Chemo-enzymatic preparation of α-6-sulfoquinovosyl-1,2-O- diacylglycerols

Article abstract of DOI:10.1016/j.tet.2012.09.100

Synthesis of α-6-sulfoquinovosyl-1,2-O-diacylglycerols is achieved by a versatile chemo-enzymatic stereoselective procedure that involves the use of α-d-glucosidase activity from the Mediterranean mollusc Aplysia fasciata. The synthetic procedure is designed to obtain a wide diversity of regio- and stereo-isomers of these compounds that have gained great interest as antineoplastic agents and potent inhibitors of DNA polymerases.

Full text of DOI:10.1016/j.tet.2012.09.100

Tetrahedron 68 (2012) 10169e10175
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Chemo-enzymatic preparation of a-6-sulfoquinovosyl-1,2-O-diacylglycerols
*
Emiliano Manzo , Annabella Tramice, Dario Pagano, Antonio Trincone, Angelo Fontana
Istituto di Chimica Biomolecolare, CNR, Via Campi Flegrei 34, I 80078 Pozzuoli (Na), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 June 2012
Received in revised form 7 September 2012
Accepted 25 September 2012
Available online 29 September 2012
Synthesis of
a
-6-sulfoquinovosyl-1,2-O-diacylglycerols is achieved by a versatile chemo-enzymatic
-glucosidase activity from the Mediterranean
stereoselective procedure that involves the use of
a-D
mollusc Aplysia fasciata. The synthetic procedure is designed to obtain a wide diversity of regio- and
stereo-isomers of these compounds that have gained great interest as antineoplastic agents and potent
inhibitors of DNA polymerases.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Sulfo-glycolipid
Sulfoquinovosyl-monoacylglycerol
Sulfoquinovosyl-diacylglycerol
a-D-Glucosidase
Chemo-enzymatic synthesis
1. Introduction
form glycosidic bonds.14 In a preliminary assessment, the prom-
inent activity found in the visceral mass of A. fasciata resulted in an
Sulfoquinovosyl-diacylglycerols (SQDGs), namely 1,2-di-O-acyl-
3-O-(60-deoxy-60-sulfo-
-glucopyranosyl)-sn-glycerols, first de-
a
-glucosidase enzyme showing a promising potential to glycosylate
a
-D
free glycerol.15 Here we report the use of A. fasciata
a-glucosidase
scribed by Benson et al. in 1959,1 are major constituents of chloro-
plast membranes of photosynthetic organisms,2,3 but have been also
reported in bacteria.4 SQDGs play a direct role in the photosynthetic
processes and, in the last years, have attracted bio-medical attention
because of their important activities as antitumors,4,5 antivirals,6,7
immunomodulators8 and inhibitors of DNA polymerase.2,9 Despite
their biological potential, SQDGs have been scarcely investigated
because of the difficulties related to their isolation from natural
sources and chemical synthesis,9b,c,e,10 mainly challenged by partial
activity as the key step of a novel and simple synthetic approach for
the preparation of SQDGs.
2. Results and discussion
As shown in Scheme 1 for the synthesis of natural sulfoquino-
vosyl-diacylglycerols,16 transglycosylation catalyzed by the
a
-glu-
cosidase activity of A. fasciata (step 1) controlled the transfer of
glucose from maltose (4-O- -glucopyranosyl- -glucose) to (rac)-
a
-D
D
loss of stereoselectivity in formation of the
a
-glycosidic bond11
1,2-O-isopropylidene glycerol. In a 10:1 molar ratio of donor/ac-
hampering the access to these products in laboratory.
ceptor the reaction gave a 1,2 glycerol acetonide conversion of 59%
Use of enzymes for the stereospecific construction of glycosidic
linkages is a new technological tool that involves the ability of
glycosyl hydrolases (endo- and exo-glycosidases)12 or glycosyl-
transferases13 to transfer a sugar unit from different donors to
specific acceptors. In particular, glycosyl hydrolases (EC 3.2.1.) are
functionally committed to the hydrolysis of glycosidic bonds but are
also able to catalyze the stereospecific formation of such linkages
(transglycosylation processes), thus finding application in the en-
zymatic synthesis of oligosaccharides and glycoconjugates.12
Recently, a study of the catalytic activity from the marine mol-
lusk Aplysia fasciata yielded a library of glycoside hydrolases able to
in 29 h. Reaction products were exclusively 3-
of 1,2-O-isopropylidene glycerol. In particular 3-
isopropylidene glycerol (1) was produced as the major component
(30% yield) together with a mixture of di- and tri-saccharide ana-
logues (23 and 6% yields, respectively).
a
-glycosyl derivatives
-glucosyl-1,2-O-
a
NMR spectroscopic data for H-10 (
C-10 (99.31 ppm) confirmed the
centre of compound 1, whereas further glucosylation in the oligo-
saccharides occurred with the preferential formation of -1/4 and
-1/6 linkages (in molar ratio 1:1), in agreement with the capa-
d
¼4.82 ppm, J¼3.29 Hz) and
a
configuration of the anomeric
a
a
bility of A. fasciata
a-glucosidase to perform polyglycosylation
reactions.17
To date few examples of enzymatic O-
a-D-glucosylation of
* Corresponding author. Tel.: þ39 (0)818675310; fax: þ39 (0)818041770; e-mail
address: emanzo@icb.cnr.it (E. Manzo).
glycerol have been reported in the literature.18e20 Interestingly, A.
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.09.100

10170
E. Manzo et al. / Tetrahedron 68 (2012) 10169e10175
OH
OH
O
HO
1) TrCl, pyridine,
DMAP, 60°C
O
O
O
O
HO
HO
HO
HO
OH
OH
OH
2.9 U α-glucosidase,
maltose in 50 mM
acetate buffer (pH 5.8),
34°C
O
O
O
HO
2) Ac2O/pyridine
O
H
OH
(30%)
1
Maltose
OH
OTr
OTr
OTr
1eq stearic acid
Dowex H+
MeOH/H2O 9:1
DCC, DMAP,
CH2Cl2, 0°C
O
OH
OH
O
O
AcO
AcO
AcO
AcO
OAc
O
OAc
O
O
(68
3
)
(65
)
2
OH
Tosyl-Cl,
pyridine, DMAP
Ostearoyl
OR
Ostearoyl
OR
O
O
AcO
AcO
I2 in MeOH
AcO
AcO
OAc
OAc
O
O
1.2 eq fatty acid
(78
)
R =H
4
R = stearoyl (79
R = oleoyl
)
6a
6b
DCC/DMAP,
CH2Cl2, r.t.
(76
)
R = stearoyl (82
R = oleoyl
)
5a
5b
(81
)
H2O2, AcOH,
KOAc
40°C
Potassium Thioacetate
2-Butanone
80°C
SAc
OTs
O
Ostearoyl
OR
Ostearoyl
OR
O
AcO
AcO
AcO
AcO
OAc
OAc
O
O
(90
(91
)
R = stearoyl
R = oleoyl
8a
8b
R = stearoyl
(82
R = oleoyl (82
7a
7b
)
)
)
SO3K
SO3K
NH2NH2, EtOH (85%)
44°C
6'
Ostearoyl
OR
Ostearoyl
O
O
1
AcO
AcO
HO
HO
1'
2
OAc
OH
3'
OR
O
O
3
(58
)
R = stearoyl
R = oleoyl
R = stearoyl (76
(73
R = oleoyl
)
9a
9b
10a
10b
(47
)
)
Scheme 1. Synthesis of natural sulfoquinovosyl-diacylglycerols.
fasciata activity was higher or at least comparable with these known
processes. In support of the synthetic potential of the mollusk en-
zyme, we also tested the reactivity of commercial glycosidases with
glycerol. Under conditions similar to those used for A. fasciata
fraction (maltose/glycerol 10:1; 24 h at 37 ꢀC), 3.75 U/mmol of
maltose of the transglycosidase of Aspergillus niger and 3.43 U/
group was largely favoured in the coupling reaction with equimolar
amounts of reagents (compound 4). Thus 2-acylglycerol derivatives
were not observed when 1 equiv of acid was used, although ester-
ification of the secondary hydroxyl group of glycerol has been
sometimes reported.23
Selective removal of the trityl group with iodine in methanol24
(6a/6b) followed by tosylation of the primary alcohol gave com-
pounds 7a/7b that, after replacement of the tosyl group with thi-
oacetate25 (8a/8b) and oxidation by hydrogen peroxide to the
sulfonic function,25 was easily converted to compounds 9a/9b. Fi-
nally, removal of the acetate groups with hydrazine hydrate com-
pleted the reaction sequence and yielded the target products 1,2-
mmol of maltose of
gave compound 1 with yields below 2%. These results are largely
lower than that obtained with the -glucosidase activity of A. fas-
a-glucosidase of Bacillus sterearothermophilus
a
ciata, suggesting that this latter enzyme may offer a viable option
for the synthesis of different types of glycolipids.
Tritylation of the primary alcohol of compound 1 followed by
peracetylation gave compound 2 that was converted to 3 by selec-
tive hydrolysis of the isopropylidene residue with Dowex Hþ in
methanol/water 9:1.21 In this last step, the reaction with common
catalysts such as HCl, HBr or TFA was hampered by the undesired
removal of the trityl group. In the 1H NMR spectrum of compound 2,
di-O-stearoyl- and 1-O-stearoyl-2-O-oleoyl-3-O-(60-
a-sulfoquino-
vosyl)glycerols (10a and 10b, respectively).22,26
The above procedure allowed both the step-wise introduction of
different acyl residues, and the control of fatty acid regiospecificity
of the resulting SQDGs. Use of palmitic, decanoic and 5-dodecenoic
acids gave a number of regioisomers, including a few natural
products (Table 1). Overall yields were good (from 14 to 23%) in-
dependent of the acyl substituents. As expected, saturated com-
pounds (e.g., 10:0/16:0) led to the best results because of the minor
susceptibility to be oxidized by hydrogen peroxide in the conver-
sion to sulfonate.
anomeric signals of two diastereoisomers at
were distinguishable in a ratio of 1:1, showing the absence of A.
fasciata -glucosidase diastereospecificity. Subsequent DCC-
d 5.18 and 5.16 ppm
a
mediated condensation with 1 or 2 equiv of free fatty acids (i.e.,
stearic or oleic) yielded diacyl derivatives 5a and 5b. In agreement
with previous observations,22 acylation of the primary hydroxyl

E. Manzo et al. / Tetrahedron 68 (2012) 10169e10175
10171
Table 1
most abundant hydrolytic enzyme in the A. fasciata visceral mass
extract was an -glucosidase, this enzymatic solution, with a total
protein content of 8.1 mg/mL and a specific activity corresponding
to 1.2 U/mg (using p-nitrophenyl -glucopyranoside as sub-
strate), was considered useful for this work without further puri-
fication. One unit of -glucosidase activity was defined as that
amount of enzyme required to catalyze the release of 1.0 mol of
p-nitrophenol per minute. Transglucosidases from A. niger and
-glucosidase from B. sterearothermophilus were purchased from
Sulfoquinovosyl-diacylglycerol derivatives prepared according to the synthetic
strategy of Scheme 1
a-D
Acyl residue C-1
Acyl residue C-2
Yielda (%)
a-D
Compound 10a
Compound 10b
Compound 11
Compound 12
Compound 13
Compound 14
Compound 15
Compound 16
C18:0
C18:0
C18:1
C16:0
C18:0
C10:0
C10:0
C12:1
C18:0
C18:1
C18:1
C18:1
C16:0
C12:1
C10:0
C12:1
w21
w16
w14
w18
w22
w17
w23
w14
a-D
m
a
Megazyme. Enzymatic reaction with A. niger was performed in
K-acetate buffer (50 mM, pH 5.5).28
a
Overall yields are calculated from the esterification reaction of compound 3.
Enzymatic reactions with A. niger and B. sterearothermophilus
were performed in K-phosphate buffer (0.1 M, pH 6.5).29
3. Conclusion
4.3. Synthetic procedures
Synthesis of 6-a-sulfoquinovosyl-diacylglycerols was achieved
4.3.1. 1,2-O-Isopropylidene glycerol. Glycerol (2.0 g, 0.022 mol) was
dissolved in N,N-dimethylformamide (4 mL); 2,2-dimethoxy-
propane (4 mL) and p-toluenesulfonic acid (300 mg) were added;
after stirring overnight at room temperature, the mixture was
portioned between water and dichloromethane; the organic phase
was purified by silica gel chromatography using a gradient of
petroleum ether/diethyl ether to give 1,2-O-isopropylidene glyc-
erol (2.0 g, 0.015 mol, 68%) as colourless oil; 1H NMR (400 MHz,
by a versatile chemo-enzymatic process. This is the first time that
an enzymatic approach has been used successfully for the prepa-
ration of this class of molecules. Avoiding procedures involving the
activation of chemical donors and using maltose as a cheap donor,
the key-intermediate 3-
obtained by a stereoselective glycosylation of the protected glycerol
by -glucosidase activity from the marine mollusc A. fasciata. The
enzymatic coupling allowed a strict control of the stereoselectivity
of the -glycosidic linkage, whereas the use of acetate as protecting
a-glucosyl-1,2-O-isopropylidene (1) was
a
CDCl3):
d
4.24 (1H, m, H-2), 4.09 (1H, dd, J¼6.7, 8.5 Hz, H-3a), 3.82
a
(1H, dd, J¼6.4, 8.5 Hz, H-3b), 3.65 (2H, m, H2-1), 1.46 (3H, s, CH3),
1.40 (3H, s, CH3); HRESIMS m/z calcd for C6H12O3Na: 155.0684;
found:155.0689.
group favoured the synthesis of saturated, unsaturated, or mixed
derivatives in high yields.22 Although both enantiopure commer-
cially available 1,2-O-isopropylidene glycerols were not tested in
this work, their use is compatible with the mild enzymatic coupling
conditions (pH 5.8).27 The proposed procedure is of general appli-
cation and allows the preparation of regio- and stereo-pure de-
4.3.2. Compound 1. 1,2-O-Isopropylidene glycerol (1.9 mmol), pre-
viously prepared and dissolved in DMSO (1 mL), was added to
a solution of maltose 0.5 M (19 mmol, 38 mL) in K-acetate buffer
rivatives of a-6-sulfoquinovosyl-1,2-O-diacylglycerols, thus giving
a realistic access to the biological use of these compounds.
(50 mM, pH 5.5) containing 600 mL of A. fasciata visceral mass en-
zymatic homogenate (5.83 U); the reaction system was put under
magnetic stirring at 34 ꢀC for 29 h up to almost total maltose con-
sumption and regularly monitored by TLC analysis (system solvent:
EtOAc/MeOH/H2O 70:20:10 vol). After 5 h 1,2-O-isopropylidene
glycerol (acceptor) was totally consumed, but being maltose still
present, a second aliquot of it (1.9 mmol) was added into the re-
action medium; a third similar aliquot of acceptor (1.9 mmol) was
added at 8 h of reaction time.
4. Experimental section
4.1. General experimental procedures
All products and synthetic intermediates structure were
assigned using 1H, 13C, COSY, TOCSY (mixing time¼68 ms), HSQC,
HMBC and NOESY (mixing time¼250 ms) NMR spectra.
Reaction was stopped after 29 h by cooling to ꢁ20 ꢀC and the
reaction mixture was fractionated by reverse phase RP-18 column
(eluting firstly with two column volumes of water and then with
1H, 13C and 2D NMR spectra were acquired by the NMR Service
of the Institute of Biomolecular Chemistry of the National Council of
Research (ICB-CNR) and recorded on a Bruker DRX-600 spectrom-
eter, equipped with a TCI CryoProbeÔ, fitted with a gradient along
the Z-axis and on Bruker instruments at 400 and/or 300 MHz.
Samples for NMR spectroscopic analysis were dissolved in the
appropriate solvent; spectra in D2O were referenced to internal
sodium 3-(trimethyl-silyl)-(2,2,3,3-2H4) propionate (Aldrich, Mil-
waukee, WI); for other solvents the downfield shift of the signal of
the solvent was used as internal standard: CDCl3 (1H, 13C): 7.26,
77.0 ppm; CD3OD (1H, 13C): 3.34, 49.0 ppm.
methanol). The product mixture, consisting of the a-glucosyl de-
rivative of 1,2-O-isopropylidene glycerol (1) and its corresponding
di- and tri-saccharides were recovered in the methanol fraction and
isolated by silica gel column chromatography (eluting with gradi-
ent of methanol in ethyl acetate); 1.71 mmol (502 mg) of pure
compound 1 (pale yellow oil) was isolated; IR (liquid film) nmax
3400, 2941, 2851, 1252, 1240 cmꢁ1; 1H NMR (400 MHz, D2O):
d 4.82
(1H, d, J¼3.29 Hz, H-10), 4.40e4.37 (1H, m, H-2), 4.07e3.73 (2H, m,
H2-1), 3.75e3.74 (1H, m, H-60a), 3.65e3.52 (2H, m, H2-3), 3.63e3.61
(1H, m, H-60b), 3.62e3.60 (1H, m, H-30), 3.57e3.55 (1H, m, H-40),
3.43 (1H, dd, H-20), 3.28 (1H, dd, H-50), 1.38 (3H, s, CH3), 1.29 (3H, s,
High resolution ESIMS were performed on
a Micromass
Q-TOF MicroÔ coupled with an HPLC Waters Alliance 2695. TLC
plates (Kieselgel 60 F254) and silica gel powder (Kieselgel 60,
0.063e0.200 mm) were from Merck.
All the reagents were purchased from SigmaeAldrich and used
without any further purification.
CH3); 13C NMR (100 MHz, D2O): 99.3 (CH, C10), 75.3 (CH, C2), 74.0
d
(CH, C30), 72.7 (CH, C40), 72.3 (CH, C20), 70.4 (CH, C50), 69.1 (CH2, C3),
66.4 (CH2, C1), 61.4 (CH2, C60), 26.5, 25.1 (acetonide methyls);
HRESIMS m/z calcd for C12H22NaO8: 317.1212; found: 317.1218.
4.2. Enzyme sources
4.3.3. Compound 2. Compound 1 (0.500 g, 0.0017 mol) was dis-
solved in pyridine (4 mL); 1.6 equiv of trityl chloride (0.756 g,
2.7 mmol) and 0.3 equiv of DMAP (57 mg, 0.51 mmol) were added;
the reaction mixture was stirred for 3 h at 60 ꢀC; subsequently,
acetic anhydride (1 mL) was added and after stirring overnight the
mixture was evaporated under a stream of nitrogen and purified by
A clear enzymatic homogenate from A. fasciata visceral mass
was prepared by homogenization in K-acetate buffer (50 mM, pH
5.5), subsequent centrifugation and dialysis procedures, and finally
concentration by ultrafiltration, as previously described.15 Since the

10172
E. Manzo et al. / Tetrahedron 68 (2012) 10169e10175
silica gel chromatography using a chloroform/methanol gradient to
give compound 2 (0.732 g, 1.1 mmol, 65%) as pale yellow oil; Rf
(light petroleum ether/diethyl ether 3:7)¼0.65; 1H NMR (400 MHz,
trityl portion), 5.39e5.37 (1H, m, H-30), 5.26e5.24 (1H, m, H-2), 5.15
and 5.14 (each 1H, br d, J¼2.7 Hz, H-10), 5.08 (1H, dd, J¼10.1, 10.1 Hz,
H-40), 4.89 (1H, dd, J¼10.5, 2.7 Hz, H-20), 4.33e4.31 (1H, m, H-1a),
4.16e4.14 (1H, m, H-1b), 3.97e3.95 (1H, m, H-50), 3.91e3.89 (1H, m,
H-3a), 3.66e3.64 (1H, m, H-3b), 3.18 (1H, br d, J¼9.8 Hz, H-60a), 3.09
CDCl3):
d
7.41e7.25 (15H, m, trityl portion), 5.41 (1H, dd, J¼9.4,
9.4 Hz, H-30), 5.18 and 5.16 (each 1H, d, J¼3.5 Hz, H-10), 5.07 (1H, dd,
J¼9.8, 9.8 Hz, H-40), 4.90 (1H, m, H-20), 4.30e4.28 (1H, m, H-2),
4.05e4.03 (2H, m, H2-1), 3.97e3.95 (1H, m, H-50), 3.79e3.77 (1H,
m, H-3a), 3.56e3.54 (1H, m, H-3b), 3.17 (1H, br d, J¼10.5 Hz, H-60a),
3.07 (1H, dd, J¼5.1, 10.5 Hz, H-60b), 2.04 (6H, s, OAc), 1.96 (3H, s,
OAc), 1.40 (3H, s, CH3), 1.32 (3H, s, CH3); 13C NMR (100 MHz, CDCl3):
128.5, 127.6, 126.9 (CH, aromatic methynes), 109.4 (C, acetonide
carbon), 95.7 (CH, C10), 70.7 (CH, C20), 74.1 (CH, C2),70.2 (CH, C30),
68.8 (CH, C40), 68.6 (CH2, C1), 68.4 (CH, C50), 66.4 (CH2, C3),61.9
(CH2, C60), 26.5, 25.5 (CH3, acetonide methyls), 20.6, 20.5, 20.4 (CH3,
acetate methyls); HRESIMS m/z calcd for C37H42NaO11: 685.2625;
found: 685.2630.
(1H, dd, J¼9.8, 4.7 Hz, H-60b), 2.31e2.27 (4H, m,
a-methylenes of
acyl portions), 2.05 (3H, s, OAc), 2.02 (3H, s, OAc), 1.98 (3H, s, OAc),
1.64e1.60 (4H, m,
b-methylenes of acyl portions), 1.38e1.23 (56H,
m, aliphatic protons), 0.92e0.90 (6H, overlapped, 2CH3); 13C NMR
(100 MHz, CDCl3): d 172.8, 172.6 (C, acyl ester carbons), 128.6, 127.7,
127.1 (CH, aromatic methynes), 96.1 (CH, C10), 71.1 (CH, C20), 70.4
(CH, C30), 70.0 (CH, C2), 69.3 (CH, C50), 68.8 (CH, C40), 66.0 (CH2, C3),
62.4 (CH2, C1), 62.2 (CH2, C60), 34.2, 34.1 (CH2,
a
-methylenes of acyl
portions), 29.8e29.0 (CH2, aliphatic methylenes), 25.2, 24.9 (CH2,
b
-
methylenes of acyl portions), 20.7, 20.5, 20.4 (CH3, acetate methyls),
14.2, 14.0 (CH3, acyl terminal methyls); HRESIMS m/z calcd for
C70H106NaO13: 1177.7531; found: 1177.7535.
4.3.4. Compound 3. Compound 2 (0.732 g, 1.1 mmol) was dissolved
in methanol/water solution (25 mL, 9:1) and Dowex 50WX8-Hþ
resin (18 g) was added; after 40 min under stirring, the reaction
mixture was filtered, evaporated and purified by silica gel chro-
matography using a gradient of light petroleum ether/diethyl ether
to give compound 3 (0.465 g, 0.748 mmol, 68%) as pale yellow oil; Rf
(light petroleum ether/diethyl ether 3:7)¼0.10; 1H NMR (400 MHz,
4.3.7. Compound 5b. Compound 4 (0.115 g, 0.130 mmol) was dis-
solved in anhydrous dichloromethane (5 mL); oleic acid (0.044 g,
0.156 mmol), dicyclohexylcarbodiimide (0.0303 g, 0.156 mmol) and
DMAP (0.00190 g, 0.0156 mmol) were added under argon; the re-
action mixture was stirred overnight at room temperature; after
evaporation under reduced pressure, the mixture was purified by
silica gel chromatography using a gradient of petroleum ether/
diethyl ether to give compound 5b (yellow oil) (0.120 g,
CDCl3):
d
7.41e7.21 (15H, m, trityl portion), 5.41 (1H, dd, J¼9.4,
9.4 Hz, H-30), 5.16 and 5.13 (each 1H, d, J¼3.5 Hz, H-10), 5.06 (1H, dd,
J¼9.8, 9.8 Hz, H-40), 4.94 and 4.92 (each 1H, dd, J¼9.39, 3.5 Hz, H-
20), 4.01e3.99 (1H, m, H-50), 3.94e3.92 (1H, m, H-2), 3.79e3.77 (1H,
m, H-3a), 3.72e3.70 (1H, m, H-1a), 3.65e3.63 (1H, m, H-3b), 3.61
(1H, m, H-1b), 3.19 (1H, br d, J¼10.5 Hz, H-60a), 3.10 (1H, br d,
J¼10.5 Hz, H-60b), 2.06 (3H, s, OAc), 1.97 (3H, s, OAc), 1.82 (3H, s,
OAc); 13C NMR (100 MHz, CDCl3): 128.5, 127.5, 126.7 (CH, aromatic
methynes), 96.3 (CH, C10), 71.0 (CH, C20), 70.4 (CH, C30), 70.3 (CH,
C2), 70.2 (CH2, C3), 69.0 (CH, C40), 68.8 (CH, C50), 63.2 (CH2, C1),62.0
(CH2, C60), 20.7, 20.6, 20.5 (CH3, acetate methyls); HRESIMS m/z
calcd for C34H38NaO11: 645.2312; found: 645.2306.
0.105 mmol, 81%). 1H NMR (400 MHz, CDCl3):
d 7.41e7.22 (15H, m,
trityl portion), 5.40e5.38 (1H, m, H-30), 5.38e5.36 (2H, m, olefinic
protons), 5.26e5.24 (1H, m, H-2), 5.16 and 5.13 (each 1H, br d,
J¼2.7 Hz, H-10), 5.08 (1H, dd, J¼10.1, 10.1 Hz, H-40), 4.89 (1H, dd,
J¼10.5, 2.7 Hz, H-20), 4.33e4.31 (1H, m, H-1a), 4.16e4.14 (1H, m, H-
1b), 3.97e3.95 (1H, m, H-50), 3.91e3.89 (1H, m, H-3a), 3.66e3.64
(1H, m, H-3b), 3.18 (1H, br d, J¼9.8 Hz, H-60a), 3.09 (1H, dd, J¼9.8,
4.7 Hz, H-60b), 2.31e2.27 (4H, m,
a
-methylenes of acyl portions),
2.04e2.02 (4H, m, allylic protons), 2.05 (3H, s, OAc), 2.02 (3H, s,
OAc), 1.98 (3H, s, OAc), 1.63e1.61 (4H, m, -methylenes of acyl
portions), 1.38e1.23 (48H, m, aliphatic protons), 0.92e0.89 (6H,
overlapped, 2CH3); 13C NMR (100 MHz, CDCl3):
172.8, 172.7 (C,
b
4.3.5. Compound 4. Compound 3 (0.20 g, 0.32 mmol) was dis-
solved in anhydrous dichloromethane (6 mL); stearic acid (0.90 g,
0.32 mmol), dicyclohexylcarbodiimide (0.0326 g, 0.32 mmol) and
DMAP (0.0038 g, 0.032 mmol) were added under argon at 0 ꢀC; the
reaction mixture was stirred overnight at 0 ꢀC; after evaporation
under reduced pressure, the mixture was purified by silica gel
chromatography using a gradient of petroleum ether/diethyl ether
to give compound 4 (0.220 g, 0.250 mmol, 78%) as a colourless oil;
d
acyl ester carbons), 131.0, 128.6, 127.7, 127.1 (CH, olefinic and aro-
matic methynes), 96.1 (CH, C10), 71.1 (CH, C20), 70.4 (CH, C30), 70.0
(CH, C2), 69.3 (CH, C50), 68.8 (CH, C40), 66.0 (CH2, C3), 62.5 (CH2, C1),
62.2 (CH2, C60), 34.2, 34.1 (CH2,
a
-methylenes of acyl portions),
29.8e29.0 (CH2, aliphatic methylenes), 27.8 (2 CH2, allylic methy-
lenes), 25.2, 24.9 (CH2, -methylenes of acyl portions), 20.7, 20.5,
b
20.4 (CH3, acetate methyls), 14.2, 14.0 (CH3, acyl terminal methyls);
HRESIMS m/z calcd for C70H104NaO13: 1175.7375; found: 1175.7381.
1H NMR (400 MHz, CDCl3):
d
7.40e7.22 (15H, m, trityl portion),
5.39e5.37 (1H, m, H-30), 5.15 and 5.11 (each 1H, br d, J¼2.7 Hz, H-
10), 5.08 (1H, dd, J¼10.1, 10.1 Hz, H-40), 4.87 (1H, dd, J¼10.5, 2.7 Hz,
H-20), 4.31e4.29 (1H, m, H-1a), 4.15e4.13 (1H, m, H-1b), 4.00e3.98
(1H, m, H-50), 3.94e3.92 (1H, m, H-3a), 3.88e3.86 (1H, m, H-2),
3.65e3.63 (1H, m, H-3b), 3.17 (1H, br d, J¼9.8 Hz, H-60a), 3.07 (1H,
4.3.8. Compound 6a. Compound 5a (0.124 g, 0.107 mmol) was
dissolved in iodineemethanol solution (5 mL, 1%); after stirring for
30 min at 60 ꢀC, the mixture was concentrated and purified by silica
gel chromatography using a gradient of petroleum ether/diethyl
ether to give compound 6a (0.077 g, 0.084 mmol, 79%) as a pale
yellow oil; Rf (light petroleum ether/diethyl ether 3:7)¼0.22; 1H
dd, J¼9.8, 4.7 Hz, H-60b), 2.31e2.28 (2H, m,
a-methylene of acyl
portion), 2.04 (3H, s, OAc), 2.02 (3H, s, OAc), 1.98 (3H, s, OAc),
1.64e1.60 (2H, m,
b
-methylene of acyl portion), 1.38e1.23 (28H, m,
NMR (400 MHz, CDCl3): d
5.52e5.50 (1H, m, H-30), 5.22e5.20 (1H,
aliphatic protons), 0.90 (3H, t, J¼6.9 Hz, CH3); HRESIMS m/z calcd
m, H-2), 5.11 (1H, br d, 2.9 Hz, H-10), 5.02e5.00 (1H, m, H-40), 4.84
(1H, dd, J¼9.9, 2.9 Hz, H-20), 4.33e4.31 (1H, m, H-1a), 4.18e4.16
(1H, m, H-1b), 3.83e3.81 (1H, m, H-3a), 3.82e3.80 (1H, m, H-50),
3.69 (1H, br d, J¼9.6 Hz, H-60a), 3.64e3.62 (1H, m, H-3b), 3.59 (1H,
for C52H72NaO12: 912.4921; found: 912.4925.
4.3.6. Compound 5a. Compound 4 (0.115 g, 0.130 mmol) was dis-
solved in anhydrous dichloromethane (5 mL); stearic acid (0.044 g,
0.156 mmol), dicyclohexylcarbodiimide (0.0303 g, 0.156 mmol) and
DMAP (0.00190 g, 0.0156 mmol) were added under argon; the re-
action mixture was stirred overnight at room temperature; after
evaporation under reduced pressure, the mixture was purified by
silica gel chromatography using a gradient of petroleum ether/
diethyl ether to give compound 5a (yellow oil) (0.122 g,
dd, J¼9.6, 4.4 Hz, H-60b), 2.31e2.27 (4H, m,
a-methylenes of acyl
portions), 2.05 (3H, s, OAc), 2.01 (6H, s, OAc), 1.63e1.60 (4H, m, b-
methylenes of acyl portions), 1.38e1.23 (56H, m, aliphatic protons),
0.91e0.89 (6H, overlapped, 2CH3); 13C NMR (100 MHz, CDCl3):
d
173.0, 172.8 (C, acyl ester carbons), 96.4 (CH, C10), 70.9 (CH, C20),
70.0 (CH, C2), 69.8 (CH, C30), 69.8 (CH, C50), 68.9 (CH, C40), 66.4 (CH2,
C3), 62.3 (CH2, C1), 61.1 (CH2, C60), 34.0, 33.9 (CH2,
-methylenes of
acyl portions), 29.9e29.0 (CH2, aliphatic methylenes), 25.2, 25.1
a
0.106 mmol, 82%). 1H NMR (400 MHz, CDCl3):
d 7.41e7.22 (15H, m,

E. Manzo et al. / Tetrahedron 68 (2012) 10169e10175
10173
(CH2,
b
-methylenes of acyl portions), 20.8, 20.7, 20.4 (CH3, acetate
30), 5.38e5.36 (2H, m, olefinic protons), 5.18e5.16 (1H, m, H-2),
5.01e4.99 (1H, m, H-40), 4.92e4.90 (1H, m, H-20), 4.73 and 4.75 (each
1H, d, J¼3.4 Hz, H-10), 4.31e4.29 (1H, m, H-1a), 4.26 (1H, br d,
J¼9.8 Hz, H-60a), 4.17e4.15 (1H, m, H-1b), 4.16e4.14 (1H, m, H-50),
4.09 (1H, dd, J¼9.8, 4.1 Hz, H-60b), 3.75e3.73 (1H, m, H-3a),
3.56e3.54 (1H, m, H-3b), 2.44 (3H, s, aromatic methyl), 2.31e2.29
methyls), 14.6, 14.1 (CH3, acyl terminal methyls); HRESIMS m/z
calcd for C51H92NaO13: 935.6436; found: 935.6441.
4.3.9. Compound 6b. Compound 5b (0.124 g, 0.107 mmol) was
dissolved in iodineemethanol solution (5 mL, 1%). After stirring for
30 min at 60 ꢀC, the mixture was concentrated and purified by silica
gel chromatography using a gradient of petroleum ether/diethyl
ether to give compound 6b (0.074 g, 0.081 mmol, 76%) as a pale
yellow oil; Rf (light petroleum ether/diethyl ether 3:7)¼0.22; 1H
(4H, m,
a-methylenes of acyl portions), 2.06e2.03 (4H, m, allylic
protons), 2.04 (6H, s, OAc), 1.98 (3H, s, OAc), 1.60e1.57 (4H, m,
b
-
methylenes of acyl portions), 1.38e1.23 (48H, m, aliphatic protons),
0.90e0.88 (6H, overlapped, 2CH3); 13C NMR (100 MHz, CDCl3):
NMR (400 MHz, CDCl3):
d
5.52e5.50 (1H, m, H-30), 5.38e5.36 (2H,
d 172.6, 172.5 (C, acyl ester carbons), 130.2, 129.6, 127.9 (CH, olefinic
m, olefinic protons), 5.22e5.20 (1H, m, H-2), 5.11 (1H, br d, 2.9 Hz,
H-10), 5.02e5.00 (1H, m, H-40), 4.84 (1H, dd, J¼9.9, 2.9 Hz, H-20),
4.33e4.31 (1H, m, H-1a), 4.18e4.16 (1H, m, H-1b), 3.83e3.81 (1H,
m, H-3a), 3.82e3.80 (1H, m, H-50), 3.69 (1H, br d, J¼9.6 Hz, H-60a),
3.64e3.62 (1H, m, H-3b), 3.59 (1H, dd, J¼9.6, 4.4 Hz, H-60b),
and aromatic methynes), 95.9 (CH, C10), 70.4 (CH, C20), 69.8 (CH, C30),
69.5 (CH, C2), 68.4 (CH, C40), 66.5 (CH2, C3), 62.0 (CH2, C1), 62.0 (CH2,
C60), 34.0, 33.9 (CH2,
a
-methylenes of acyl portions), 29.9e29.4 (CH2,
aliphatic methylenes), 27.9 (2CH2, allylic methylenes), 24.8, 24.6
(CH2, -methylenes of acyl portions), 20.6, 20.5, 20.4 (CH3, acetate
b
2.31e2.27 (4H, m,
a-methylenes of acyl portions), 2.05 (3H, s, OAc),
methyls),14.1,13.9 (CH3, acyl terminal methyls); HRESIMS m/z calcd
2.04e2.01 (4H, m, allylic protons), 2.01 (6H, s, OAc), 1.64e1.60 (4H,
m, -methylenes of acyl portions), 1.38e1.23 (48H, m, aliphatic
protons), 0.92e0.89 (6H, overlapped, 2CH3); 13C NMR (100 MHz,
for C58H96NaO15S: 1099.6368; found: 1099.6373.
b
4.3.12. Compound 8a. Compound 7a (0.076 g, 0.073 mmol) was
dissolved in 2-butanone (7 mL) and potassium thioacetate (0.021 g,
0.183 mmol) was added. The reaction mixture was stirred at 80 ꢀC
2.5 h, and after evaporation under reduced pressure, the mixture
was purified by silica gel chromatography using a light petroleum
ether/diethyl ether gradient to give compound 8a (0.064 g,
0.065 mmol, 90%) as a colourless oil; Rf (light petroleum ether/
CDCl3):
d 172.9, 172.8 (C, acyl ester carbons), 131.0 (2CH, olefinic
methynes), 96.3 (CH, C10), 70.9 (CH, C20), 70.0 (CH, C2), 69.8 (CH,
C30), 69.8 (CH, C50), 68.9 (CH, C40), 66.4 (CH2, C3), 62.3 (CH2, C1),
61.1 (CH2, C60), 34.0, 33.9 (CH2,
a
-methylenes of acyl portions),
29.8e29.0 (CH2, aliphatic methylenes), 27.9 (2CH2, allylic methy-
lenes), 25.2, 25.1 (CH2, -methylenes of acyl portions), 20.8, 20.7,
b
20.4 (CH3, acetate methyls), 14.7, 14.1 (CH3, acyl terminal methyls);
HRESIMS m/z calcd for C51H90NaO13: 933.6279; found: 933.6275.
diethyl ether 1:1)¼0.60; 1H NMR (400 MHz, CDCl3):
d 5.41e5.39
(1H, m, H-30), 5.20e5.18 (1H, m, H-2), 5.04e5.02 (1H, m, H-40),
4.94e4.92 (1H, m, H-20), 4.82 and 4.79 (each 1H, br d, J¼3.9 Hz, H-
10), 4.33e4.31 (1H, m, H-1a), 4.19 (1H, br d, m, H-1b), 3.83e3.81
(1H, m, H-50), 3.80e3.78 (1H, m, H-3a), 3.60e3.58 (1H, m, H-3b),
3.19e3.18 (1H, m, H-60a), 3.12e3.10 (1H, m, H-60b), 2.34 (3H, s,
4.3.10. Compound 7a. Compound 6a (0.081 g, 0.089 mmol) was
dissolved in anhydrous pyridine (3 mL). p-Tosylchloride (0.169 g,
0.888 mmol) and DMAP (0.010 g, 0.089 mmol) were added at 0 ꢀC
under argon and the reaction mixture was stirred overnight at room
temperature. After evaporation under a stream of nitrogen, the
mixture was purified by silica gel chromatography using a gradient
of petroleum ether/diethyl ether to give compound 7a (0.076 g,
0.073 mmol, 82%) as a pale yellow oil; Rf (light petroleum ether/
SCH3), 2.32e2.30 (4H, m,
a-methylenes of acyl portions), 2.07 (3H,
s, OAc), 2.05 (3H, s, OAc), 1.99 (3H, s, OAc), 1.60e1.58 (4H, m,
b
-
methylenes of acyl portions), 1.37e1.23 (56H, m, aliphatic protons),
0.90e0.87 (6H, overlapped, 2CH3); 13C NMR (100 MHz, CDCl3):
d
172.8, 172.6 (C, acyl ester carbons), 95.8 (CH, C10), 70.6 (CH, C40),
diethyl ether 1:1)¼0.28; 1H NMR (400 MHz, CDCl3):
d
7.76 (2H, d,
70.7 (CH, C20), 69.7 (CH, C30), 69.4 (CH, C2), 66.1 (CH2, C3), 62.0
(CH2, C1), 34.1, 34.0 (CH2, -methylenes of acyl portions), 30.5 (CH3,
thioacetate methyl), 29.1e29.7 (CH2, aliphatic methylenes), 29.7
(CH2, C60), 24.9, 24.7 (CH2,
-methylenes of acyl portions), 20.6,
J¼8.3 Hz, Ar-H), 7.34 (2H, d, J¼8.3 Hz, Ar-H), 5.40e5.38 (1H, m, H-
30), 5.18e5.16 (1H, m, H-2), 5.02e5.00 (1H, m, H-40), 4.92e4.90 (1H,
m, H-20), 4.73 and 4.75 (each 1H, d, J¼3.4 Hz, H-10), 4.31e4.29 (1H,
m, H-1a), 4.26 (1H, br d, J¼9.8 Hz, H-60a), 4.17e4.15 (1H, m, H-1b),
4.16e4.14 (1H, m, H-50), 4.09 (1H, dd, J¼9.8, 4.1 Hz, H-60b),
3.75e3.73 (1H, m, H-3a), 3.56e3.54 (1H, m, H-3b), 2.44 (3H, s, ar-
a
b
20.5, 20.5 (CH3, acetate methyls), 14.0, 13.9 (CH3, acyl terminal
methyls); HRESIMS m/z calcd for C53H94NaO13S: 993.6313 found:
993.6316.
omatic methyl), 2.31e2.29 (4H, m,
2.04 (6H, s, OAc), 1.98 (3H, s, OAc), 1.60e1.58 (4H, m,
of acyl portions), 1.38e1.23 (56H, m, aliphatic protons), 0.91e0.88
(6H, overlapped, 2CH3); 13C NMR (100 MHz, CDCl3):
172.7,172.5 (C,
a
-methylenes of acyl portions),
b
-methylenes
4.3.13. Compound 8b. Compound 7b (0.076 g, 0.073 mmol) was
dissolved in 2-butanone (7 mL) and potassium thioacetate (0.021 g,
0.183 mmol) was added. The reaction mixture was stirred at 80 ꢀC
for 2.5 h, and after evaporation under reduced pressure, the mix-
ture was purified by silica gel chromatography using a light pe-
troleum ether/diethyl ether gradient to give compound 8b (0.065 g,
0.066 mmol, 91%) as a pale yellow oil; Rf (light petroleum ether/
d
acyl ester carbons), 129.6, 127.9 (CH, aromatic methynes), 95.8 (CH,
C10), 70.4 (CH, C20), 69.8 (CH, C30), 69.5 (CH, C2), 68.4 (CH, C40), 66.5
(CH2, C3), 62.0 (CH2, C1), 62.0 (CH2, C60), 34.0, 33.9 (CH2,
a
-meth-
ylenes of acyl portions), 29.9e29.4 (CH2, aliphatic methylenes),
24.8, 24.6 (CH2, -methylenes of acyl portions), 20.6, 20.5, 20.4 (CH3,
b
diethyl ether 1:1)¼0.60; 1H NMR (400 MHz, CDCl3):
d 5.41e5.39
acetate methyls), 14.0, 13.9 (CH3, acyl terminal methyls); HRESIMS
m/z calcd for C58H98NaO15S: 1089.6524; found: 1089.6521.
(1H, m, H-30), 5.38e5.36 (2H, m, olefinic protons), 5.20e5.18 (1H, m,
H-2), 5.04e5.02 (1H, m, H-40), 4.94e4.92 (1H, m, H-20), 4.82 and
4.80 (each 1H, br d, J¼3.9 Hz, H-10), 4.33e4.31 (1H, m, H-1a), 4.19
(1H, br d, m, H-1b), 3.83e3.80 (1H, m, H-50), 3.80e3.78 (1H, m, H-
3a), 3.60e3.58 (1H, m, H-3b), 3.19e3.17 (1H, m, H-60a), 3.12e3.11
4.3.11. Compound 7b. Compound 6b (0.081 g, 0.089 mmol) was
dissolved in anhydrous pyridine (3 mL). p-Tosylchloride (0.169 g,
0.888 mmol) and DMAP (0.010 g, 0.089 mmol) were added at 0 ꢀC
under argon and the reaction mixture was stirred overnight at room
temperature. After evaporation under a stream of nitrogen, the
mixture was purified by silica gel chromatography using a gradient
of petroleum ether/diethyl ether to give compound 7b (0.076 g,
0.073 mmol, 82%) as a colourless oil; Rf (light petroleum ether/
(1H, m, H-60b), 2.34 (3H, s, SCH3), 2.32e2.30 (4H, m,
of acyl portions), 2.07 (3H, s, OAc), 2.02e2.00 (4H, m, allylic pro-
tons), 2.05 (3H, s, OAc), 1.99 (3H, s, OAc), 1.61e1.59 (4H, m,
a-methylenes
b
-
methylenes of acyl portions), 1.37e1.23 (48H, m, aliphatic protons),
0.91e0.88 (6H, overlapped, 2CH3); 13C NMR (100 MHz, CDCl3):
d
172.7, 172.6 (C, acyl ester carbons), 130.1 (2CH, olefinic methynes),
95.8 (CH, C10), 70.6 (CH, C40), 70.7 (CH, C20), 69.7 (CH, C30), 69.4 (CH,
C2), 66.1 (CH2, C3), 62.0 (CH2, C1), 34.1, 34.0 (CH2, -methylenes of
diethyl ether 1:1)¼0.28; 1H NMR (400 MHz, CDCl3):
d 7.77 (2H, d,
J¼8.3 Hz, AreH), 7.34 (2H, d, J¼8.3 Hz, AreH), 5.40e5.38 (1H, m, H-
a

10174
E. Manzo et al. / Tetrahedron 68 (2012) 10169e10175
acyl portions), 30.5 (CH3, thioacetate methyl), 29.1e29.7 (CH2, ali-
H-2), 4.81 and 4.79 (each 1H, br d, J¼3.3 Hz, H-10), 4.35 (1H, dd,
J¼12.2, 7.3 Hz, H-1a), 4.22 (1H, dd, J¼12.2, 6.6 Hz, H-1b), 4.15e4.13
(1H, m, H-40), 4.11e4.08 (1H, m, H-50), 4.08e4.06 (1H, m, H-3a),
3.69e3.67 (1H, m, H-30), 3.67e3.65 (1H, m, H-3b), 3.46e3.44 (1H,
m, H-20), 3.12 (1H, dd, J¼9.3, 9.3 Hz, H-60a), 2.95 (1H, dd, J¼14.4,
phatic methylenes), 29.7 (CH2, C60), 27.8 (2CH2, allylic methylenes),
24.9, 24.7 (CH2,
b-methylenes of acyl portions), 20.6, 20.5, 20.5
(CH3, acetate methyls), 14.1, 13.9 (CH3, acyl terminal methyls);
HRESIMS m/z calcd for C53H92NaO13S: 991.6156 found: 991.6159.
9.3 Hz, H-60b), 2.38e2.34 (4H, m,
a
-methylenes of acyl portions),
-methylenes of acyl portions), 1.39e1.27 (56H,
m, aliphatic protons), 0.91e0.89 (6H, overlapped, 2CH3); 13C NMR
(100 MHz, CD3OD): 175.2, 174.9 (C, acyl ester carbons), 99.1 (CH,
C10), 74.8 (CH, C40), 74.1 (CH, C30), 72.9 (CH, C20), 70.8 (CH, C2), 66.4
(CH2, C3), 63.1 (CH2, C1), 53.8 (CH2, C60), 34.1, 34.0 (CH2,
-methy-
lenes of acyl portions), 29.4e29.9 (CH2, aliphatic methylenes), 24.9,
24.7 (CH2, -methylenes of acyl portions), 14.0, 13.8 (CH3, acyl ter-
4.3.14. Compound 9a. Compound 8a (0.065 g, 0.0.066 mmol) was
dissolved in a mixture of potassium acetate (0.032 g, 0.323 mmol),
aq H2O2 (0.161 mL) (34% w/v) and acetic acid (1.9 mL); the reaction
mixture was stirred overnight at 40 ꢀC; after evaporation under
a stream of nitrogen, the mixture was purified by silica gel chro-
matography using a gradient of chloroform/methanol to give com-
pound 9a (0.038 g, 0.038 mmol, 58%) as a colourless oil; Rf
(chloroform/methanol 9:1)¼0.32; 1H NMR (400 MHz, CDCl3):
1.64e1.62 (4H, m, b
d
a
b
minal methyls); HRESIMS m/z calcd for C45H85NaO12KS: 911.5297;
found: 911.5300.
d
5.41e5.39 (1H, m, H-30), 5.23e5.21 (1H, m, H-2), 5.15e5.13 (1H,
overlapped, H-10), 5.01e4.99 (1H, m, H-40), 4.93e4.91 (1H, m, H-20),
4.33e4.31 (1H, m, H-1a), 4.27e4.25 (1H, m, H-1b), 4.26e4.24 (1H, m,
H-50), 3.72e3.70 (1H, m, H-3a), 3.64e3.62 (1H, m, H-3b), 3.23e3.21
4.3.17. Compound 10b. Compound 9b (0.042 g, 0.042 mmol) was
dissolved in aq ethanol (85%) (4.7 mL), hydrazine monohydrate
(0.064 g, 1.28 mmol) was added, and the reaction mixture was
stirred for 3 h at 44 ꢀC. After evaporation under a stream of nitro-
gen, the mixture was purified by silica gel chromatography using
a gradient of chloroform/methanol to give compound 10b (0.026 g,
0.030 mmol, 73%) as a white solid, mp 92e98 ꢀC; Rf (chloroform/
methanol 7:3)¼0.15; IR (liquid film) nmax 3400, 2940, 2829, 1750,
(1H, m, H-60a), 3.12e3.10 (1H, m, H-60b), 2.31e2.29 (4H, m,
a-
methylenes of acyl portions), 2.04 (6H, s, OAc), 1.99 (3H, s, OAc),
1.60e1.57 (4H, m, -methylenes of acyl portions),1.37e1.23 (56H, m,
aliphatic protons), 0.90e0.87 (6H, overlapped, 2CH3); 13C NMR
(100 MHz, CDCl3): 172.7, 172.5 (C, acyl ester carbons), 95.5 (CH,
C10), 70.9 (CH, C40), 70.4 (CH, C30), 70.3 (CH, C2), 70.2 (CH, C20), 66.4
(CH2, C3), 62.9 (CH2, C1), 51.0 (CH2, C60), 34.1, 34.0 (CH2,
-methy-
lenes of acyl portions), 29.4e29.9 (CH2, aliphatic methylenes), 24.9,
24.7 (CH2, -methylenes of acyl portions), 20.6, 20.5 (CH3, acetate
b
d
a
1352, 1340 cmꢁ1; 1H NMR (400 MHz, CD3OD):
d 5.39e5.36 (2H, m,
olefinic protons), 5.34e5.32 (1H, m, H-2), 4.81 and 4.79 (each 1H, br
d, J¼3.3 Hz, H-10), 4.36 (1H, dd, J¼12.2, 7.3 Hz, H-1a), 4.22 (1H, dd,
J¼12.2, 6.6 Hz, H-1b), 4.15e4.13 (1H, m, H-40), 4.11e4.09 (1H, m, H-
50), 4.08e4.06 (1H, m, H-3a), 3.69e3.67 (1H, m, H-30), 3.67e3.65
(1H, m, H-3b), 3.46e3.44 (1H, m, H-20), 3.12 (1H, dd, J¼9.3, 9.3 Hz,
b
methyls),14.0,13.8 (CH3, acyl terminal methyls); HRESIMS m/z calcd
for C51H91NaO15KS: 1037.5613; found: 1037.5616.
4.3.15. Compound 9b. Compound 8b (0.065 g, 0.0.066 mmol) was
dissolved in a mixture of potassium acetate (0.032 g, 0.323 mmol),
aq H2O2 (0.161 mL) (34% w/v) and acetic acid (1.9 mL); the reaction
mixture was stirred overnight at 40 ꢀC; after evaporation under
a stream of nitrogen, the mixture was purified by silica gel chro-
matography using a gradient of chloroform/methanol to give
compound 9b (0.031 g, 0.031 mmol, 47%) as a colourless oil; Rf
(chloroform/methanol 9:1)¼0.32; IR (liquid film) nmax 3400, 2940,
H-60a), 2.95 (1H, dd, J¼14.4, 9.3 Hz, H-60a), 2.36e2.32 (4H, m,
a-
methylenes of acyl portions), 2.03e2.01 (4H, m, allylic protons),
1.65e1.62 (4H, m,
b-methylenes of acyl portions), 1.39e1.27 (48H,
m, aliphatic protons), 0.90e0.88 (6H, overlapped, 2CH3); 13C NMR
(100 MHz, CD3OD): d 175.1, 174.9 (C, acyl ester carbons), 130.5 (2CH,
olefinic methynes), 99.1 (CH, C10), 74.8 (CH, C40), 74.1 (CH, C30), 72.9
(CH, C20), 70.8 (CH, C2), 66.4 (CH2, C3), 63.1 (CH2, C1), 53.7 (CH2,
C60), 34.1, 34.0 (CH2,
a
-methylenes of acyl portions), 29.4e29.9
(CH2, aliphatic methylenes), 27.6 (CH2, allylic methylene), 24.9, 24.7
(CH2, -methylenes of acyl portions), 14.1, 13.8 (CH3, acyl terminal
2829, 1748, 1200, 1081 cmꢁ1
d
;
1H NMR (400 MHz, CDCl3):
5.41e5.39 (1H, m, H-30), 5.38e5.36 (2H, m, olefinic protons),
b
5.23e5.21 (1H, m, H-2), 5.15e5.13 (1H, m, H-10), 5.01e5.00 (1H, m,
H-40), 4.93e4.91 (1H, m, H-20), 4.33e4.31 (1H, m, H-1a), 4.27e4.25
(1H, m, H-1b), 4.26e4.24 (1H, m, H-50), 3.72e3.70 (1H, m, H-3a),
3.63e3.62 (1H, m, H-3b), 3.23e3.21 (1H, m, H-60a), 3.11e3.10 (1H,
methyls); HRESIMS m/z calcd for C45H83NaO12KS: 909.5140; found:
909.5144.
Acknowledgements
m, H-60b), 2.31e2.29 (4H, m,
a
-methylenes of acyl portions), 2.04
(6H, s, OAc), 2.03e2.00 (4H, m, allylic protons), 1.99 (3H, s, OAc),
1.59e1.57 (4H, m, -methylenes of acyl portions), 1.37e1.23 (48H,
m, aliphatic protons), 0.89e0.87 (6H, overlapped, 2CH3); 13C NMR
The authors thank Mrs. D. Melck and Alessandro Esposito of
ICB-NMR service, M. Zampa of ‘Servizio di Spettrometria di Massa’
for HRESIMS, and C. Iodice for spectrophotometric measurements.
The authors thank the support of the CNR Project on ‘Identification
and function of natural products of interest for human wellbeing’
(DPM 01.20).
b
(100 MHz, CDCl3):
d 172.7, 172.6 (C, acyl ester carbons), 130.2 (2CH,
olefinic methynes), 95.5 (CH, C10), 70.9 (CH, C40), 70.4 (CH, C30), 70.3
(CH, C2), 70.2 (CH, C20), 66.4 (CH2, C3), 62.9 (CH2, C1), 51.0 (CH2,
C60), 34.1, 34.0 (CH2,
a
-methylenes of acyl portions), 29.4e29.9
(CH2, aliphatic methylenes), 27.7 (2CH2, allylic methylenes), 24.9,
24.7 (CH2, -methylenes of acyl portions),20.6, 20.5 (CH3, acetate
References and notes
b
methyls),14.1,13.8 (CH3, acyl terminal methyls); HRESIMS m/z calcd
for C51H89NaO15KS: 1035.5457; found: 1035.5461.
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