epimerization,22 b-elimination23). Moreover, a second
step of epimerization of the a/b mixture into the b epimer
was required consequently (Scheme 4).24
Compared to the ®rst pathway (Scheme 1), the phosphonate
condensation constitutes a much shorter route to the key
compound 4 although the yield remains very low.
The ®rst condensations of phosphonate 7 were tested on
2,3,4,6-tetra-O-benzyl-d-glucopyranose. This approach led
to poor results, whatever base (NaH, nBuLi, iPr2NEt/
This poor reactivity could be attributed to the steric
Scheme 2.
Scheme 3. Methods. A: (i) nBuLi, THF; (ii) CuI; (iii) PhCH2COCl (0,yield,62%); B: (i) LDA 2 equiv., THF; (ii) PhCH2COOMe (31%); C: (i) nBuLi, THF;
(ii) PhCH2CON(OMe)Me; (iii) H2O, HCl (79%).

V. Aucagne et al. / Tetrahedron 56 (2000) 2647±2654
2649
Scheme 4.
hindrance of the benzyl group in 2-position.25 In order to
tide this problem over, we then tried to condense phos-
phonate 7 with 4,6-O-benzylidene-d-glucopyranose (8),
which can be readily prepared in one step from d-glucose.26
As a matter of fact, this reaction proved to be much more
ef®cient and did not require any heating. In addition, the use
of sodium methoxide in methanol allowed us to skip the
epimerization step. The C-glycoside 9 was concomitantly
deprotected and converted into the oxime sulfonate in a one-
pot procedure using hydroxylamine-O-sulfonic acid. The
diastereomeric ratio observed for C-glucotropaeolin (6)
was similar to that previously obtained (3:2) (Scheme 5).
glucosinolates in rapeseed consists in an enzymatic
desulfation on an ion exchange column, followed by the
HPLC of the desulfated products on a C18 column (Scheme
7).27,28 This preliminary desulfation allows to separate
glucosinolates from the complex vegetable matrix.
We have tried to apply this technique to C-glucotropaeolin
(6). Unexpectedly, the chromatogram showed only one
notable peak which might be either both stereoisomers, or
only Z or E. In order to remove this ambiguity, we have
performed the chemical synthesis of the non-sulfated
diastereoisomers of C-glucotropaeolin (14) as standard
compounds.
In order to check a possible general application of this
method, we have made use of the same reaction protocol
with 8 using commercially available diethyl 2-oxopropane-
phosphonate. By this way, we could obtain the C-glycosidic
analog 11 of glucocapparinÐa glucosinolate present in
Boscia senegalensis and capers (Capparidaceae)Ðin
comparable yields (Scheme 6).
The direct conversion of ketone 5 into oxime 14 was
ineffective. Therefore 5 was peracetylated and converted
into its oxime derivative. A ®nal transesteri®cation step
afforded the desired product 14 as a 3:2 diastereomeric
1
mixture (ratio determined by H NMR) (Scheme 8).
Co-injection of the diastereomeric mixture 14 together with
the product resulting from enzymatic desulfation proved
that only one isomer of the oxime was obtained in the
enzymatic process. Moreover, a short study of the time-
dependent evolution of 14 demonstrated that the two
isomeric oximes were engaged in a slow equilibrium
process, accompanied by hydrolysis to the corresponding
Analysis of C-glucosinolates
HPLC experiments on intact C-glucotropaeolin (11) have
already been performed:9 it was thus shown that an equi-
librium existed between E and Z isomers under acidic
conditions. The EU homologated analytical method for
Scheme 5.
Scheme 6.
Scheme 7.

2650
V. Aucagne et al. / Tetrahedron 56 (2000) 2647±2654
Scheme 8.
Figure 1. (a) Time-dependent evolution of the desulfated mixture. A: immediately after desulfation; B: 2 days after desulfation; C: 1 month after desulfation.
Column: Spherisorb ODS-2 (250£4.6 mm); Mobile phase: CH3CN in H2O, 0±80 gradient in 20 min. (b) UV spectra of constituents 2, 3 and 4. Peak 1:
unidenti®ed impurity; Peak 2 and 4: desulfated isomers 14 of C-GTL; Peak 3: ketone 5.
ketone (5) (Fig. 1a). UV absorption spectra of these three
compounds are reported in Fig. 1b.
generalized, allows to synthesize C-glucosinolates in three
steps, starting from d-glucose. Within this framework, we
have developed an ef®cient route to dimethyl 2-oxo-3-
phenylpropane phosphonate by use of a Weinreb amide.
We have shown that the sulfatase which is currently used
for the characterization of natural glucosinolates can
recognise and desulfate our C-analogs, to afford only one
of the two possible diastereoisomers. However, introductory
inhibition experiments demonstrated that C-glucotropaeolin
(6) was not recognized by myrosinase. Further work is in
progress to ®nd other analogs to explain the mechanism of
myrosinase hydrolysis.
Myrosinase inhibition experiments
Preliminary investigation of the inhibition properties of the
C-analogs have been undertaken. No activity against myro-
sinase has been detected so far: the diastereomeric mixture
of C-glucosinolates is not degraded by the enzyme, and
displays no inhibitory activity at concentrations up to
20 mM against sinigrin as substrate. These negative results
seem to indicate that the C-glucosinolates do not bind to the
enzyme. This is to our knowledge the ®rst report showing
that a glycosylhydrolase does not recognize the C-analog of
its natural substrate.
Experimental
General methods: Melting points were determined on a
Conclusion
Ko¯er hot-stage apparatus and are uncorrected. 1H and
È
13C NMR spectra were recorded on a Bruker Avance
DPX250 at 250 and 62.89 MHz, respectively, except for
1H NMR spectra of compound 6 which were recorded on
We have described a simple access to C-glycosidic analogs
of glucosinolates. This method, which can most likely be

V. Aucagne et al. / Tetrahedron 56 (2000) 2647±2654
2651
a Bruker Avance DPX501 at 500 MHz. The chemical
shifts (d) are reported in ppm down®eld from tetra-
methylsilane as the internal standard. Speci®c rotations
were measured at 208C using a Perkin±Elmer 141 polari-
meter . IR spectra were measured using a Perkin±Elmer
FT Paragon 1000 PC spectrophotometer. HRMS spectra
were recorded on a VG analytical 70 SV. Evaporation,
in vacuo, was conducted with a BuÈchi rotary evaporator.
Analytical TLC was carried out on precoated silica gel
60F-254 plates (E. Merck) and spots were detected by
UV light (254 nm). Flash column chromatography was
performed on Kieselgel 60 (230±400 mesh) silica gel
(E. Merck).
The mixture was further diluted with a saturated aqueous
solution of sodium bicarbonate and the aqueous layer
extracted with ethyl acetate (£3). The combined organic
extracts were dried (MgSO4), concentrated in vacuo and
the residue was recrystallized in ethyl acetate to give pure
4 (477 mg, 96%) as a white solid.
Method B: To a solution of phosphonate 7 (111 mg,
2.3 equiv.) in dry THF at 08C was added nBuLi (0.58 mL,
1.6 M in hexane, 2 equiv.). The solution was stirred at this
temperature for 30 min and 2,3,4,6 tetra-O-benzyl-d-gluco-
pyranose (110 mg, 0.20 mmol) was added. The reaction
mixture was re¯uxed for 5 h, quenched by water (10 mL)
and extracted with CH2Cl2 (£3). The combined organic
extracts were washed with brine, dried (MgSO4), concen-
trated in vacuo and the residue was puri®ed by ¯ash
column chromatography (petroleum ether±AcOEt) 9:1,
followed by a recrystallization from ethyl acetate to
afford ketone 4 (40 mg, 30%). Mp 74±768C; [a]Dˆ23
1-C-(2,3,4,6-tetra-O-benzyl-b-d-glucopyranosyl)-3-
phenylpropan-2-ol (3). A solution of the diastereomeric
mixture 2 (600 mg, 1.03 mmol)12 in dry THF (50 mL) was
cooled to 2788C and phenyllithium (1.14 mL, 1.8 M in
hexane, 2 equiv.) then BF3´Et2O (0.27 mL, 2 equiv.) were
added dropwise. The solution was stirred at 2788C for
35 min, quenched by the addition of methanol (30 mL)
and was then allowed to warm to room temperature. The
mixture was poured into a saturated aqueous solution of
sodium bicarbonate and extracted with ethyl acetate (£3).
The combined organic fractions were dried (MgSO4),
concentrated in vacuo and the residue puri®ed by ¯ash
column chromatography (petroleum ether±AcOEt, 8:2) to
afford a diastereomeric mixture of 3 (583 mg, 86%) as a
white amorphous solid. Pure fractions of both isomers
were isolated for characterization. Mp 108±1108C (3a),
68±708C (3b); [a]Dˆ16 (3a), 113 (3b) (c 1.0, CHCl3);
1H NMR (CDCl3); diastereomer 3a: d 1.48 (ddd, 1H,
1
(c 1.0, CHCl3); H NMR (CDCl3): d 2.60 (dd, 1H, H-10b,
J1 b-1 aˆ15.3 Hz, J1 b-1ˆ8.3 Hz), 2.77 (dd, 1H, H-10a,
0
0
0
0
J1 a-1ˆ5.2 Hz), 3.33 (dd, 1H, H-2, J1-2ˆ9.4 Hz, J2-3
ˆ
9.4 Hz), 3.44 (dt, 1H, H-5, J4-5ˆ9.6 Hz, J5-6ˆ3.0 Hz),
3.62±3.86 (m, 5H, H-1, H-3, H-4, H-6a, H-6b), 3.72 (s,
2H, H-30), 4.47±5.00 (m, 8H, CH2Ph), 7.10±7.48 (m,
13
25H, HAr); C NMR (CDCl3): d 44.6 C-10 51.3 C-30 69.2
C-6, 73.9, 75.4, 75.9 CH2Ph; 76.0 C-1; 78.8, 87.6 C-3 and
C-4; 79.3 C-5; 81.5 C-2; 127.3, 128.1, 128.2, 128.3, 128.4,
128.8, 128.9, 129.0, 130.0, 134.4, 138.4, 138.5, 138.9 CAr;
206.4 CO; HRMS: calcd for C43H44O6 (656.3138), found
(656.3149).
H-10b, J1 b-1 aˆ14.5 Hz, J1 b-1ˆ9.7 Hz, J1 b-2 ˆ9.7 Hz), 2.06
1-C-(b-d-glucopyranosyl-3)-phenylacetone (5). Method
A: A suspension of compound 4 (100 mg, 0.15 mmol) and
10% Pd/C (20 mg) in acetic acid (7 mL) was purged twice
with hydrogen and stirred at room temperature for 22 h
under 3 bar. The reaction mixture was then ®ltered through
a pad of Celite, washed with acetic acid, concentrated in
vacuo (coevaporation with toluene) and the residue was
puri®ed by ¯ash column chromatography (AcOEt±
MeOH±water, 90:8:2) to give ketone 5 (27 mg, 60%) as a
colorless syrup.
0
0
0
0
0
(ddd, 1H, H-10a, J1 a-1ˆ2.8 Hz, J1 a-2 ˆ2.8 Hz), 2.64 (dd,
0
0
0
1H, H-30b, J3 b-3 aˆ13.5 Hz, J3 b-2 ˆ7.3 Hz), 2.91 (dd, 1H,
0
0
0
0
H-30a. J3 a-2 ˆ6.2 Hz), 3.24 (dd, 1H, H-2, J1-2ˆ9.1 Hz,
J2-3ˆ9.1 Hz), 3.38±3.84 (m, 6H, H-1, H-3, H-4, H-5, H-
6a, H-6b), 4.01±4.17 (m, 1H, H-20), 4.40±5.00 (m, 8H,
CH2Ph), 7.03±7.53 (m, 25H, HAr); diastereomer 3b: d
0
0
1.77 (ddd, 1H, H-10b, J1 b-1 aˆ14.5 Hz, J1 b-1ˆ7.2 Hz,
0
0
0
J1 b-2 ˆ2.5 Hz), 1.91 (ddd, 1H, H10a, J1 a-1ˆ3.5 Hz,
0
0
0
0
0
0
0
0
J1 a-2 ˆ8.8 Hz), 2.72 (dd, 1H, H-3 b, J3 b-3 aˆ13.5 Hz,
0
0
0
0
0
J3 b-2 ˆ6.3 Hz), 2.83 (dd, 1H, H-3 a, J3 a-2 ˆ6.6 Hz), 3.34
(dd, 1H, H-2, J1-2ˆ8.8 Hz, J2-3ˆ8.8 Hz), 3.39±3.80 (m,
6H, H-1, H-3, H-4, H-5, H-6a, H-6b), 4.07±4.22 (m, 1H,
H-20), 4.32±4.98 (m, 8H, CH2Ph), 7.05±7.45 (m, 25H,
HAr);13C NMR (CDCl3); diastereomer 3a: d 37.6 C-1044.3
C-30 69.5 C-6; 73.7 C-20 74.0, 75.5, 76.0 CH2Ph; 78.8, 79.1,
80.9, 87.2 C-1, C-3, C-4 and C-5; 82.6 C-2; 126.3, 126.7,
127.7, 128.1, 128.3, 128.4, 128.8, 128.9, 129.9, 138.0,
138.3, 138.9, 139.0 CAr; diastereomer 3b: d 37.2 C-10
44.3 C-3069.3 C-6; 70.2 C-20 73.9, 75.4, 75.5, 76.0
CH2Ph; 78.0, 78.7, 79.1, 87.6 C-1, C-3, C-4 and C-5; 81,4
C-2; 126.8, 128.1, 128.2, 128.3, 128.4, 128.8, 128.9, 129.9,
138.3, 138.4, 138.5, 138.9, 139.0 CAr; HRMS: calcd for
C43H46O6 (658.3294), found (658.3303).
Method B: Compound 9 (115 mg, 0.30 mmol) was
dissolved in a 7:3 mixture of acetic acid and water
(10 mL) and the resulting solution was heated at 608C for
10 h. The yellowish oil obtained after evaporation of the
solvent in vacuo was puri®ed by ¯ash column chromato-
graphy to afford ketone 5 (64 mg, 72%) as a colorless syrup.
[a]Dˆ221 (c 1.0, MeOH);1H NMR (CD3OD): d 2.64 (dd,
1H, H-10b, J1 b-1 aˆ15.8 Hz, J1 b-1ˆ8.9 Hz), 2.90 (dd, 1H,
0
0
0
H-10a, J1b a-1ˆ2.8 Hz), 3.06 (dd, 1H, H-4, J3-4ˆ9.0 Hz,
0
J4-5ˆ9.0 Hz), 3.17±3.39 (m, 2H, H-2 and H-3), 3.61±3.77
(m, 3H, H-1, H-6a and H-6b), 3.81 (dd, 1H, H-5,
0
J5-6ˆ2.9 Hz), 3.88 (s, 2H, H3 ), 7.17±7.37 (m, 5H, HAr);
13C NMR (CD3OD): d 48.1 C-10; 49.3 C-30; 60.9 C-6;
69.8 C-2; 73.2 C-4; 75.4 C-1; 77.7 C-3; 79.7 C-5; 125.9,
127.6, 128.9, 133.9 CAr; 207.5 CO; HRMS: calcd for
C15H20O6 (296.1260), found (296.1247).
1-C-(2,3,4,6 -tetra-O-benzyl-b-d-glucopyranosyl)-3-phenyl-
acetone (4). Method A: To a solution of the diastereomeric
mixture 3 (500 mg, 0.76 mmol) in methanol (28 mL) cooled
at 08C Jones reagent (1.20 mL, 2.1 equiv.)12 was added. The
reaction was stirred at 08C for 1 h and quenched by the
addition of a 5% aqueous solution of sodium metabisul®te.
1-C-b-d-glucopyranosyl-3-phenylacetone oxime O-sulfo-
nate potassium salt (6). Method A: To a solution of
compound 5 (100 mg, 0.34 mmol) in a 1:1 mixture of

2652
V. Aucagne et al. / Tetrahedron 56 (2000) 2647±2654
dichloromethane and methanol (2 mL) at 2108C was added
hydroxylamine-O-sulfonic acid (41 mg, 2.8 equiv.). The
reaction was allowed to warm to room temperature, stirred
for 15 min more and then hydrolyzed by a sodium bicarbo-
nate solution (2.8 equiv.). The solvents were evaporated in
vacuo and the residue was puri®ed by ¯ash column chro-
matography (AcOEt±MeOH±water, 80:15:5) then freeze-
dried to give the E1Z diastereomeric mixture 6 (145 mg,
quantitative) as a white amorphous solid.
by the addition of a 1 M aqueous solution of HCl until
neutral and extracted with ethyl acetate (£3). The combined
organic extracts were washed with brine, dried (MgSO4),
concentrated in vacuo and the residue was puri®ed by distil-
lation under reduced pressure to afford phosphonate 7
(1.20 g, 31%) as a yellowish oil.
Method C: A solution of nBuLi (0.65 mL, 2.05 M in hexane,
0.9 equiv.) in dry THF (1.5 mL) was cooled to 2788C and
dimethyl methanephosphonate (0.158 mL,1.46 mmol) in
solution in dry THF (0.5 mL) was added. The deprotonation
reaction was allowed to continue at 08C for 20 min then the
mixture was cooled again to 2788C. N-methoxy-N-methyl-
phenylacetamide (240 mg, 1.34 mmol) was added and the
solution was stirred at 2788C while monitoring by TLC.
After 30 min, the mixture was hydrolyzed by the addition
of 1 M aqueous HCl (5 mL) and extracted with CH2Cl2
(£3). The combined organic extracts were dried (MgSO4),
concentrated in vacuo and the residue was puri®ed by ¯ash
column chromatography (petroleum ether±AcOEt, 3:7) to
afford phosphonate 3 (255 mg, 79%) as a yellowish oil. IR
Method B: Using the same procedure, compound 9 (200 mg,
0.52 mmol) was converted into the desired product 6
(150 mg, 67%). Mp 100±1028C (decomposition); 1H
NMR (CD3OD): major isomer (M) 6a: d 2.39 (dd, H-10;b,
J1 b-1 aˆ14.7 Hz, J1 b-1ˆ9.9 Hz), 2.81 (dd, H-10a,
0
0
0
0
J1 a-1ˆ2.9 Hz), 2.87±2.94 (m, H-5), 3.06±3.16 (m, H-2),
3.25±3.35 (m, H-3 and H-4), 3.39 (ddd, H-1,
J1-2ˆ9.9 Hz), 3.54 (dd, H-6b, J5-6bˆ4.8 Hz, J6a-6bˆ12.9),
3.63 (dd, H-6a, J5-6aˆ2.1 Hz), 3.77 (d, H-30b,
J3 b-3 aˆ14.2 Hz), 3.93 (d, H-30a), 7.25±7.40 (m, HAr);
0
0
minor isomer (m) 6b: d 2.55 (dd, H-10b, J1 b-1 aˆ14.2 Hz,
0
0
1
J1 b-1ˆ10.1 Hz), 2.74 (dd, H-10a, J1 a-1ˆ2.9 Hz), 3.06±3.16
(m, H-2 and H-5), 3.25±3.35 (m, H-3 and H-4), 3.44 (ddd,
H-1, J1-2ˆ10.1 Hz), 3.60 (dd, H-6b, J6b-6aˆ12.3 Hz,
(KBr) 1721 cm21 (CO); H NMR (CDCl3): d 3.11 (d, 2H,
0
0
2
3
CH2PO, JH±Pˆ22.6 Hz), 3.78 (d, 6H, OCH3, JH±P
ˆ
11.3 Hz), 3.90 (s, 2H, CH2Ph), 7.17±7.40 (m, 5H, HAr);
1
J5-6bˆ5.3 Hz), 3.68 (d, H-30b, J3 b-3 aˆ14.9 Hz), 3.72 (dd,
13C NMR (CDCl3): d 40.5 (d, CH2PO, JC±Pˆ123 Hz),
0
0
3
2
H-6a, J5-6aˆ2.1 Hz), 3.74 (d, H-30a), 7.25±7.40 (m, HAr);
51.2 (d, CH2Ph, JC±Pˆ1.8 Hz), 53.3 (d, MeO, JC±P
ˆ
13C NMR (CDCl3): d 29.9 C-10m; 33.5 C-10M; 35.3 C-30M
;
6.7 Hz), 126.8, 129.2, 130.0, 131.1 CAr, 199.9 (d, CO,
2JC±Pˆ6.1 Hz); HRMS: calcd for C11H15O4P (242.0708),
found (242.0720).
38.9 C-30;m; 59.3, 59.6 C-6; 68.3, 68.5, 76.1 C-3 and C-4;
72.3, 72.8 C-2; 74.6, 75.1 C-1; 78.1, 78.3 C-5; 126.0, 126.2,
127.8, 127.9, 128.1, 132.6, 134.0, 134.5 CAr; 164.6, 165.5
C-20 HRMS: calcd for C15H20KNO9S (429.0496), found
(429.0488).
1-C-(4,6-O-benzylidene-b-d-glucopyranosyl)-3-phenyl-
acetone (9). To a solution of phosphonate 7 (600 mg,
2.2 equiv.) in methanol (3.7 mL) at room temperature was
added sodium methoxide (2.26 mL, 1 M in methanol,
2 equiv.). After stirring for 1 h, 4,6-O-benzylidene-d-gluco-
pyranose (8) (300 mg, 1.12 mmol)26 was added. The result-
ing mixture was stirred 17 h more, hydrolyzed (10 mL) and
extracted with CH2Cl2 (£3). The combined organic extracts
were washed with brine, dried (MgSO4) and concentrated in
vacuo. The residue was puri®ed by ¯ash column chromato-
graphy (petroleum ether±AcOEt, 3:7) then recrystallized
from ethyl acetate to give ketone 9 (273 mg, 63%) as a
Dimethyl (2-oxo-3-phenyl)propane phosphonate (7).
Method A: A solution of nBuLi (10.3 ml, 1.6 M in hexane,
1.1 equiv.) in dry THF (10 mL) was cooled to 2788C and
dimethyl methanephosphonate (1.63 mL, 15 mmol)
dissolved in dry THF (5 mL) was slowly added. The solu-
tion was stirred for 20 min at this temperature then freshly
puri®ed dry CuI (2.13 g, 1.13 equiv.) was added and the
mixture was slowly warmed to 2308C. The dark suspension
was stirred for 90 min then cooled to 2408C. Phenylacetyl
chloride (2.12 mL, 1.05 equiv.) was then added and the
reaction mixture was allowed to reach room temperature
overnight. After dilution with water (15 mL) and CH2Cl2
(30 mL), the mixture was ®ltered through a pad of Celite
(£2) and extracted with CH2Cl2 (£3). The combined organic
extracts were dried (MgSO4), concentrated in vacuo and the
residue was puri®ed by distillation under 1 Torr pressure
(Tebˆ1208C) to give phosphonate 7 (2.25 g, 62%) as a
yellowish oil.
1
white solid. Mp 182±1848C; [a]Dˆ16 (c 1.0, CHCl3); H
NMR (CDCl3): d 2.66 (dd, 1H, H-10b, J1 b-1 aˆ16.2 Hz,
0
0
J1 b-ˆ1ˆ7.9 Hz), 2.88 (dd, 1H, H-10a, J1 a-1ˆ3.8 Hz), 3.33
(dd, 1H, H-2, J1-2ˆ9.6 Hz, J2-3ˆ9.0 Hz), 3.37±3.50 (m,
1H, H-4), 3.54±3.79 (m, 3H, H-3, H-5 and H-6b), 3.74 (s,
2H, CH2Ph), 3.84 (ddd, 1H, H-1), 4.25 (dd, 1H, H-6a,
J6a-5ˆ4.3 Hz, J6a-6bˆ10.4 Hz), 5.48 (s, 1H, CHPh), 7.15±
0
0
13
7.55 (m, 10H, HAr); C NMR (CDCl3): d 45.0 C-10; 51.3
C-30; 69.1 C-6; 70.7 C-2; 74.6 C-5; 75.6 C-3; 76.3 C-1; 81.4
C-5; 102.3 CHPh; 126.7, 127.5, 128.8, 129.1, 129.7, 130.0,
134.1 and 137.4 CAr; 206.5 CO; HRMS: calcd for C22H24O6
(384.1573), found (384.1579).
Method B: A solution of freshly distilled diisopropylamine
(4.71 mL, 2.1 equiv.) in dry THF (40 mL) was cooled to
2788C and nBuLi (20 mL, 1.6 M in hexane, 2.0 equiv.)
was added slowly. The reaction mixture was warmed up
to 08C, stirred at this temperature for 40 min then cooled
to 2788C. Dimethyl methanephosphonate (1.71 mL,
16 mmol) was added and the resulting solution was stirred
at 08C for 30 min then cooled again to 2788C. Methyl
phenylacetate (2.53 mL, 1.1 equiv.) dissolved in dry THF
(5 mL) was added over a period of 30 min, then the solution
was allowed to stir at room temperature for 18 h, quenched
1-C-(4,6-O-benzylidene-b-d-glucopyranosyl) acetone (10).
To a solution of diethyl 2-oxopropane phosphonate
(832 mg, 2.3 equiv.) in methanol (3.7 mL) at room tempera-
ture was added sodium methoxide (3.9 mL, 1 M in
methanol, 2.1 equiv.). After stirring for 1 h, 4,6-O-benzyl-
idene-d-glucopyranose (8) (500 mg, 1.86 mmol) was added.
The resulting mixture was stirred for 21 h more, hydrolyzed
(15 mL) and extracted with CH2Cl2 (£3). The combined

V. Aucagne et al. / Tetrahedron 56 (2000) 2647±2654
2653
organic extracts were washed with brine, dried (MgSO4) and
concentrated in vacuo. The residue was puri®ed by ¯ash
column chromatography (petroleum ether±AcOEt, 3:7) to
afford ketone 10 (361 mg, 63 %) as a white solid. Mp 142±
1448C; [a]Dˆ223 (c 1.0, CHCl3) 1H NMR (CDCl3): d 2.20
white precipitate was ®ltered then recrystallized in ethanol
to give a diastereomeric mixture of oximes 13 (105 mg,
88%) as a white solid. Mp 70±728C; H NMR (CDCl3): d
1.92±2.32 (m, CH3CO, H-10a and H-10b), 3.45±4.23 (m,
H-1, H-5, H-6a and H-6b), 4.66 (2 dd, H-4, J3-4ˆ9.3 Hz,
J4-5ˆ9.3 Hz), 4.87 (dd, H-2, J1-2ˆ9.3, J2-3ˆ9.3 Hz), 5.20
(dd, H-3), 7.13±7.39 (m, HAr), 10.69 and 10.79 (2 bs,
NOH); HRMS: calcd for C23H29NO10 (479.1791), found
(479.1802).
1
(s, 3H,CH3), 2.64 (dd, 1H, H-10b, J1 b-1 aˆ16.2 Hz, J1 b-
0
0
0
ˆ7.9 Hz), 2.89 (dd, 1H, H-10a, J1 a-1ˆ3.6 Hz), 3.36 (dd,
0
1
1H, H-2, J1-2ˆ9.6 Hz, J2-3ˆ 9.0 Hz), 3.42±3.53 (m, 1H,
H-4), 3.59±3.78 (m, 3H, H-3, H-5 and H-6b), 3.86 (ddd,
1H, H-1), 4.29 (dd, 1H, H-5, J4-5ˆ10.3 Hz, J5-6ˆ4.0 Hz),
5.50 (s, 1H, CHPh), 7.33±7.53 (m, 10H, HAr); 13C NMR
(CDCl3): d 30.9 CH3; 46.1 C-10; 68.7 C-6; 70.4 C-5; 74.2
C-4; 75.2 C-2; 75.9 C-1; 81.0 C-3; 101.9 CHPh; 126.3,
128.4, 129.3, 137.0 CAr; 206.7 CO; HRMS: calcd for
C16H20O6 (308.1260), found (308.1247).
1-C-b-d-glucopyranosyl-3-phenylacetone oxime (14). To
a solution of compound 13 (180 mg, 0.37 mmol) dissolved
in 2 mL methanol at room temperature was added sodium
methoxide (370 mL, 1 M in methanol, 0.1 equiv.). The reac-
tion mixture was stirred for 20 h then made neutral with
Amberlite IR-120 (H1) and the solvent was removed in
vacuo. The residue was puri®ed by ¯ash column chromato-
graphy (AcOEt/MeOH/water 85/10/5) to afford a 4:3
diastereomeric mixture of oximes 14 (82 mg, 70%) as a
1-C-b-d-glucopyranosylacetone
oxime
O-sulfonate
potassium salt (11). Starting from ketone 10 (104 mg,
0.337 mmol), the procedure used for compound 6 produced
a E1Z diastereomeric mixture of oxime sulfonates 11
(63 mg, 53%) as a white amorphous solid. Mp 130±1348C
1
white amorphous solid. Mp 70±728C; H RMN (CD3OD):
major isomer 14a: d 2.30 (dd, H-10b, J1 b-1 aˆ14.6 Hz,
0
0
1
J1 b-1ˆ10.0 Hz), 2.62±2.80 (m, H-10a), 2.91±3.01 (m,
0
(decomposition); H NMR (CD3OD): d 1.89 (s, CH3m),
1.93 (s, CH3M), 2.33 (dd, H-10bm, J1 b-1 aˆ14.5 Hz,
0
0
H-4), 3.05±3.20 (m, H-2), 3.27±3.40 (m, H-1, H-5, H-6a
and H-6b), 3.51±3.68 (m, H-3), 3.69±3.81 (m, H-30b), 3.89
J1 b-1ˆ9.8 Hz), 2.50 (dd, H-10 bM, J1 b-1 aˆ14.6 Hz,
0
0
0
J1 b-1ˆ9.8 Hz), 2.70 (dd, H-10 am, J1 a-1ˆ2.7 Hz), 2.79 (dd,
(d, J3 b-3 aˆ14.8 Hz, H-30a), 7.23±7.48 (m, 10H, HAr); minor
isomer 14b: d 2.62±2.80 (m, H-10a and H-10b), 3.05±3.20
(m, H-2 and H-4), 3.27±3.40 (m, H-3), 3.42±3.50 (m, H-1),
3.51±3.68 (m, H-5 and H-6b), 3.69±3.81 (m, H-30b and
0
0
0
0
H-10 aM, J1 a-1ˆ2.5 Hz), 3.08 (dd, H-2M, J1-2ˆ9.8 Hz,
0
J2-3ˆ9.8 Hz), 3.11 (dd, H-2m, J1-2ˆ9.8 Hz, J2-3ˆ9.8 Hz),
3.20±3.77 (m, H-1, H-3, H-4, H-5, H-6a and H-6b); 13C
NMR (CD3OD): d 14.9 C-10M; 20.1 C-10m; 32.8 C-30m;
37.9 C-30M; 61.1 C-6; 70.1, 77.6,1 C-3 and C-4; 73.4,
73.8 C-2; 76.3, 76.6 C-1; 79.9 C-5; 166.3, 166.6 C-20
HRMS: calcd for C9H16KNO9S (353.0183), found
(353.0192).
H-6a),3.89 (d, H-30b, J3 b-3 aˆ14.8 Hz), 7.23±7.48 (m,
0
0
13
10H, HAr); C RMN (CDCl3): d 29.4, 33.1 C-10 36.3,
39.5 C-30 60.3, 60.5 C-6; 69.2, 69.4 C-2; 73.3, 73.7
C-4; 75.8, 75.9 C-1; 77.0, 77.1 C-3; 78.9, 79.2 C-5;
125.8, 126.5, 126.8, 128.6, 128.7, 128.8, 139.9, 136.5
CAr; HRMS: calcd for C15H21NO6 (311.1369), found
(311.1372).
1-C-(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)-3-phenyl-
acetone (12). A solution of compound 5 (151 mg,
0.510 mmol) in pyridine (4 mL) and acetic anhydride
(2 mL, 40 equiv.) was stirred for 15 h at room temperature.
The solvent was then removed in vacuo (coevaporation with
toluene) and the residue puri®ed by ¯ash column chromato-
graphy (petroleum ether±AcOEt, 7:3) to yield ketone 12
(187 mg, 79%) as a white solid. Mp 90±928C; [a]Dˆ215
(c 1.0, CHCl3); 1H NMR (CDCl3): d 1.94, 1.97, 2.00, 2.04 (4
Myrosinase inhibition assays: A couple of hexokinase/
glucose 6-phosphate dehydrogenase was used to assay the
formation of glucose during the hydrolysis of glucosinolate
(sinigrin) with myrosinase. The transformation was checked
at 340 nm. Reference assays without C-glucosinolate was
realized with solutions of sinigrin (0.3 mM), myrosinase
(6.1024 U/mL), MES buffer (pH 6.5), MgCl2 (3 mM),
ATP (0.55 mM), NADP (0.72 mM), hexokinase (0.56 U/
mL), glucose 6-phosphate dehydrogenase (0.35 U/mL) at
258C. For inhibtion assays, C-glucosinolate was added to
attain 10 and 20 mM in ®nal solutions. Results are reported
in the text discussion.
s, 12H, CH3CO), 2.47 (dd, 1H, H-10b, J1 b-1 aˆ16.4 Hz,
0
0
0
J1 b-1ˆ3.2 Hz), 2.75 (dd, 1H, H-10a, J1 a-1ˆ8.7 Hz), 3.63
0
(ddd, 1H, H-5, J5-4ˆ9.8 Hz, J5-6bˆ4.9 Hz, J5-6aˆ2.3 Hz),
3.70 (s, 2H, CH2Ph), 3.98 (ddd, 1H, H-1, J1-2ˆ10.0 Hz),
4.04 (dd, 1H, H-6b, J6b-6aˆ12.3 Hz), 4.20 (dd, 1H, H-6a),
4.84 (dd, 1H, H-4, J4-3ˆ9.4 Hz), 5.00 (dd, 1H, H-2,
J2-3ˆ9.4 Hz), 5.16 (dd, 1H, H-3), 7.12±7.33 (m, 5H, HAr);
13C NMR (CDCl3): d 22.85, 22.98 CH3CO; 46.0 C-10; 53.2
C-30; 64.3 C-6; 70.7 C-2; 73.8 C-4; 76.2 C-1; 76.3 C-3; 78.0
C-5; 129.4; 131.0; 131.7, 135.6 CAr; 171.8; 172.1; 172.5;
172.9 COCH3; 206.9 CO; HRMS: calcd for C23H28O10
(464.1682), found (464.1675).
Analysis of C-glucosinolates: The ISO 9167-1 standardized
method used to analyze glucosinolates in rapeseed was
slightly modi®ed for C-glucosinolates and applied in the
following way. After binding on a DEAE Sephadex A-25
exchanger column (15£8 mm), the C-glucosinolates were
desulfated by a sulfatase (type H1, Sigma), eluted by
water and analyzed by reverse phase liquid chromatography
with a Spherisorb ODS 2, 5 mm, 250£4.6 mm column. The
elution gradient was carried out from 0 to 80% of aceto-
nitrile in water in 15 min and the C-desulfoglucosinolates
were detected by UV absorption at 230 nm. The UV spectra
(200±350 nm) were collected on a diode-array type spectro-
meter (Thermo-Quest, Focus) and treated by the Spectacle
(Thermo-Quest) software.
1-C-(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)-3-phenyl-
acetone oxime (13). Hydroxylamine hydrochloride
(240 mg, 14 equiv.) and sodium acetate (480 mg,
23 equiv.) were dissolved in water (2 mL) and ketone 12
(116 mg, 0.25 mmol) was added. The suspension was
diluted with ethanol (4 mL) until the mixture was homo-
geneous. After 45 min stirring at room temperature, a

2654
V. Aucagne et al. / Tetrahedron 56 (2000) 2647±2654
12. Howard, S.; Withers, S. G. J. Am. Chem. Soc. 1998, 120,
10326±10331.
Acknowledgements
13. Rychnovsky, S. D.; Griesgraber, G.; Zeller, S.; Skalitzky, D. J.
J. Org. Chem. 1991, 56, 5161±5169.
The authors thank the MENRT for ®nancial support (V. A.
and D. G.). We also thank Dr Hugues Driguez and Dr
Sylvain Cottaz (CERMAV, Grenoble, France) and Dr
Renato Iori (ISCI, Bologna, Italy) for myrosinase inhibition
experiments. We are indebted to Dr Sandro Palmieri (ISCI)
for a generous gift of myrosinase.
14. Wallace, R. G. Org. Prep. Proced. Int. 1982, 14, 265±307.
15. Werner, R. M.; Williams, L. M.; Davis, J. T. Tetrahedron Lett.
1999, 39, 9135±9138.
16. Riglowski, A.; Kafarski, P. Tetrahedron 1989, 52, 10685±
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Lett. 1989, 30, 4787±4790.
18. Mathey, F.; Savignac, P. Tetrahedron 1978, 34, 649±654.
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