Sweet Poisons: Synthetic Strategies towards Tutin Glycosides

Article abstract of DOI:10.1071/CH16429

The polycyclic, polyoxygenated picrotoxane tutin was subjected to various glycosylation reaction conditions in an effort to synthesise β-linked tutin glycosides, recently found in toxic honeys. Cationic palladium-mediated glycosylation of tutin was succes

Full text of DOI:10.1071/CH16429

CSIRO PUBLISHING
Aust. J. Chem. 2017, 70, 301–306
Full Paper
http://dx.doi.org/10.1071/CH16429
Sweet Poisons: Synthetic Strategies towards
Tutin Glycosides
A
A B
,
A B
,
A
Duong Nhu, Lesley Larsen,
Nigel B. Perry,
David S. Larsen,
A C
,
and Bill C. Hawkins
A
Department of Chemistry, University of Otago, PO Box 56, Dunedin 9054,
New Zealand.
B
The New Zealand Institute for Plant and Food Research Limited, Department of
Chemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
C
Corresponding author. Email: bhawkins@chemistry.otago.ac.nz
The polycyclic, polyoxygenated picrotoxane tutin was subjected to various glycosylation reaction conditions in an effort
to synthesise b-linked tutin glycosides, recently found in toxic honeys. Cationic palladium-mediated glycosylation of tutin
was successful; however, the a-linked tutin tetrabenzyl glucoside was obtained as the major product (5 : 1, a : b).
Hydrogenolysis of the benzyl ether protecting groups resulted in concomitant tutin double-bond migration. Epoxide
opening and rearrangement were observed upon acetylation of the tutin glucoside.
Manuscript received: 22 July 2016.
Manuscript accepted: 23 August 2016.
Published online: 15 September 2016.
Introduction
We are not aware of any previous reports of synthetic glycosyl-
ation of tutin or of any other picrotoxanes;[1b] however, acyla-
tion of the secondary alcohol 2-OH has been achieved,[8] and
double acetylation at both the 2-OH and C6-OH has been
reported.[9] The highly strained skeleton of tutin (1), including
two epoxide rings and a lactone, was expected to be susceptible
to various rearrangements, as found in previous attempts to
solve and interrelate picrotoxane structures.[10]
Assembly of the tutin glucoside 2 could be achieved via the b-
O-glycosylation reaction between tutin (1) and an activated sugar
donor of the general structure 3 (Scheme 1). Numerous methods
of O-glycosylation have been reported towards the synthesis
of complex glycosides with anomeric b-stereoselectivity.[11]
Strategies to achieve selectivity include using silyl tethering
groups,[12] and judicious choice of leaving groups and/or neigh-
bouring group participation,[11e,13] catalyst,[11a] and conforma-
tional rigidity[13c,14] of the glycosyl donor.
The neurotoxin tutin (1),[1] a polyoxygenated, polycyclic ses-
quiterpene from the picrotoxane family,[1b] has been shown to be
present in New Zealand toxic honey (Scheme 1).[2] In fact, until
recently, it was thought to be the major toxic component in honey
samples and as such its concentration was carefully moni-
tored.[3,4] Concerns about the presence of other unmonitored
tutin-based metabolites[2] led to a human pharmacokinetic study
of tutin from honey. The study indicated that unknown conjugates
of tutin were also present.[4] The structures of two main tutin
conjugates were later elucidated to be 2-(b-D-glucopyranosyl)-
tutin (2; Scheme 1) and 2-[60-(a-D-glucopyranosyl)-b-D-gluco-
pyranosyl]-tutin (not shown).[5] Due to the difficulty in isolating
useful quantities of these compounds from honeys, we sought to
develop a synthesismethod for preparing tutin glycoside 2. Larger
amounts of this tutin glycoside would allow further examination
of its toxicity and pharmacokinetics, and the compound could
potentially be used as a standard for food safety analyses.
There has been only one total synthesis study of tutin (1)
reported to date.[6] Our approach was to use natural tutin (1),
available in reasonable yields from leaves of the plant tutu,
Coriaria species,[7] as the substrate for glycosylation efforts.
Results and Discussion
Our first glycosylation efforts, subjecting tutin (1) to epoxide 4
(obtained from dimethyldioxirane (DMDO)-mediated glycal
OH
OPG
OH
O
β-O-glycosylation
2
OH
HO
HO
O
PGO
PGO
HO
O
O
ϩ
1Ј
HO
LG
PGO
O
O
O
O
O
O
O
2
1
3
Scheme 1. PG, protecting group; LG, leaving group.
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302
D. Nhu et al.
oxidation), activated with ZnCl2 under the conditions reported
by Halcomb and Danishefsky[13b] or Bronsted acid–base
pair,[15] were unsuccessful (Scheme 2). Our next choice of sugar
donor was the known tetraacetylated trichloroacetimidate 6
(Scheme 3).[16] It was anticipated that the neighbouring acetyl
group would stabilise the reactive oxocarbenium species 7 and
direct the tutin 1 nucleophile to the b face, thereby conferring
b-selectivity.
Preparation of the acetimidate 6 was performed using a
literature procedure wherein tetra-O-acetyl-D-glucose (5) was
treated with trichloroacetonitrile[17] to provide a mixture of
anomers (1 : 2, a : b), which was used without further purification.
To our disappointment, despite screening several commonly used
Lewis acids (Table 1, entries 1–4) at low-to-ambient tempera-
tures, no evidence for the formation of the desired glycoside was
found. In the case of trimethylsilyl trifluoromethanesulfonate
(TMSOTf), decomposition of tutin (1) was observed. Only start-
ing materials were recovered upon treatment with AuCl3 and
ZnCl2, whereas boron trifluoride diethyl etherate (BF3ꢀEt2O) led
to a Chapman rearrangement of the sugar donor.[18]
corresponding oxocarbenium ion. Thus, we applied the in situ
generated Pd(PPh3)2(OTf)2 species[19b] and the commercially
available Pd(MeCN)4(BF4)[219a] (Table 1, entries 5–7) to our
system. Pleasingly, traces of a glycosylated tutin derivative were
observed when conducting the reaction in dichloromethane
(DCM; Table 1, entry 5), as indicated by electrospray ionisation
mass spectrometry (ESI-MS). The spectrum featured a peak at
m/z 839.3372, assigned as the [M þ Na]þ. Notably, when MeCN
was used as the solvent, no traces of the product were found
(Table 1, entry 6). This is consistent with the proposed mecha-
nism as a coordinating solvent is expected to affect the binding
of the metal complex to the glycosyl donor. Regardless, no
conditions were found to generate the corresponding glycosy-
lated tutin in an appreciable amount. This low conversion could
be attributed to the stabilising effect of the acyl-protecting groups,
which deactivate the oxacarbenium ion towards glycosylation
with the sterically crowded secondary alcohol on tutin (1).
These preliminary results prompted us to investigate the
transformation using the tetrabenzylated trichloroacetimidate 9
bearing armed protecting groups (Scheme 4).[20] The glycosyl
donor 9 was obtained in three steps as described in the literature
(Scheme 4).[21] With the more reactive glycosyl donor in hand,
we first examined the traditional Lewis acid-mediated process
with BF3ꢀEt2O as this was previously shown to be compatible
with tutin (1) (Table 2, entry 1). As the glycosyl donor 9 does not
possess a participating group, poor stereoselectivity in the
glycosylation step may be expected. Furthermore, solvent
choice is also well known to affect selectivity. Thus, we
examined various reaction conditions to account for such effects
(Table 2, entries 1–3).[22] The use of diethyl ether (Et2O)
achieved the highest conversion (10 % by NMR), with the least
amount of competing processes such as trichloroacetimidate
hydrolysis and diglucoside formation (Table 2, entry 3).
Recently, reports on the use of cationic metal catalysts to
facilitate glycosylation have emerged.[19] These metal com-
plexes are proposed to coordinate and promote the departure
of the leaving group on the glycosyl donor to generate the
OTBS
ZnCl2
or CSA and thiourea
O
TBSO
TBSO
ϩ
1
2
O
4
Scheme 2. TBS, tert-butyldimethylsilyl; CSA, camphorsulfonic acid.
OAc
OAc
OAc
1
, Lewis acid
or catalyst
ϩ
see ref. 17 for
reaction conditions
AcO
AcO
O
AcO
AcO
O
NH
AcO
AcO
O
O
AcO
OH
O
AcO
CCl3
O
7
2
6
(1:2, α :β)
5
O
H2N
CCl3
Scheme 3.
Table 1. Screening of catalysts for the glycosylation of tutin 1 with sugar donor 6
cat., catalytic amount; NP, no product
Sugar donor
Entry
Conditions
Solvent
Temperature
ConversionA [%]
6
1
2
3
4
5
6
7
TMSOTf
DCM
08C–RT
RT
Decomposition
AuCl3 (cat.)
DCM
NP
ZnCl2 (cat. or 1–2.1 equiv.)
BF3ꢀEt2O (cat.)
DCM
ꢁ788C to RT
08C or RT
RT
NP
DCM/Et2O/MeCN
DCM
NP
Pd(MeCN)4(BF4)2 (cat.)
Pd(MeCN)4(BF4)2 (cat.)
PdCl2(PPh3)2, AgOTf
Trace
NP
MeCN
RT
DCM
RT
NP
ADetermined by NMR.

Sweet Poisons: Strategies towards Tutin Glycosides
303
The early observation of glycosylation when Pd(MeCN)4
(BF4)2 was used (see above) prompted us to further investigate
the use of palladium-mediated catalysis. Furthermore, to coun-
teract diglucoside formation, dried powdered molecular sieves
were added to the reaction mixture.[23] Though no traces of the
desired product were detected with Pd(MeCN)4(BF4)2 (Table 2,
entry 4), treatment of tutin (1) and the glycosyl donor 9 with
Pd(PPh3)2(OTf)2 was encouraging (Table 2, entries 6–7).
Gratifyingly, 20-% conversion was achieved with the more
nucleophilic [Pd] species Pd(PhCN)2(OTf)2 with double
catalyst loading (10 mol-% [Pd] and 20 mol-% AgOTf, Table 2,
entry 8).
Purification of the tutin tetrabenzyl glucoside product was
complicated by co-elution with the hydrolysis product tetrabenzyl
glucose(indicatedbyNMRand ESI-MS)and required acetylation
and multiple chromatographic separations. Eventually, we were
able to isolate the tutin tetrabenzyl glucoside in a disappointing
9-% yield (41 % based on recovered tutin) as a ,5 : 1 mixture of
anomers. The NMR spectra indicated the main anomer to be tutin
tetrabenzyl-a-glucoside 2a (Table 3), as evident by the 4.1-Hz
coupling constant of the resonance, attributable to the anomeric
H10 by the 2D NMR correlations (Table 3, the corresponding H10
in 2-(b-D-glucopyranosyl)-tutin (2) showed 8-Hz coupling[5]).
We next examined the effect of the hindered base 2,6-
lutidine, which can act as scavenger of the triflic acid generated
during the course of the reaction. Although we observed 30-%
conversion at best with this modification (Table 2, entry 9), as
indicated by NMR analysis of the crude reaction mixture, the
procedure was not reproducible on a larger scale. Increasing
the amount of catalyst to 20 mol-% did not appear to benefit the
transformation (Table 2, entry 10).
The subsequent removal of the benzyl ethers via hydroge-
nolysis was initially carried out in the presence of Pd/C, under an
atmosphere of hydrogen gas, at ambient temperatures
(Scheme 5). Monitoring of the reaction with thin layer chroma-
tography (TLC) and mass spectrometry indicated the complete
consumption of the starting material, with the emergence of two
products after 48 h. The mass spectrum of the crude reaction
mixture indicated the presence of a deprotected tutin glucoside
as well as traces of the over-reduced dihydrotutin glucoside 11
(Scheme 5). However, the 1H NMR spectrum did not show the
coupled H13 olefinic methylene signals of a tutin (Table 3).
Instead, the spectrum featured two allylic methyl signals, also
observed in the 13C NMR spectrum.[24] Therefore, the structure
was assigned as a-neotutin glucoside (10) with double-bond
isomerisation resulting from the formation of the p-allyl com-
plex with [Pd] catalyst, which has been documented for tutin (1)
treated with the same catalyst.[9,25] Double-bond isomerisation
to the tetra-substituted alkene 10 in conjunction with the
considerable steric bulk of the tutin scaffold account for
the absence of concomitant double-bond reduction during the
hydrogenolysis of the benzyl ethers.
Purification of 10 via silica gel chromatography proved
difficult. Thus, the mixture was treated with pyridine and acetic
anhydride, with the aim to isolate and characterise the perace-
tylated neotutin glycoside derivative. This reaction proceeded
smoothly, however, affording the unexpected glycoside 12. The
1H NMR spectrum (Table 4) did not show the coupled H11
epoxide methylene signals of a tutin derivative (Table 3), and
the heteronuclear multiple-bond correlation (HMBC) spectrum
did not show a correlation between H2 and the anomeric carbon
C10 of the sugar moiety. Instead, a glycosidic linkage was
OBn
OH
OBn
O
BnO
BnO
see Table 2
for reaction
conditions
see ref. 20 for
reaction conditions
2
NH
BnO
OH
HO
HO
O
O
BnO
BnO
O
HO
OMe
O
BnO
CCl3
O
O
O
O
8
9
2a
Scheme 4.
Table 2. Screening of catalysts for the glycosylation of tutin 1 with donor 9
NP, no product
Sugar
donor
Entry
Conditions
Solvent
ConversionA [%]
9
1
2
3
4
5
BF3ꢀEt2O (0.1 equiv.), 08C–RT or ꢁ788C to RT
BF3ꢀEt2O (0.1 equiv.), 08C–RT or ꢁ788C to RT
BF3ꢀEt2O (0.1 equiv.), 08C–RT or ꢁ788C–RT
Pd(MeCN)4(BF4)2 (cat.)
DCM
MeCN
Et2O
Trace
NP
,10
NP
DCM
DCM
PdCl2(PPh3)2 (2.5 mol-%), AgOTf (2.5 mol-%), RT, 24 h
NP, hydrolysis and
dimerisation of 9
˚
PdCl2(PPh3)2 (2.5 mol-%), AgOTf (2.5 mol-%), 4-A sieves, RT, 24 h
6
DCM
,5, hydrolysis and
dimerisation of 9
˚
PdCl2(PPh3)2 (5 mol-%), AgOTf (5 mol-%), 4-A sieves, RT, 24–48 h
˚
PdCl2(PhCN)2 (10 mol-%), AgOTf (20 mol-%), 4-A sieves, RT, 24–48 h
˚
PdCl2(PhCN)2 (10 mol-%), AgOTf (20 mol-%), 4-A sieves, 2,6-lutidine, RT, 24–48 h
7
8
DCM
DCM
DCM
DCM
E10
20
9
30
˚
10
PdCl2(PhCN)2 (20 mol-%), AgOTf (40 mol-%), 4-A sieves, (þ/–)-2,6 lutidine, RT, 24–48 h
Trace
ADetermined by NMR.

304
D. Nhu et al.
Table 3. NMR data for tutin tetrabenzyl-a-glucoside 2a
hindered tutin using palladium catalysis to obtain the tutin tet-
rabenzylated a-glycoside 2a, albeit in low yields. Removal of
the benzyl ethers resulted in concomitant double-bond migra-
tion to produce the tutin analogue 10. Under standard acetylation
reaction conditions, the neotutin glycoside 10 was found to
rearrange to provide C11-glycoside 12.
OBn
d4.91
H
H
H
d4.71
BnO
BnO
O
H
H
1Ј
H
H
BnO
H
O
OH
O
H
H
1
H
Experimental
O
O
O
General Experimental Methods
d3.81
H
11
TLC was performed on ALUGRAMꢁ aluminium-backed
UV254 silica gel 60 (0.2 mm) plates. Compounds were visua-
lised with either p-anisaldehyde or 20 % w/w phosphomolybdic
acid in ethanol. Column chromatography was performed using
silica gel 60. Infrared spectra were recorded on a Bruker Optics
Alpha attenuated total reflectance Fourier transform infrared
spectrometer. High-resolution mass spectrometry (HRMS) was
conducted on a Bruker microTOFQ mass spectrometer using an
ESI source in either the positive or the negative mode. 1H NMR
spectra were recorded at either 400 MHz on a Varian 400-MR
NMR system or 500 MHz on a Varian 500 MHz AR premium
shielded spectrometer. All spectra were recorded from samples
in CDCl3 at 258C in 5-mm NMR tubes. Chemical shifts (d) were
reported relative to the residual chloroform singlet at 7.26 ppm.
Resonances were assigned as follows: chemical shift (number of
protons, multiplicity, coupling constant(s), assigned proton(s)).
Multiplicity abbreviations are reported by the conventions:
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and
br (broad). Proton decoupled 13C NMR spectra were recorded at
either 100 MHz on a Varian 400-MR NMR system or 125 MHz
on a Varian 500 MHz AR premium shielded spectrometer under
the same conditions as those used to record the 1H NMR spectra.
Chemical shifts were reported relative to the CDCl3 triplet at
77.16 ppm. DCM was dried using a PURE SOLV MD-6 solvent
purification system. All other solvents and reagents were used as
received. Tutin (1) used in this study was isolated from leaves of
the plant tutu, Coriaria species.[7,26]
H
d2.82
H
2a
#
d
H [ppm]
d
C [ppm] #
d
H [ppm]
dC [ppm]
1
45.78 10
82.91 20
81.71 30
4.80 (d, J 4.1)
3.51 (dd, J 9.9, 4.8) 80.11
3.84 (t, J 9.8, 8.9) 81.41
101.71
2 4.02 (s)
3 5.17
(br d, J 4.8)
4 2.92 (br m)
5 3.05
49.34 40
3.50 (dd, J 9.6, 8.6) 77.81
48.8
50
3.94 (m)
71.36
(dd, J 4.5, 0.9)
6
78.05 60
3.64 (2H, m)
69.18
73.53
7 3.73 (d, J 3.1)
8 3.18 (d, J 3.1)
9
60.72 60-CH2Ph 4.53 (d, J 11.9)
60.32 4.42 (d, J 11.9)
64.91 40-CH2Ph 4.70 (2H, m)
20.44 30-CH2Ph 4.80 (2H, m)
52.42 20-CH2Ph 4.48 (d, J 11.0)
4.87 (d, J 11.0)
73.39
75.73
75.27
10 1.45 (s)
11 2.82 (d, J 5.3)
3.81 (d, J 5.3)
12
140.91 Aromatics 7.17–7.40 (m)
112.51
138.51
138.41
13 4.71
(br d, J 2.6)
4.91 (br m)
14 1.86 (s)
15
138.21
137.91
128.51
128.41A
128.01
127.91A
127.81A
22.83
174.71
a-2,3,4,6-Tetra-O-benzyl Tutin Glucoside (2a)
Grey arrow: ROESY correlations; black arrow: HMBC correlations;
Amultiple signals.
A solution of the anomeric mixture of 2,3,4,6-tetra-O-benzyl-D-
glucopyranosyl trichloroacetimidate (465 mg, 0.680 mmol, 1.0
equiv.) in anhydrous DCM (3.0 mL) was transferred via a
syringe to an oven-dried reaction flask containing pre-activated
indicated between C10 and an acetal CH (1H: d 5.73 ppm; 13C: d
106.6 ppm; Table 4). This acetal proton was further vicinally
homonuclear coupled to a neighbouring CH proton (determined
by correlation spectroscopy) and heteronuclear coupled to an
epoxy CH corresponding to tutin C8 (determined by HMBC).
Overall, the 1D and 2D NMR data led us to propose the structure
14, the aglycone portion of which was previously assigned to an
˚
4-A sieves (1.0 g, 500 wt-%). To this suspension was added tutin
(200 mg, 0.680 mmol, 1 equiv.), and the light yellow suspension
was stirred at RT for 10 min. To a separate flask containing a
solution of Pd(PhCN)2Cl2 (26 mg, 0.07 mmol, 0.1 equiv.) in
DCM (1 mL) was added AgOTf (35 mg, 0.14 mmol, 0.2 equiv.),
and the resulting dark red suspension was allowed to stir at RT
for 10 min. This suspension of pre-formed Pd(PhCN)2(OTf)2
was then transferred dropwise to the reaction flask and the light
yellow suspension was allowed to stirred at RT for 48 h. Then,
the reaction mixture was passed through a plug of Celite, rinsed
with ethyl acetate (EtOAc), and the solvent was removed under
vacuum. The crude residue was purified by silica gel chroma-
tography (0–30 % EtOAc/petroleum ether) to remove the
unreacted acetimidate 9 and tutin (1) (16–20-% conversion) to
afford the title compound as a (5 : 1, a/b) mixture of anomers.
The fractions containing the product and co-eluted hydrolysed
sugar were redissolved in pyridine (2 mL), and acetic anhydride
(3 drops) was added and the mixture was stirred overnight at
RT. Then, the solvent was removed under vacuum, and the
residue was subjected to silica gel chromatography to separate
1
acid rearrangement product of dihydrotutin.[25] Our H NMR
data (Table 4) agreed with those previously reported,[25] and the
couplings and NOESY correlations supported the stereochem-
istry shown.
The exhaustive debenzylation of 2a using Pearlman’s catalyst
was found to proceed at a faster rate, with complete consumption
of the starting material after 16 h at room temperature (RT);
however, it did not suppress the unwanted double-bond
isomerisation.
Conclusions
Though the desired tutin b-glycosides could not be obtained,
we have successfully achieved glycosylation of the sterically

Sweet Poisons: Strategies towards Tutin Glycosides
305
OH
O
OH
O
OBn
HO
HO
HO
HO
BnO
BnO
O
Pd/C or Pd(OH)2,
H2, EtOH
RT
OH
OH
HO
HO
BnO
OH
O
O
ϩ
O
O
O
O
O
O
O
O
O
O
O
O
O
10
11
2a (5:1, α:β)
Ac2O, pyridine,
RT, overnight
OAc
O
OH
O
AcO
AcO
AcO
O
O
O
O
H
H
H
12
Scheme 5.
Table 4. NMR data for acetylated rearranged neotutin glucoside 12
a-Neotutin Glucoside (10)
A solution of the anomeric mixture of a-2,3,4,6-tetra-O-benzyl
tutin glucoside (2a) (a : b, 5 : 1) (15.0 mg, 0.018 mmol) was
dissolved in ethanol (EtOH; 2 mL), followed by the addition of a
catalytic amount of Pd(OH)2 (5 mg). The reaction flask was
evacuated and refilled with N2 twice before it was purged with
H2. The reaction mixture was allowed to stir overnight (16 h) at
RT. TLC analysis indicated the complete consumption of the
starting material. The reaction mixture was filtered through a
plug of Celite to remove the catalyst, rinsed with EtOAc, and
concentrated. The resultant colourless oil was taken up in D2O,
and the aqueous layer was washed once with CDCl3. The
aqueous layer was freeze-dried overnight to obtain the title
compound 10 as a colourless oil (8 mg, quantitative, but with
minor impurities of reduced product 11). nmax (film)/cmꢁ1 3422,
2954, 1736, 1056. dH (D2O, 400 MHz) 5.45 (1H, s, H3), 4.89
(1H, d, J 3.9, H10), 3.87 (1H, d, J 3.2, H8), 3.81 (2H, m, H60),
3.72 (1H, J 4.5, H11), 3.66 (2H, m, overlap, H30, H40), 3.63 (1H
s, H5), 3.49 (1H, br s, H2), 3.39 (1H, d, J 3.8, H20), 3.29 (1H,
J 3.2, overlap, H7), 3.29 (1H, m, overlap, H50), 2.97 (1H, d, J 4.5,
H11), 1.65 (6H, s, 13-CH3, 14-CH3, overlap), 1.25 (3H, s,
10-CH3). dC (D2O, 125 MHz) 179.47, 104.82, 89.30, 85.96,
82.39, 76.04, 75.15, 74.40, 72.25, 69.36, 63.26, 61.60, 61.42,
56.17, 53.93, 48.39, 22.45, 22.14, 21.57. m/z (HRMS ESI)
479.1524; [M þ Na]þ requires 479.1529.
OAc
O
13
H
14
AcO
AcO
H
OH
O
O
8
1Ј
H
O
H
AcO
O
9
11
HMBC
O
H
H
H
14 (proposed structure)
#
d
H [ppm]
d
C [ppm]
#
d
H [ppm]
d
C [ppm]
1
55.21 10
89.94 20
77.55 30
124.02 40
49.45 50
81.44 60
65.93
58.39 20-OAc 1.98 (s)
60.03 30-OAc 2.03 (s)
30.33 40-OAc 2.03 (s)
60-OAc 2.08 (s)
130.21 20-(CO)
20.21 30-(CO)
20.69 40-(CO)
172.43 60-(CO)
5.24 (d, J 3.8)
97.61
71.68
70.49
68.59
68.32
62.05
2
3
4
5
6
7
8
9
3.87 (d, J 4.7)
5.17 (d, J 4.7)
4.95 (dd, J 3.9, 10.2)
5.74 (t, J 9.8)
5.11 (dd, J 9.6, 10.2)
4.76 (br m)
3.54 (s)
4.27 (dd, J 3.9, 12.4)
4.15 (dd, J 2.3, 12.5)
4.04 (d, J 2.6)
3.59 (d, J 2.5)
2.87 (d, J 7.3)
20.9
20.95
10 1.07 (s)
11 5.42 (d, J 7.3) 106.6
20.95
20.99
12
169.81
169.83
170.51
171.02
13 1.77 (s)
14 1.82 (s)
15
2,3,4,6-Tetra-O-acetyl-a-neotutin Acetal-Glucoside (12)
A solution of the a-2,3,4,6-tetra-O-benzyl tutin glucoside
(20 mg, 0.018 mmol) was dissolved in EtOH (2 mL), followed
by the addition of a catalytic amount of Pd/C (5 mg). The
reaction flask was evacuated and refilled with N2 twice before it
was purged with H2 for 48 h at RT. TLC analysis indicated the
complete removal of the benzyl groups. The reaction mixture
was filtered through a plug of Celite, rinsed with EtOAc, and
concentrated to give a colourless gum. Mass spectrometry
analysis indicated the presence of tutin glycoside and dihy-
drotutin glucoside. The crude residue was taken up in D2O and
washed with CDCl3. The sample in D2O was freeze-dried (5 mg)
and the resultant white solid crude residue was dissolved in
Grey arrow: NOESY correlations; black arrow: HMBC correlations.
1-O-acetyl-2,3,4,6-tetra-O-benzyl-D-glucopyranoside from the
product, which was eluted with 25–30 % EtOAc/petroleum
ether as a colourless gum (55 mg, 9 %, 41 % based on recovered
tutin). NMR assignments for the major isomer 2a are provided in
Table 3. nmax (film)/cmꢁ1 3449, 3062, 2918, 2865, 1780, 1496,
1068, 735, 696. m/z (HRMS ESI) 839.3372; [M þ Na]þ requires
839.3407.

306
D. Nhu et al.
pyridine (1 mL). Acetic anhydride (2 drops) was added, and the
reaction mixture was stirred overnight at RT. Then, pyridine was
removed under vacuum, and the residue was purified with a small
plug of silica (gradient elution, 10–60 % EtOAc/petroleum
ether) to give the glycoside with the proposed structure 12
(1 mg). nmax (film)/cmꢁ1 2958, 2924, 1744, 1367, 1224, 1035,
996. The NMR data of the isolated glycoside are provided in
Table 4. m/z HRMS ESI 647.2009; [M þ Na]þ requires
647.1952.
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