Synthesis of the allelochemical alliarinoside present in garlic mustard (Alliaria petiolata), an invasive plant species in North America

Article abstract of DOI:10.1016/j.carres.2014.05.006

The allelochemical alliarinoside present in garlic mustard (Alliaria petiolata), an invasive plant species in North America, was chemically synthesized using an efficient and practical synthetic strategy based on a simple reaction sequence. Commercially available 1,2,3,4,6-penta-O-acetyl- β-d-glucopyranose was converted into prop-2-enyl 2,3,4,6-tetra-O-acetyl- β-d-glucopyranoside and subjected to epoxidation. In a one-pot reaction, ring-opening of the epoxide using TMSCN under solvent free conditions followed by treatment of the formed trimethylsilyloxy nitrile with pyridine and phosphoryl chloride, afforded the acetylated β-unsaturated nitriles (Z)-4-(2,3,4,6-tetra-O-β-d-glucopyranosyloxy)but-2-enenitrile and its isomer (E)-4-(23,4,6-tetra-O-β-d-glucopyranosyloxy)but-2-enenitrile. Deacetylation of Z- and/or E-isomers afforded the target molecules alliarinoside and its isomer.

Full text of DOI:10.1016/j.carres.2014.05.006

Carbohydrate Research 394 (2014) 13–16
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis of the allelochemical alliarinoside present in garlic
mustard (Alliaria petiolata), an invasive plant species in North
America
Carl Erik Olsen a,b, Birger Lindberg Møller a,b,c,d, Mohammed Saddik Motawia a,b,c,
⇑
a Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark
b VILLUM Research Center for ‘Plant Plasticity’, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark
c Center for Synthetic Biology ‘bioSYNergy’, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark
d Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
The allelochemical alliarinoside present in garlic mustard (Alliaria petiolata), an invasive plant species in
North America, was chemically synthesized using an efficient and practical synthetic strategy based on a
Received 14 January 2014
Received in revised form 2 May 2014
Accepted 10 May 2014
simple reaction sequence. Commercially available 1,2,3,4,6-penta-O-acetyl-b-
D-glucopyranose was con-
verted into prop-2-enyl 20,30,40,60-tetra-O-acetyl-b-
D-glucopyranoside and subjected to epoxidation. In a
Available online 17 May 2014
one-pot reaction, ring-opening of the epoxide using TMSCN under solvent free conditions followed by
treatment of the formed trimethylsilyloxy nitrile with pyridine and phosphoryl chloride, afforded the
Keywords:
Allyl b-D-glucopyranoside tetra-acetate
Epoxidation
Trimethylsilyl cyanide
acetylated b-unsaturated nitriles (Z)-4-(20,30,40,60-tetra-O-b-
D
-glucopyranosyloxy)but-2-enenitrile and
-glucopyranosyloxy)but-2-enenitrile. Deacetylation of Z- and/or
E-isomers afforded the target molecules alliarinoside and its isomer.
Ó 2014 Elsevier Ltd. All rights reserved.
its isomer (E)-4-(20,30,40,60-tetra-O-b-
D
a,b-Unsaturated nitriles
Deprotection
Plants produce a large number of allellochemicals that when
introduced into North America in the 1860s. Unfortunately it has
now spread as an invasive species on most of the North American
continent where it is established as a predominant plant in numer-
ous different environments.7 Mustard garlic produces allellochem-
icals that prevent the growth of the mycorrhizal fungi species that
facilitate the ability of other plant species to take-up phosphorous
ions from the soil.8,9 This enables garlic mustard to slow down the
growth of native plants. Likewise, the insect herbivores feeding on
garlic mustard in its original native environments are not present
in North America which further adds to its property as an invasive
species.
liberated in the environment affect the growth, survival, and repro-
duction of other organisms to the plants own benefit.1,2 They may
exert their effects, for example, by preventing seeds from other
plant species to germinate or by inhibiting the growth of other
plants.3 Allellochemical production may thus offer a plant species
unique opportunities to obtain higher abundance and to spread
to different types of plant communities. Many of these allello-
chemicals are produced in low amounts or as exudates from roots
and thus difficult to isolate in amounts required for studies of the
effect of the specific allellochemical in different biological systems.
In many of such cases chemical synthesis of the allellochemical
constitutes the obvious alternative.
In this study we report an easy and cheap route to the synthesis
of alliarinoside (5) and its l stereoisomer 6. Alliarinoside is an all-
ellochemical produced by garlic mustard (Alliaria petiolata).4 It
was initially isolated from the foliage of garlic mustard5 and has
not been described to be present in any other plant species. Garlic
mustard is a common native plant at most continents and one of
the oldest known herbs in Europe selected based on its mild garlic
and mustard flavor.6 For the very same reason garlic mustard was
Garlic mustard belongs to the mustard family, Brassicaceae.4
The main class of secondary metabolites produced by the mustard
family is the glucosinolates and garlic mustard produces glucosin-
olates derived from homomethionine, dihomomethionine, phenyl-
alanine, homophenylalanine, and tryptophan.10,11 2-Propenyl
glucosinolate (sinigrin) derived from homomethionine is the most
dominant glucosinolate in aerial tissues of garlic mustard and is
the glucosinolate showing the closest structural similarity to allia-
rinoside. However, alliarinoside is not a glucosinolate and belongs
to the related class of compounds denoted as hydroxynitrile glyco-
sides.12 Except for Carica papaya,13 other glucosinolate producing
plants are not known to produce hydroxynitrile glucosides.
Hydroxynitrile glucosides are widespread in the plant kingdom
⇑
Corresponding author. Tel.: +45 35333305.
E-mail address: mosm@life.ku.dk (M.S. Motawia).
http://dx.doi.org/10.1016/j.carres.2014.05.006
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

14
C. E. Olsen et al. / Carbohydrate Research 394 (2014) 13–16
with cyanogenic glucosides (
a
-hydroxynitrile glucosides) being
in 1,2-disubstituted double bonds are around 17 Hz (the range
from 12 to 18). The observed value of 11.1 and 11.4 Hz for the ole-
finic protons of 4a and 5, respectively did not allow for unambigu-
ous assignment of their cis-configuration. However, the observed
value of 16.4 Hz for the olefinic protons of 4b and 6 confirmed
the assignment of their trans-configuration and thus the cis-config-
uration of the olefinic protons in 4a and 5 as previously shown.5
In conclusion, an efficient, practical, and stereospecific synthe-
sis of alliarinoside 5 and its isomer 6 was developed starting from
present in pteridophytes, gymnosperms and angiosperms as well
as in a few arthropods.12,14,15 Alliarinoside belongs to the
c
-
hydroxynitriles which together with b-hydroxynitriles typically
co-occur with cyanogenic glucosides but in a restricted number
of plant species.12,16
The only previously reported chemical synthesis of 5 and 6 is the
small scale synthesis by Haribal and co-worker,5 based on ozonolysis
of the allyl glucoside 2 to produce the corresponding aldehyde which
ina Horner–Emmonsreaction withdiethylcyanomethylenephospho-
nateafforded the protectedglucoside4a and4bwhich provided 5 and
6 after deprotection. However, our need for alliarinoside (5) and its
stereoisomer 6 for use in large scale biological experiments prompted
us to develop an easier synthetic approach. The synthetic strategy is
outlined in Scheme 1.
b-
D-glucopyranose pentaacetate via a simple reaction sequence
(allyl glucosidation, epoxidation, one-pot
a,b-unsaturated nitrile
formation and deacetylation). The method offers a straightforward
route to the synthesis and isolation of alliarinoside (5) and its
stereoisomer 6.
Commercially available 1,2,3,4,6-penta-O-acetyl-b-
D-glucopyranose
1. Experimental
(1) was converted into prop-2-enyl 20,30,40,60-tetra-O-acetyl-b-
D-
glucopyranoside (2) using ZnCl2 as described by Yuasa and Yuasa17
as the most convenient procedure among several alternative
options.17–20 Epoxidation of 2 was accomplished using m-chloro-
perbenzoic acid (m-CPBA) according to reported procedures21–23
except for use of 2 equiv of m-CPBA instead of 3 equiv22 and reflux
in C2H2Cl2 instead of CHCl3 or CH2Cl2 for 30 min to obtain (2R)- and
1.1. General methods
Melting points were determined using a Mettler FP81 MBC Cell
connected to a Mettler FP80 Central processor unit. Optical rota-
tions were measured at 21 2 °C with an Optical Activity Ltd AA-
1000 Polarimeter. All reactions were monitored by TLC on alumi-
num sheets coated with silica gel 60F254 (0.2 mm thickness, E.
Merck, Darmstadt, Germany) and the components present were
detected by charring with 10% H2SO4 in MeOH. Column chroma-
tographies were carried out using silica gel 60 (particle size
0.040–0.063 mm, 230–400 mesh ASTM, E. Merck). Solvent extracts
were dried with anhyd MgSO4 unless otherwise specified. The 1H
and 13C NMR spectra were recorded on a Bruker Avance 400 spec-
trometer at 400 and 101 MHz, respectively. d-Values are relative to
internal TMS; coupling constants (J) are given in Hz. HR-ESI-MS
(2S)-2,3-epoxypropyl 20,30,40,60-tetra-O-acetyl-b-
D-glucopyranoside
(3) in near 100% yield and an epimeric ratio of 3:2. In a one-pot
reaction, ring-opening of epoxide 3 using TMSCN under solvent
free conditions24 followed by treatment of the formed trimethylsi-
lyloxy nitrile with pyridine and phosphoryl chloride25 afforded
the acetylated
-glucopyranosyloxy)but-2-enenitrile (4a), (33% yield) and, (E)-
4-(20,30,40,60-tetra-O-b-
-glucopyranosyloxy)but-2-enenitrile (4b),
a
,b-unsaturated nitriles (Z)-4-(20,30,40,60-tetra-O-b-
D
D
(43% yield) which were separated using chromatography on silica
in a total yield of 77% (Z/E = 3:4). Deprotection of 4a and/or 4b with
triflic acid in MeOH afforded the target molecules 5 and 6 after
chromatographic purification on silica gel. The overall yield of 5
and 6 was 14% and 19%, respectively, calculated from 1.
All the synthesized compounds were characterized and their
identity verified using electrospray ionization mass spectrometry
and 1H and 13C NMR. Typical coupling constant values for cis
protons in 1,2-disubstituted double bonds are known to be around
10 Hz (the range is from 6 to 12), whereas those for trans protons
spectra were recorded on
spectrometer.
a
Bruker micrOTOF-Q mass
1.2. Prop-2-enyl 20,30,40,60-tetra-O-acetyl-b-
D-glucopyranoside
(2)17
b-D-Glucose pentaacetate 1 (15.0 g, 38.4 mmol), allyl alcohol
(10.0 mL, 117.1 mmol), and ZnCl2 (5.3 g, 38.9 mmol) in toluene
(120.0 mL) were reacted as previously described.17 The product
OAc
OAc
OAc
O
O
O
O
lit. 17
AcO
AcO
AcO
AcO
AcO
AcO
O
a
O
OAc
OAc
OAc
OAc
3
1
2
OAc
OH
O
O
AcO
AcO
HO
HO
O
O
c
c
HO
OAc
OH
CN
CN
b
4a
5
3
+
HO
OAc
O
CN
O
CN
O
AcO
AcO
O
HO
OH
OAc
6
4b
Scheme 1. Reagents and conditions: (a) m-CPBA, DCE, 85 °C, 1 h, quant.; (b) TMS-CN, LiClO4, 3H2O, 85 °C 1 h, Py/POCl3, 85 °C, 2 h, 77% yield; (c) TfOH, MeOH, rt, 3 h,
Amberlyst-A26 (OHꢀ form), 85% yield.

C. E. Olsen et al. / Carbohydrate Research 394 (2014) 13–16
15
was purified by column chromatography on silica gel with 60–80%
Et2O/n-pentane as eluent to give 2 as colorless needles from Et2O/
n-pentane (7.5 g, 19.2 mmol, 50% yield). Mp 90 °C (lit.19 89–90 °C,
on silica gel (150 g) with 60–80% Et2O/n-pentane as eluent to give
4a and 4b as colorless crystals from Et2O/n-pentane. Compound
4a: Rf = 0.23, 80% Et2O/n-pentane (2.7 g, 6.5 mmol, 33% yield). Mp
24
23
24
lit.17 85–86 °C). [
(c 1, CHCl3) and lit.19
a
]
ꢀ25.3° (c 3.8, CHCl3) [(lit.17
[
a
]
ꢀ25.0°
96 °C. [
a]
ꢀ4.6° (c 2.5, CHCl3). 1H NMR (CDCl3): 2.10, 2.06,
D
D
D
25
ꢀ25.8° (c 5, CHCl3)]. 1H NMR (CDCl3):
2.03, 2.01 (4s, 12H, CH3CO), 3.73 (ddd, 1H, J5 ,4 = 10.0, J5 ,6 b = 4.9,
0
0
0
0
[
a
]
D
2.09, 2.05, 2.03, 2.01 (4s, 12H, CH3CO), 3.69 (ddd, 1H, J5 ,4 = 10.0,
J5 ,6 a = 2.4, H-50), 4.17 (dd, 1H, J6 a,6 b = 12.4, J6 a,5 = 2.4, H-60a), 4.26
0
0
0
0
0
0
0
0
J5 ,6 b = 4.8, J5 ,6 a = 2.5, H-50), 4.10 (ddt, 1H, J = 13.2, 6.2, 1.4, H-1a),
(dd, 1H, J6 b,6 a = 12.4, J6 b,5 = 4.9, H-60b), 4.52 (ddd, 1H, J = 14.3,
0
0
0
0
0
0
0
0
4.15 (dd, 1H, J6 a,6 b = 12.3, J6 a,5 = 2.4, H-60a), 4.27 (dd, 1H,
6.8, 1.7, H-4b), 4.57 (d, 1H, J1 ,2 = 7.8, H-10), 4.59 (ddd, 1H, J = 14.3,
0
0
0
0
0
0
J6 b,6 a = 12.3, J6 b,5 = 4.8, H-60b), 4.34 (ddt, 1H, J = 13.2, 4.9, 1.6, H-
5.7, 1.7, H-4a), 5.02 (dd, 1H, J2 ,3 = 9.6, J2 ,1 = 8.0, H-20), 5.09 (dd,
0
0
0
0
0
0
0
0
1b), 4.56 (d, 1H, J1 ,2 = 7.9, H-10), 5.03 (dd, 1H, J2 ,3 = 9.6,
1H, J4 ,5 = 10.0, J4 ,3 = 9.4, H-40), 5.21 (t, 1H, J3 ,2 = J3 ,4 = 9.5, H-30),
5.48 (dt, 1H, J = 11.2, 1.7, H-2), 6.58 (ddd, 1H, J = 11.2, 6.7, 5.7,
H-3). 13C NMR (CDCl3): 21.1, 21.1, 21.0, 21.0 (4s, CH3CO), 61.8
(C-60), 67.4 (C-4), 68.2 (C-40), 71.1 (C-20), 72.2 (C-30), 72.7 (C-50),
100.4 (C-10), 101.3 (C-2), 114.8 (CN), 149.5 (C-3), 171.0, 170.6, 169.8,
169.7 (4s, CH3CO). HR-ESI-MS: m/z calcd for [C11H23NO10+Na]+
= 436.1214; found m/z [M+Na]+ = 436.1217. Compound 4b:
0
0
0
0
0
0
0
0
0
0
0
0
J2 ,1 = 7.9, H-20), 5.10 (dd, 1H, J3 ,2 = 10.0, J3 ,4 = 9.4, H-40), 5.21
0
0
0
0
0
0
(dq, 1H, J = 10.5, 1.5, H-3a), 5.21 (t, 1H, J4 ,3 = J4 ,5 = 9.5, H-30),
5.28 (dq, 1H, J = 17.2, 1.6, H-3b), 5.85 (dddd, 1H, J = 17.2, 10.6,
6.1, 4.8, H-2). 13C NMR (CDCl3): 20.7, 20.6, 20.6, 20.6 (4s, CH3CO),
62.0 (C-60), 71.9 (C-50), 70.0 (C-1), 68.5 (C-40), 72.9 (C-30), 71.3
(C-20), 99.6 (C-10), 117.7 (C-3), 133.3 (C-2), 170.7, 170.3, 169.3,
169.3 (4s, CH3CO). ESIMS: m/z [M+Na]+: 411.
0
0
0
0
Rf = 0.17, 80% Et2O/n-pentane (3.6 g, 8.7 mmol, 44% yield) as color-
less crystals. Mp 107 °C. [
a
]
ꢀ29.2° (c 6, CHCl3). 1H NMR (CDCl3):
24
D
1.3. (2R)- and (2S)-2,3-Epoxypropyl 20,30,40,60-tetra-O-acetyl-O-b-
2.10, 2.06, 2.03, 2.02 (4s, 12H, CH3CO), 3.72 (ddd, 1H, J5 ,4 = 10.0,
0
0
J5 ,6 b = 4.7, J5 ,6 a = 2.4, H-50), 4.15 (dd, 1H, J6 a,6 b = 12.4, J6 a,5 = 2.5,
0
0
0
0
0
0
0
0
D
-glucopyranoside (3)
H-60a), 4.23 (ddd, 1H, J = 16.9, 3.9, 2.3, H-4b), 4.26 (dd,
1H, J6 b,6 a = 12.4, J6 b,5 = 4.7, H-60b), 4.52 (ddd, 1H, J = 16.9, 3.6,
0
0
0
0
A solution of 2 (10.0 g, 28.3 mmol) and m-CPBA 70% (14.0 g,
2.4, H-4a), 4.56 (d, 1H, J1 ,2 = 7.9 Hz, H-10), 5.05 (dd, 1H, J2 ,3 = 9.6,
0
0
0
0
56.6 mmol) in dry DCE (100 mL) was refluxed for 30 min and reac-
tion was worked-up as described.9 The product was purified by
column chromatography on silica gel with 60–80% Et2O/n-pentane
as eluent to give 3 (11.1 g, 27.5 mmol, 97% yield). An analytical
J2 ,1 = 7.9, H-20), 5.09 (t, 1H, J4 ,3 = J4 ,5 = 9.8, H-40), 5.22 (t, 1H,
0
0
0
0
0
0
J3 ,2 = J3 ,4 = 9.5, H-30), 5.63 (dt, 1H, J = 16.3, 2.4, H-2), 6.70 (dt, 1H,
J = 16.3, 3.7, H-3). 13C NMR (CDCl3): 21.1, 21.1, 21.0, 21.0
(4s, CH3CO), 61.7 (C-60), 67.3 (C-4), 68.2 (C-40), 71.0 (C-20), 72.1
(C-30), 72.5 (C-50), 100.3 (C-10), 101.3 (C-2), 116.9 (CN), 148.8
(C-3), 171.0, 170.6, 169.8, 169.7 (4s, CH3CO). HR-ESI-MS: m/z
calcd for [C11H23NO10+Na]+ = 436.1214; found m/z [M+Na]+ =
436.1211.
0
0
0
0
sample of 3 was obtained as colorless needles from Et2O/n-pen-
tane. Mp 116 °C (lit.22 105–106 °C and lit.21 115–117 °C). [
a]
24
D
ꢀ18.8° (c 4.2, CHCl3) [(lit.22
[a
]
D
ꢀ19.0° (c 1, CHCl3) and lit.21
30
[
a]
D
ꢀ18.9° (c 4.0, CHCl3)]. A-epimer (major): 1H NMR (CDCl3):
2.09, 2.7 2.03, 2.01 (CH3CO), 2.55 (dd, J3a,3b = 4.9, J3a,2 = 2.7, H-3a),
2.80 (dd, J3a,3b = 4.9, J3b,2 = 4.2, H-3b), 3.14 (m, H-2), 3.48 (dd,
J1a,1b = 11.9, J1b,2 = 6.8, H-1b), 3.71 (m, H-50), 4.05 (dd, J1a,1b = 11.9,
1.5. (Z)-4-(b-D-Glucopyranosyloxy)but-2-enenitrile (5) and (E)-4-
J1a,2 = 2.9, H-1a), 4.15 (dd, J6 a,6 b = 12.3, J6 a,5 = 2.4, H-60a), 4.26
(b- -Glucopyranosyloxy)but-2-enenitrile (6)
D
0
0
0
0
(dd, J6 a,6 b = 12.3, J6 b,5 = 4.7, H-60b), 4.65 (d, J1 ,2 = 8.0, H-10), 5.02
0
0
0
0
0
0
(dd, J2 ,3 = 9.6, J1 ,2 = 8.0, H-20), 5.10 (t, J4 ,3 = J4 ,5 = 9.7, H-40), 5.22
0
0
0
0
0
0
0
0
(t, J3 ,2 = J3 ,4 = 9.5, H-30). 13C NMR (CDCl3): 20.7, 20.7, 20.6, 20.6
(4s, CH3CO), 44.1 (C-3), 50.5 (C-2), 61.9 (C-60), 68.4 (C-40), 70.7
(C-1), 71.2 (C-20), 71.9 (C-50), 72.8 (C-30), 100.5 (C-10), 170.6,
170.2, 169.4, 169.4 (4s, CH3CO). B-epimer (minor): 1H NMR
(CDCl3): 2.09, 2.7, 2.03, 2.01 (CH3CO), 2.65 (dd, J3a,3b = 5.1,
J3a,2 = 2.7, H-3a), 2.78 (dd, J3a,3b = 5.1, J3b,2 = 4.1, H-3b), 3.14 (m,
H-2), 3.71 (m, H-50), 3.50 (dd, J1a,1b = 12.3, J1b,2 = 4.2, H-1b), 3.87
1.5.1. General procedure
0
0
0
0
Trifluoromethanesulfonic acid (triflic acid:TfOH) (0.65 mL,
7.34 mmol) was added in one portion at room temperature to a
stirred solution of 4a or 4b (1.18 mmol) in dry CH3OH (5 mL) under
Ar. Stirring was continued at room temperature for 3 h after which
the reaction mixture was supplemented with CH3OH (25 mL), neu-
tralized with Amberlyst-A26 (OHꢀ form) resin, filtered, and evapo-
rated to dryness. The residue was then chromatographed on silica
gel with 10% MeOH/CH2Cl2 as eluent to afford compound 5 as col-
0
0
(dd, J1a,1b = 12.3, J1a,2 = 3.2, H-1a), 4.14 (dd, J6 a,6 b = 12.3,
J6 a,5 = 2.4, H-60a), 4.27 (dd, J6 a,6 b = 12.3, J6 b,5 = 4.7, H-60b), 4.56
0
0
0
0
0
0
(d, J1 ,2 = 8.0, H-10), 5.00 (dd, J2 ,3 = 9.6, J1 ,2 = 8.0, H-20), 5.09 (t,
orless gum or 6 white solid. Compound 5: (0.25 g, 1.0 mmol, 85%);
0
0
0
0
0
0
J4 ,3 = J4 ,5 = 9.7, H-40), 5.21 (t, J3 ,2 = J3 ,4 = 9.5, H-30). 13C NMR
(CDCl3): 20.7, 20.7, 20.6, 20.6 (4s, CH3CO), 44.1 (C-3), 50.3 (C-2),
61.9 (C-60), 68.4 (C-40), 69.1 (C-1), 71.2 (C-20), 71.9 (C-50), 72.7
(C-30), 101.0 (C-10), 170.6, 170.2, 169.4, 169.4 (4s, CH3CO).
[
a
]
ꢀ16.7° (c 2.6, CH3OH). 1H NMR (CD3OD): 3.22 (dd, 1H,
24
0
0
0
0
0
0
0
0
D
J2 ,3 = 9.0, J2 ,1 = 7.7, H-20), 3.31 (m, 1H, H-40), 3.1 (m, 1H, H-50),
0
0
0
0
3.37 (t, 1H, J3 ,2 = J3 ,4 = 9.1, H-30), 3.68 (m, 1H, H-60a), 3.88 (m,
0
0
0
0
1H, H-60b), 4.33 (d, 1H, J1 ,2 = 7.8 , H-10), 4.51 (ddd, 1H, J = 14.6,
6.4, 1.8, H-4b), 4.65 (ddd, 1H, J = 14.6, 5.8, 1.8, H-4a), 5.65 (dt,
1H, J = 11.3, 1.7, H-2), 6.77 (ddd, 1H, J = 11.3, 6.4, 5.8, H-3). 13C
NMR (CD3OD): 62.6 (C-60), 68.5 (C-4), 71.5 (C-40), 75.0 (C-20), 78.0
(C-50), 78.1 (C-30), 101.1 (C-2), 104.2 (C-10), 116.4 (CN), 152.1
(C-3). HR-ESI-MS: m/z calcd for [C10H15NO6+Na]+ = 268.0792;
0
0
1.4. (Z)-4-(20,30,40,60-Tetra-O-acetyl-b-
D-glucopyranosyloxy)but-
2-enenitrile (4a) and (E)-4-(20,30,40,60-tetra-O-acetyl-b-
D-
glucopyranosyloxy)but-2-enenitrile (4b)
A mixture of epoxide 3 (8.0 g, 20.0 mmol), LiClO4ꢁ3H2O (0.32 g,
10.0 mol%), and TMSCN (3.0 mL, 24.0 mmol) was heated at 85 °C
for 1 h under nitrogen. The reaction mixture was cooled to room
temperature, pyridine (40 mL), and POCl3 (6.0 mL, 64.4 mmol) were
added and the reaction mixture heated at 85 °C for 1 h under nitro-
gen. When cooled to room temperature, Et2O (200 mL) was added
and the mixture was filtered through a layer of sand and silica
gel. The silica gel was washed several times with Et2O
(5 ꢂ 25 mL), and the collected filtrate was washed thoroughly with
satd aq NaHCO3(3 ꢂ 50 mL), water (3 ꢂ 50 mL), and brine (50 mL),
dried and evaporated to dryness. The residue was chromatographed
found m/z [M + Na]+ = 268.0787. Compound 6: (0.25 g, 1.0 mmol,
24
85%). Mp 92 °C. [
a
]
ꢀ39.2° (c 2.2, CH3OH). 1H NMR (CD3OD):
D
3.22 (dd, 1H, J2 ,3 = 9.1 Hz, J2 ,1 = 7.8 Hz, H-20), 3.36 (t, 1H, J3 ,2
=
0
0
0
0
0
0
J3 ,4 = 9.0, H-30), 3.27 (m, 1H, H-40), 3.27 (m, 1H, H-50), 3.65 (m,
0
0
1H, H-60a), 3.86 (m, 1H, H-60b), 4.29 (d, 1H, J1 ,2 = 7.7, H-10), 4.32
(ddd, 1H, J = 17.4, 3.9, 2.3, H-4b), 4.54 (ddd, 1H, J = 17.4, 3.7, 2.4,
H-4a), 5.94 (dt, 1H, J = 16.3, 2.3, H-2), 6.88 (dt, 1H, J = 16.3, 3.8,
0
0
13
H-3). C NMR (CD3OD): 62.7 (C-60), 68.6 (C-4), 71.6 (C-40), 75.1
(C-20), 78.0 (C-50), 78.1 (C-30), 101.2 (C-2), 104.1 (C-10), 118.5 (CN),
152.7 (C-3). HR-ESI-MS: m/z calcd for [C10H15NO6+Na]+ = 268.0792;
found m/z [M+Na]+ = 268.0794.

16
C. E. Olsen et al. / Carbohydrate Research 394 (2014) 13–16
6. Saul, H.; Madella, M.; Fischer, A.; Glykou, A.; Hartz, S.; Craig, O. E. PLoS ONE
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Financial support from the VILLUM Research Center ‘Plant
Plasticity’, from the UNIK Center for Synthetic Biology and Center
for Synthetic Biology: ‘bioSYNergy’ from the University of Copen-
hagen Excellence Program for Interdisciplinary Research, and from
the Center for Biosustainability supported from the Novo Nordisk
Foundation is greatly appreciated.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2014.05.
006.
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