Short regioselective chemoenzymatic synthesis and biological evaluation of 1- O-Acyl-2- O-(β-D-sulfoquinovopyranosyl)-sn-glycerols

Article abstract of DOI:10.1002/ejoc.200900943

A convenient chemoenzymatic synthesis of a new class of non-natural sulfo-glycolipids - 2-O-(β-D-sulfoquinovosyl)monoacylglycerols (2-O-β-D-SQMG) - derived from 2-O-(βD-glucopyranosyl)glycerol and carrying acyl chains of various lengths at the 1-position

Full text of DOI:10.1002/ejoc.200900943

FULL PAPER
DOI: 10.1002/ejoc.200900943
Short Regioselective Chemoenzymatic Synthesis and Biological Evaluation of
1-O-Acyl-2-O-(β- -sulfoquinovopyranosyl)-sn-glycerols
D
Milind Dangate,[a] Laura Franchini,[a] Fiamma Ronchetti,[a] Takanari Arai,[b] Akira Iida,[c]
Harukuni Tokuda,[d] and Diego Colombo*[a]
Keywords: Glycolipids / Enzyme catalysis / Antitumor agents
A convenient chemoenzymatic synthesis of a new class of
non-natural sulfo-glycolipids 2-O-(β- -sulfoquinovosyl)-
monoacylglycerols (2-O-β- -SQMG) – derived from 2-O-(β-
-glucopyranosyl)glycerol and carrying acyl chains of
various lengths at the 1-position of the sn-glycerol moiety,
was performed with the aid of a key step involving regiose-
lective lipase-catalyzed acylation of 2-O-(6-deoxy-6-tosyl-β-
through thioacetate substitution of the selectively inserted to-
syl group with subsequent Oxone® oxidation in the presence
of unprotected primary and secondary hydroxy groups ef-
ficiently afforded the target compounds, the hexanoyl, do-
decanoyl, and octadecanoyl derivatives 1a–c, which were
active when tested in the EBV-EA in vitro assay for antitumor
promoters.
–
D
D
D
D
-glucopyranosyl)-sn-glycerol (4) at its 1-position, reported
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
here for the first time. Elaboration of the sugar moiety
recently shown high in vitro and in vivo antitumor-promot-
ing activities[7] and, in particular, the in vitro activities of
fatty acid monoesters of 2-O-(β--glucopyranosyl)-sn-gly-
cerol[8] [namely 1-O-acyl-2-O-(β--glucopyranosyl)-sn-gly-
cerols] proved to be strictly related to the length of the acyl
chain.[9]
Introduction
Sulfoquinovosylacylglycerols are a family of sulfoglyco-
lipids in which 6-deoxy-6-sulfoglucose (sulfoquinovose) is
α-linked to the sn-3 position of an acylglycerol.[1,2] Among
them, sulfoquinovosyldiacylglycerols (SQDGs, Figure 1),
generally extracted from green plants or photosynthetic mi-
croorganisms together with glycoglycerolipids (lacking the
characteristic SQDG sulfonate), are characterized by the
presence of pairs of fatty acid acyl chains. Conversely, the
presence of only single chains is distinctive of synthetic sul-
foquinovosylmonoacylglycerols (SQMGs). Recently, both
SQDGs and SQMGs have come to be considered promising
compounds for cancer therapy and prevention, due to their
noteworthy biological activities.[3–6] With regard to cancer
prevention, 2-O-(β--glycopyranosyl)-sn-glycerol-related li-
pids (synthetic analogues of natural glycoglycerolipids) have
Figure 1. Structures of SQDGs (R and RЈ = acyl chain) and
SQMGs (R = acyl chain, RЈ = H).
In view of these results we set out to synthesize a new
class of SQMGs derived from 2-O-β--glucopyranosyl-sn-
glycerol in which acyl chains of different length would be
linked to one of the two free hydroxy groups in the glycerol
part and to test their in vitro antitumor-promoting activi-
ties. Here we report a rapid chemoenzymatic synthesis of
[a] Dipartimento di Chimica, Biochimica e Biotecnologie per la
Medicina, Università di Milano,
Via Saldini 50, 20133 Milano, Italy
Fax: +39-0250316036
E-mail: diego.colombo@unimi.it
[b] Endowed Center for Advancement of Pregnancy, Perinatal and the sulfoquinovosides 1a–c (Scheme 1, below) – 1-O-octa-
Infant Care, Graduate School of Medical Science, Kanazawa
University,
decanoyl, -dodecanoyl, and -hexanoyl-2-O-(β--sulfoquino-
vopyranosyl)-sn-glycerols – in which the key steps were se-
lective 6Ј-O-tosylation of the glucose moiety of a suitable
starting compound, selective lipase-catalyzed 1-O-
monoacylation of the glycerol moiety, and a final oxidation
reaction without any protection of the sugar unit. The in-
vitro antitumor activities of the obtained compounds 1a–c
are also reported.
13-1 Takara-machi, Kanazawa 920-8640, Japan
[c] School of Agriculture, Kinki University,
Nara, 631-8505, Japan
[d] Department of Complementary and Alternative Medicine,
Clinical R&D Graduate School of Medical Science, Kanazawa
University,
13-1 Takara-machi, Kanazawa 920-8640, Japan
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200900943.
Eur. J. Org. Chem. 2009, 6019–6026
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
6019

D. Colombo et al.
FULL PAPER
Results and Discussion
Synthesis
5a–c were obtained in yields of about 70% (see Exp. Sec-
tion). The 2S configurations were assigned to 5a–c on the
basis of chemical correlation with the known 1-O-octade-
canoyl, -dodecanoyl, and -hexanoyl-2-O-(β--glucopyran-
osyl)-sn-glycerols 6a–c, as discussed in the next section.[8,11]
Compounds 5a–c were then converted into the corre-
sponding 1-O-acyl-2-O-(6-deoxy-6-thioacetyl-β--glucopyr-
anosyl)-sn-glycerols 7a–c by potassium thioacetate treat-
ment in DMF in 64–67% yields. The final oxidation step
was carried out with Oxone® (potassium peroxymonosulf-
ate) in acetic acid[10] directly on the unprotected compounds
7a–c. Although 4Ј-O-acetylated sulfoquinovosides were
To perform the synthesis of the target monoesters 1a–c
we decided to exploit the convenient known regioselectivity
of lipase from Pseudomonas cepacia (LPS) towards 2-O-(β-
-glucopyranosyl)-sn-glycerol.[8] In particular, in transester-
ification reactions carried out in pyridine with 2,2,2-trifluoro-
ethyl alkanoates as acyl carriers, LPS had selectively acyl-
ated this polyhydroxylated compound at the 1-O position
in its sn-glycerol moiety.[8] The selectivity was especially
high (94%) with short-chain acyl carriers (2,2,2-trifluoro-
ethyl butanoate) and decreased (80%) with the lengthening
of the chain (2,2,2-trifluoroethyl octadecanoate), becoming
accompanied by increased acylation (15%) of the glucose
primary hydroxy group.[8] In view of these previous results,
our strategy was to use a tosyl group to protect the glucose
6Ј-OH of 2-O-(β--glucopyranosyl)-sn-glycerol before the
enzymatic regioselective acylation of the 1-OH group in the
sn-glycerol moiety and, in the meantime, also to use it as a
leaving group for insertion of the desired sulfonate group
into the molecule by means of thioacetate substitution and
final oxidation (Scheme 1).[10]
1
formed in a side reaction (diagnostic H NMR signals for
the dodecanoyl derivative in CDCl3/CD3OD/D2O: H-4Ј,
dd, at δ = 4.66 ppm and CH3CO, s, at δ = 2.08 ppm), this
procedure allowed us to obtain the target 1-O-acyl-2-O-(β-
-sulfoquinovopyranosyl)-sn-glycerols 1a–c directly in 35–
45% yields, avoiding the need for protecting group manipu-
lation.
Configuration Assignment
The 6Ј-tosyl derivatives 5a–c obtained from enzymatic
transesterification were transformed into the corresponding
ditosyl derivatives 8a–c (Scheme 2) by selective ZnBr2-cata-
lyzed tosylation under the conditions reported above (see
Exp. Section). When the known 1-O-octadecanoyl, -do-
decanoyl, and -hexanoyl-2-O-(β--glucopyranosyl)-sn-glyc-
erols 6a–c (2S configurations)[8,11] were subjected to selec-
tive3,6Ј-ditosylation,1-O-octadecanoyl,-dodecanoyl,and-hex-
anoyl-3-O-tosyl-2-O-(6-O-tosyl-β--glucopyranosyl)-sn-gly-
cerol (8a–c) were also obtained (Scheme 2). The complete
superimposability of the H and 13C NMR spectra of the
1
two batches in [D5]pyridine (see Exp. Section) allowed us
to assign 2R configurations to 8a–c and, consequently, 2S
configurations to 5a–c.
Scheme 1. Synthesis of SQMG analogues 1a–c.
The starting 1,3-di-O-benzyl-2-O-(β--glucopyranosyl)-
sn-glycerol (2) was prepared from glucose pentaacetate and
1,3-dibenzylglycerol as previously reported.[11] ZnBr2-cata-
lyzed selective tosylation[12] of the primary 6Ј-hydroxy
group of 2 in pyridine at –20 °C then yielded 1,3-di-O-ben-
zyl-2-O-(6-O-tosyl-β--glucopyranosyl)-sn-glycerol (3) in
high yields. Pd/C-catalyzed benzyl hydrogenolysis removed
Scheme 2. Configuration assignment of compounds 8a–c.
To validate this assignment we considered performing an
the benzyl groups, quantitatively affording 2-O-(6-O-tosyl- enzymatic transesterification (Scheme 3) on the 6Ј-tosylate
β--glucopyranosyl)-sn-glycerol (4), which was the sub- 4 with the aid of lipase from Candida antarctica (LCA) as
strate for the LPS-catalyzed transesterifications.[8]
the catalyst, because it is reported that this enzyme shows
The enzymatic reactions were run in pyridine with the an opposite, although lower, regioselectivity to that of LPS
appropriate 2,2,2-trifluoroethyl ester as acyl carrier until al- in the acylation of the glycerol moiety of 2-O-(β--gluco-
most complete disappearance of the starting material. In pyranosyl)-sn-glycerol, affording both the 2S and the 2R
this way, the pure 1-O-octadecanoyl, -dodecanoyl, and stereoisomers (in a 25:75 ratio in the presence of trifluoro-
-hexanoyl-2-O-(6-O-tosyl-β--glucopyranosyl)-sn-glycerols methyl decanoate as acyl carrier).[8] Here we decided to pre-
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2-O-(β--Sulfoquinovosyl)-monoacylglycerols
pare only the C-18 derivatives (Scheme 3), by treatment of tory effects of the already studied 1-O-acyl-2-O-(β--gluco-
4 with trifluoroethyl octadecanoate in pyridine in the pres- pyranosyl)-sn-glycerols 6a–c[9] are also reported for com-
ence of LCA. A mixture of the two monotosyl octade- parison.
1
canoates 5a and 9a in a 47:53 ratio (by H NMR) was ob-
Only weak cytotoxicities against Raji cells were observed
tained, with 5a proving to be the less polar compound by for compounds 1a–c (50% viability at 1000 mol ratio/TPA,
TLC analysis and 9a (easily purified by column chromatog- Table 1) and, in general, all compounds were active as indi-
raphy) being established to be its 2-diastereomer, because cated by their percentages to control values (53.6–61.3 at
its acyl chain was unambiguously shown by 1H NMR 500 mol ratio/TPA, Table 1). As already reported for com-
analysis to be linked to one of the two primary positions in pounds 6a–c,[9] shortening of the acyl chain, from C18 (1a)
the sn-glycerol component (see Exp. Section). Finally, 3-O- to C6 (1c), resulted in increasing inhibitory activity (IC50
octadecanoyl-2-O-(6-O-tosyl-β--glucopyranosyl)-sn-gly- 543 for 1a vs. 417 for 1c; see Table 1). In general, the sulfo-
cerol (9a) was converted into the 1,6Ј-ditosyl derivative 3- nate group seems to have little influence on the antitumor-
O-octadecanoyl-1-O-tosyl-2-O-(6-O-tosyl-β--glucopyrano- promoting activities of glycoglycerolipids bearing medium
syl)-sn-glycerol (10a, 2S configuration). This showed dif- or long chains (IC50 543 and 463 for 1a and 1b vs. IC50 550
1
ferent H and 13C NMR spectra from those of the 1-O- and 504 for 6a and 6b; Table 1) in this in vitro experimental
octadecanoyl derivative 8a (2R configuration; see Exp. Sec- model. However, when a short acyl chain is present, the
tion), further confirming the previously performed configu- sulfonate exerts a strong negative effect on the activity (IC50
ration assignment.
417 for 1c vs. 29.3 for 6c; Table 1).
Conclusions
A new class of sulfoquinovosylmonoacylglycerols based
on 2-O-(β--glucopyranosyl)-sn-glycerol – 1-O-acyl-2-O-(β-
-sulfoquinovopyranosyl)-sn-glycerols
1a–c
(2-O-β--
SQMG) – were prepared by means of a short chemoenzym-
atic procedure using the tosyl group as a convenient tempo-
rary protecting group and LPS as an efficient and selective
biocatalyst for polyhydroxylated substrates. It is noteworthy
that, due to the practicability of chromatographic separa-
tion of the two 2-diastereomers 5a and 9a, the same pro-
cedure could in principle also be exploited to synthesize 3-
O-acyl-2-O-(β--sulfoquinovopyranosyl)-sn-glycerols sim-
ply by changing the employed biocatalyst, as in the case of
Scheme 3. LCA-catalyzed synthesis of 10a (diastereomer of 8a).
EBV-EA Assay for Antitumor Promoters
Epstein–Barr Virus (EBV) is known to be activated by LCA vs. LPS. A preliminary bioassay was carried on the
tumor promoters to produce viral early antigens (EAs), and obtained compounds, showing their antitumor-promoting
an evaluation of its inhibition is often used as a primary activities, and some new structure–activity relationship con-
screening for in vitro antitumor-promoting activities.[13] The clusions on the influence of the sulfonate group could also
inhibitory effects of the 2-O-β--SQMGs 1a–c were assayed be drawn. In particular, a highly negative effect of this
in a short-term in vitro assay for EBV-EA activation in- group was observed for the short-chain compound 1c in
duced by the tumor promoter 12-O-tetradecanoylphorbol- relation to the corresponding non-sulfonated 6c. In con-
13-acetate (TPA) in Raji cells, as described in ref.[6,14] (see trast, when a long acyl chain is present on the glycerol moi-
Exp. Section). Table 1 shows the in vitro tumor-promoting ety (see compound 1a vs. 6a), the simultaneous presence of
inhibitory activities of compounds 1a–c; the in vitro inhibi- a sulfonate moiety does not significantly induce variation
Table 1. Inhibitory effects of 1a–c and 6a–c on TPA-induced EBV-EA activation.
Concentration (mol ratio/TPA)
1000
500
100
10
% to controlϮSE (n = 3)[a]
IC50
1a
1b
22.5Ϯ1.6 (50)[b]
17.0Ϯ1.5 (50)
14.3Ϯ1.2 (50)
19.6Ϯ0.7 (60)
0.0Ϯ0.4 (60)
0.0Ϯ0.0 (70)
61.3Ϯ2.2 (70)
57.2Ϯ2.1 (70)
53.6Ϯ2.0 (70)
57.4Ϯ2.2 (70)
52.5Ϯ2.3 (70)
10.6Ϯ0.6 (70)
83.5Ϯ1.7 (80)
81.5Ϯ1.9 (80)
80.8Ϯ1.9 (80)
90.6Ϯ0.5 (80)
82.5Ϯ1.6 (80)
30.9Ϯ1.8 (80)
100Ϯ0.4 (80)
100Ϯ0.4 (80)
100Ϯ0.5 (80)
100Ϯ0.0 (80)
100Ϯ0.2 (80)
68.2Ϯ2.5 (80)
543
463
417
550
504
29.3
1c
6a[c]
6b[c]
6c[c]
[a] Values are EBV-EA activation (%) in the presence of the test compound relative to the control (100%). Activation was achieved by
treatment with TPA (32 pmol). IC50 represents the mol ratio to TPA that inhibits 50% of positive control (100%) activated with 32 pmol
TPA. [b] Values in parentheses are viability percentages of Raji cells. [c] See ref.[9]
Eur. J. Org. Chem. 2009, 6019–6026
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D. Colombo et al.
Synthesis of 1,3-Di-O-benzyl-2-O-(6-O-tosyl-β- -glucopyranosyl)-
FULL PAPER
D
in activity, showing that the effects of the “negative” groups
(sulfonate and long chain) are not additive. This conclusion
is strengthened by the recent observation that 1,3-di-O-oc-
tadecanoyl-2-O-(β--sulfoquinovopyranosyl)-sn-glycerol,
which contains three different “negative” factors (i.e., sulfo-
nate, long chain, and double acylation of glycerol) has an
activity similar to those of compounds 1a and 6a (IC50 543
and 550, Table 1, vs. 632, ref.[15]). Work to further charac-
terize the biological potential of these new synthetically
available sulfoquinovosyl-monoacylglycerols is in progress.
sn-glycerol (3): Zinc bromide (6.21 g, 27.6 mmol) was added at
–20 °C to a solution of compound 2 (3.00 g, 6.90 mmol) in dry
pyridine (240 mL). After the addition of tosyl chloride (6.58 g,
34.5 mmol) the reaction mixture was stirred at –20 °C for 0.5 h and
monitored by TLC (EtOAc/CH3OH, 95:5 v/v). After quenching
with CH3OH, the solvent was evaporated under vacuum, the resi-
due was dissolved in EtOAc (450 mL), the organic solution was
washed with HCl (1 , 2ϫ120 mL), H2O (120 mL), saturated
NaHCO3 solution (120 mL), and H2O (2ϫ120 mL), and the aque-
ous phases were extracted again with EtOAc (2ϫ360 mL). The col-
lected organic layers were dried with Na2SO4, concentrated under
vacuum, and purified by flash column chromatography with EtOAc
to provide the tosyl derivative 3 (3.65 g, 6.20 mmol, 90% yield) as
a gel-like compound. [α]2D0 = –6.3 (CHCl3). 1H NMR (CDCl3): δ =
2.39 (s, 3 H, CH3), 3.33 (dd, J1Ј,2Ј = 7.8, J2Ј,3Ј = 8.4 Hz, 1 H, 2Ј-
H), 3.39–3.49 (m, 3 H, 3Ј-H, 4Ј-H, 5Ј-H), 3.50–3.64 (m, 4 H, 1a-
H, 1b-H, 3a-H, 3b-H), 4.02 (m, 1 H, 2-H), 4.21 (dd, J6Јa,5Ј = 4.6,
J6Јa,6Јb = 10.9 Hz, 1 H, 6Јa-H), 4.26 (dd, J6Јb,5Ј Ͻ 1.0 Hz, 1 H, 6Јb-
H), 4.40 (d, 1 H, 1Ј-H), 4.46–4.54 (m, 4 H, 2ϫCH2), 7.23–7.34 (m,
12 H, Ph), 7.76 (d, J = 8.2 Hz, 2 H, Ph) ppm. 13C NMR (CDCl3):
δ = 21.64 (PhCH3), 68.65 (C6Ј), 69.44 (C4Ј or C5Ј), 70.25 and 70.61
(C1 or C3), 73.09 (C2Ј), 73.49 and 73.51 (2ϫPhCH2), 73.70 (C4Ј
or C5Ј), 75.98 (C3Ј), 77.60 (C2), 103.04 (C1Ј), 127.5–128.6 (10 C,
2ϫPh), 127.95 and 129.85 (4 C, Ts), 132.83 (Ts), 137.60 and 137.74
(2 C, Ph), 144.91 (Ts) ppm. ESI-MS (CH3OH, positive-ion mode,
relative intensity): m/z (%) = 611.1 (100) [M + Na]+. C30H36O10S
(588.67): calcd. C 61.21, H 6.16, S 5.45; found C 61.30, H 6.18, S
5.44.
Experimental Section
General: Pseudomonas cepacia lipase (LPS, lipase PS, specific ac-
tivity 30.5 triacetin units/mg solid), from Amano Pharmaceuti-
cal Co (Mitsubishi Italia), was supported on celite;[8] Candida ant-
arctica lipase SP 435 L, immobilized on a macroporous acrylic
resin (Novozym® 435, LCA, specific activity 9.5 PL units/mg solid)
was purchased from Novo Nordisk. LPS and LCA were kept under
vacuum overnight prior to use. Pyridine was distilled from calcium
hydride prior to use. The acyl carriers – 2,2,2-trifluoroethyl esters –
were synthesized as described.[8] 1,3-Di-O-benzyl-2-O-(β--gluco-
pyranosyl)-sn-glycerol (2) was synthesized by a literature pro-
cedure.[11] Optical rotations were determined with a Perkin–El-
mer 241 polarimeter in 1% CHCl3, CH3OH or CHCl3/CH3OH/
H2O (65:25:4) solutions at 20 °C, in a 1 dm cell. Melting points
(m.p.) were recorded on a Büchi 510 capillary melting point appa-
ratus and are uncorrected. All reagents and solvents used were rea-
gent grade and were purified before use by standard methods. Dry
solvents and liquid reagents were distilled prior to use or were dried
with molecular sieves (4 Å). Column chromatography was carried
out on flash silica gel (Merck 230–400 mesh). TLC analysis was
carried out on silica gel plates (Merck 60F254) with development
with 50% sulfuric acid or anisaldehyde-based reagent. Evaporation
under reduced pressure was always performed with a bath tempera-
ture below 40 °C. The structures of all the new synthesized com-
Synthesis of 2-O-(6-O-Tosyl-β-D-glucopyranosyl)-sn-glycerol (4):
Compound 3 (3.00 g, 5.10 mmol) was dissolved in methanol
(160 mL), and Pd on activated carbon (10%, 1.0 g) was added. The
mixture was shaken under hydrogen for 2 h with monitoring by
TLC (EtOAc/CH3OH 100:1 v/v) and was then filtered through a
celite bed to afford the debenzylated 4 (2.02 g, 4.95 mmol, 97%
1
yield); m.p. 168 °C (amorphous solid). [α]2D0 = +4.1 (CH3OH). H
NMR (CD3OD): δ = 2.45 (s, 3 H, CH3), 3.18 (dd, J1Ј,2Ј = 7.8, J2Ј,3Ј
= 9.2 Hz, 1 H, 2Ј-H), 3.21 (dd, J3Ј,4Ј = 9.5, J4Ј,5Ј = 9.5 Hz, 1 H, 4Ј-
H), 3.32 (dd, 1 H, 3Ј-H), 3.45 (ddd, J5Ј,6Јa = 6.0, J5Ј,6Јb = 1.7 Hz, 1
H, 5Ј-H), 3.55–3.72 (m, 5 H, 1a-H, 1b-H, 2-H, 3a-H, and 3b-H),
4.12 (dd, J6Јa,6Јb = 10.7 Hz, 1 H, 6Јa-H), 4.31 (dd, 1 H, 6Јb-H), 4.38
(d, 1 H, 1Ј-H), 7.45 (d, J = 8.2 Hz, 2 H, Ph), 7.80 (d, 2 H, Ph) ppm.
13C NMR (CD3OD): δ = 21.58 (PhCH3), 62.67 and 62.98 (C1 or
C3), 70.67 (C6Ј), 71.05 (C4), 74.96 (C2Ј), 75.05 (C5Ј), 77.56 (C3Ј),
82.95 (C2), 104.37 (C1Ј), 129.08 and 131.11 (4 C, Ph), 134.26 and
146.57 (2 C, Ph) ppm. ESI-MS (CH3OH, positive-ion mode, rela-
tive intensity): m/z (%) = 431.1 (100) [M + Na]+. C16H24O10S
(408.42): calcd. C 47.05, H 5.92, S 7.85; found C 47.19, H 5.94, S
7.88.
pounds were confirmed through full H and 13C NMR characteri-
zation and mass spectroscopy. H NMR analysis were performed
1
1
at 500 MHz with a Bruker FT-NMR AVANCETM DRX 500 spec-
trometer with a 5 mm z-PFG (pulsed field gradient) broadband
reverse probe at 298 K unless otherwise stated, and 13C NMR spec-
tra of all the new compounds were measured at 125.76 MHz. The
signals were unambiguously assigned by 2D COSY and HSQC ex-
periments (standard Bruker pulse program). Chemical shifts are
reported as δ (ppm) relative to residual CHCl3, pyridine, or
CH3OH (when CDCl3/CD3OD/D2O solvent mixture was used)
fixed at 7.24, 7.19 (higher field signal), and 3.30 ppm, respectively,
General Procedure for the Enzymatic Synthesis of Monoesters 5a–
c: Tosyl derivative 4 (0.600 g, 1.47 mmol) was dissolved in pyridine
(10 mL) and the appropriate trifluoroethyl ester (8.82 mmol) and
LPS (3.6 g) were added in that order. The suspension was stirred
at 45 °C overnight and monitored by TLC (CH2Cl2/CH3OH,
90:10 v/v). The reaction was stopped by filtering off the enzyme,
which was washed with pyridine. After removal of the solvent un-
der vacuum, the resulting crude material was subjected to flash
column chromatography (CH2Cl2/CH3OH, 95:5 v/v) to yield the
pure 1-O-monoesters 5a–c.
1
for H NMR spectra and relative to CDCl3 fixed at δ = 77.0 ppm
(central line), [D5]pyridine 123.0 (higher field signal, central line),
or CD3OD at δ = 49.00 ppm (central line) for 13C NMR spectra;
scalar coupling constants are reported in Hz. Mass spectra were
recorded in negative- or positive-ion electrospray (ESI) mode with
a Thermo Quest Finnigan LCQTM DECA ion-trap mass spectrom-
eter equipped with a Finningan ESI interface; sample solutions
were injected with a ionization spray voltage of 4.5 or 5.0 kV (posi-
tive- and negative-ion mode, respectively), a capillary voltage of
32 V or –15 V (positive- and negative-ion mode, respectively), and
capillary temperature of 250 °C. Data were processed with the aid
of the Finnigan Xcalibur software system.
1-O-Octadecanoyl-2-O-(6-O-tosyl-β-D-glucopyranosyl)-sn-glycerol
(5a): Reaction time 21 h, yield 72%; m.p. 96 °C (amorphous solid).
[α]2D0 = –11.3 (CHCl3). 1H NMR ([D5]pyridine, 312 K): δ = 0.87 (t,
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Eur. J. Org. Chem. 2009, 6019–6026

2-O-(β--Sulfoquinovosyl)-monoacylglycerols
J = 7.0 Hz, 3 H, CH3), 1.17–1.35 (m, 28 H, 14ϫCH2), 1.66 (m, 2
rated with cyclohexane at reduced pressure at 45 °C and subse-
quent flash column chromatography (CH2Cl2/CH3OH, 98:2–
H, CH2), 2.18 (s, 3 H, CH3), 2.37 (m, 2 H, CH2), 3.86 (dd, J1Ј,2Ј
=
7.8, J2Ј,3Ј = 8.8 Hz, 1 H, 2Ј-H), 3.94 (dd, J3Ј,4Ј = 8.8, J4Ј,5Ј = 9.7 Hz, 95:5 v/v) of the crude residue yielded the desired derivative 7.
1 H, 4Ј-H), 4.00 (ddd, J5Ј,6Јa = 6.0, J5Ј,6Јb = 1.6 Hz, 1 H, 5Ј-H), 4.10
2-O-(6-Deoxy-6-thioacetyl-β-D-glucopyranosyl)-1-O-octadecanoyl-
(dd, 1 H, 3Ј-H), 4.11–4.18 (m, 2 H, 3a-H, 3b-H), 4.38 (m, 1 H, 2-
H), 4.62–4.73 (m, 3 H, 1a-H, 1b-H, and 6Јa-H), 4.91 (dd, J6Јa,6Јb
sn-glycerol (7a): Reaction time 17 h, yield 64%; m.p. 90 °C (brown
=
1
amorphous solid). [α]2D0 = –20.8 (CHCl3). H NMR (CDCl3): δ =
10.4 Hz, 1 H, 6Јb-H), 4.98 (d, 1 H, 1Ј-H), 7.24 (d, J = 8.1 Hz, 2 H,
Ph), 8.00 (d, 2 H, Ph) ppm. 13C NMR ([D5]pyridine, 312 K): δ =
13.76 (CH3), 20.79 (PhCH3), 22.44 (CH2), 24.77 (CH2), 28.90–
29.60 (12ϫCH2), 31.64 (CH2), 33.92 (CH2CO), 62.23 (C3), 63.87
(C1), 70.41 (C6Ј), 70.46 (C4Ј), 74.23 (C2Ј), 74.52 (C5Ј), 77.53 (C3Ј),
78.71 (C2), 104.20 (C1Ј), 127.84 and 129.82 (4 C, Ph), 133.42 and
145.55 (2 C, Ph), 173.14 (CO) ppm. ESI-MS (CH3OH, positive-
ion mode, relative intensity): m/z (%) = 697.3 (100) [M + Na]+.
C34H58O11S (674.88): calcd. C 60.51, H 8.66, S 4.75; found C 60.68,
H 8.60, S 4.81.
0.85 (t, J = 7.1 Hz, 3 H, CH3), 1.17–1.31 (m, 28 H, 14ϫCH2), 1.58
(m, 2 H, CH2), 2.30 (m, 2 H, CH2), 2.36 (s, 3 H, –SCOCH3), 3.20
(dd, J6Јa,5Ј = 6.2, J6Јa,6Јb = 14.4 Hz, 1 H, 6Јa-H), 3.29 (dd, J3Ј,4Ј
=
9.3, J4Ј,5Ј = 9.3 Hz, 1 H, 4Ј-H), 3.33 (dd, J6Јb,5Ј = 2.8 Hz, 1 H, 6Јb-
H), 3.37 (dd, J1Ј,2Ј = 7.7, J2Ј,3Ј = 8.2 Hz, 1 H, 2Ј-H), 3.46 (ddd, 1
H, 5Ј-H), 3.54 (dd, 1 H, 3Ј-H), 3.57–3.70 (m, 2 H, 3a-H, 3b-H),
3.88 (m, 1 H, 2-H), 4.13 (dd, J1a,2 = 5.7, J1a,1b = 11.7 Hz, 1 H, 1a-
H), 4.24 (dd, J1b,2 = 5.0 Hz, 1 H, 1b-H), 4.36 (d, 1 H, 1Ј-H) ppm.
13C NMR (CDCl3): δ = 14.15 (CH3), 22.72 (CH2), 24.90 (CH2),
29.16–29.78 (12 ϫ CH2), 30.57 (SCOCH3), 30.67 (C6Ј), 31.96
(CH2), 34.22 (CH2CO), 62.61 (C3), 63.03 (C1), 72.02 (C4Ј), 73.31
(C2Ј), 74.49 (C5Ј), 75.45 (C3Ј), 80.34 (C2), 103.01 (C1Ј), 174.11
(CO), 197.40 (SCO) ppm. ESI-MS (CH3OH, positive-ion mode,
relative intensity): m/z (%) = 601.3 [M + Na]+ (100). C29H54O9S
(578.8): calcd. C 60.18, H 9.40, S 5.54; found C 60.07, H 9.36, S
5.59.
1-O-Dodecanoyl-2-O-(6-O-tosyl-β-
D-glucopyranosyl)-sn-glycerol
(5b): Reaction time 21 h, yield 68%; gel-like compound. [α]2D0
=
–13.0 (CHCl3). 1H NMR ([D5]pyridine, 312 K): δ = 0.86 (t, J =
7.0 Hz, 3 H, CH3), 1.17–1.30 (m, 16 H, 8ϫCH2), 1.65 (m, 2 H,
CH2), 2.18 (s, 3 H, CH3), 2.37 (m, 2 H, CH2), 3.86 (dd, J1Ј,2Ј = 7.8,
J2Ј,3Ј = 8.8 Hz, 1 H, 2Ј-H), 3.94 (dd, J3Ј,4Ј = 8.8, J4Ј,5Ј = 9.7 Hz, 1
H, 4Ј-H), 4.00 (ddd, J5Ј,6Јa = 5.9, J5Ј,6Јb = 1.7 Hz, 1 H, 5Ј-H), 4.10
(dd, 1 H, 3Ј-H), 4.11–4.18 (m, 2 H, 3a-H, 3b-H), 4.38 (m, 1 H, 2-
2-O-(6-Deoxy-6-thioacetyl-β-D-glucopyranosyl)-1-O-dodecanoyl-sn-
glycerol (7b): Reaction time 17 h, yield 65%; brown, gel-like com-
H), 4.65–4.72 (m, 3 H, 1a-H, 1b-H, 6Јa-H), 4.90 (dd, J6Јa,6Јb
=
1
pound. [α]2D0 = –30.8 (CHCl3). H NMR (CDCl3): δ = 0.84 (t, J =
10.4 Hz, 1 H, 6Јb-H), 4.98 (d, 1 H, 1Ј-H), 7.24 (d, J = 8.3 Hz, 2 H,
Ph), 8.00 (d, 2 H, Ph) ppm. 13C NMR ([D5]pyridine, 312 K): δ =
13.76 (CH3), 20.79 (PhCH3), 22.43 (CH2), 24.76 (CH2), 28.94
(CH2), 29.09 (2CH2), 29.26 (CH2), 29.37 (2ϫCH2), 31.62 (CH2),
33.92 (CH2CO), 62.23 (C3), 63.87 (C1), 70.41 (C6Ј), 70.46 (C4Ј),
74.22 (C2Ј), 74.51 (C5Ј), 77.53 (C3Ј), 78.71 (C2), 104.19 (C1Ј),
127.83 and 129.82 (4 C, Ph), 133.41 and 145.55 (2 C, Ph), 173.14
(CO) ppm. ESI-MS (CH3OH, positive-ion mode, relative intensity):
m/z (%) = 613.2 [M + Na]+ (100). C28H46O11S (590.72): calcd. C
56.93, H 7.85, S 5.43; found C 57.10, H 7.93, S 5.37.
7.0 Hz, 3 H, CH3), 1.16–1.30 (m, 16 H, 8ϫCH2), 1.56 (m, 2 H,
CH2), 2.28 (m, 2 H, CH2), 2.33 (s, 3 H, –SCOCH3), 3.07 (dd, J6Јa,5Ј
= 7.5, J6Јa,6Јb = 14.2 Hz, 1 H, 6Јa-H), 3.29 (dd, J3Ј,4Ј = 9.0, J4Ј,5Ј
=
9.0 Hz, 1 H, 4Ј-H), 3.35–3.45 (m, 3 H, 2Ј-H, H5Ј-H, 6Јb-H), 3.51
(dd, J2Ј,3Ј = 8.5 Hz, 1 H, 3Ј-H), 3.56–3.73 (m, 2 H, 3a-H, 3b-H),
3.89 (m, 1 H, 2-H), 4.12–4.21 (m, 2 H, 1a-H, 1b-H), 4.38 (d, J1Ј,2Ј
= 7.7 Hz, 1 H, 1Ј-H) ppm. 13C NMR (CDCl3): δ = 14.05 (CH3),
22.62 (CH2), 24.82 (CH2), 29.13 (CH2), 29.28 (2 ϫ CH2), 29.46
(CH2), 29.58 (2ϫCH2), 30.50 (SCOCH3), 30.73 (C6Ј), 31.85 (CH2),
34.12 (CH2CO), 62.48 (C3), 62.77 (C1), 72.52 (C4Ј), 73.08 (C2Ј),
74.58 (C5Ј), 75.57 (C3Ј), 79.05 (C2), 102.48 (C1Ј), 173.94 (CO),
196.71 (SCO) ppm. ESI-MS (CH3OH, positive-ion mode, relative
intensity): m/z (%) = 517.1 (100) [M + Na]+. C23H42O9S (494.64):
calcd. C 55.85, H 8.56, S 6.48; found C 56.03, H 8.61, S 6.39.
1-O-Hexanoyl-2-O-(6-O-tosyl-β-D-glucopyranosyl)-sn-glycerol (5c):
Reaction time 21 h, yield 70%; oil. [α]2D0 = –16.4 (CHCl3). 1H NMR
([D5]pyridine, 312 K): δ = 0.78 (t, J = 7.0 Hz, 3 H, CH3), 1.19 (m,
4 H, 2ϫCH2), 1.60 (m, 2 H, CH2), 2.18 (s, 3 H, CH3), 2.33 (m, 2
H, CH2), 3.85 (dd, J1Ј,2Ј = 7.7, J2Ј,3Ј = 8.8 Hz, 1 H, 2Ј-H), 3.93 (dd,
J3Ј,4Ј = 8.8, J4Ј,5Ј = 9.7 Hz, 1 H, 4Ј-H), 4.00 (ddd, J5Ј,6Јa = 5.9, J5Ј,6Јb
= 1.7 Hz, 1 H, 5Ј-H), 4.09 (dd, 1 H, 3Ј-H), 4.10–4.17 (m, 2 H, 3a-
H, 3b-H), 4.37 (m, 1 H, 2-H), 4.59–4.71 (m, 3 H, 1a-H, 1b-H, 6Јa-
H), 4.91 (dd, J6Јa,6Јb = 10.4 Hz, 1 H, 6Јb-H), 4.96 (d, 1 H, 1Ј-H),
7.24 (d, J = 8.3 Hz, 2 H, Ph), 8.00 (d, 2 H, Ph) ppm. 13C NMR
([D5]pyridine, 312 K): δ = 13.49 (CH3), 20.78 (PhCH3), 22.02
(CH2), 24.36 (CH2), 30.98 (CH2), 33.83 (CH2CO), 62.22 (C3),
63.89 (C1), 70.42 (C6Ј), 70.46 (C4Ј), 74.22 (C2Ј), 74.51 (C5Ј), 77.53
(C3Ј), 78.69 (C2), 104.19 (C1Ј), 127.83 and 129.82 (4 C, Ph), 133.40
and 145.56 (2 C, Ph), 173.09 (CO) ppm. ESI-MS (CH3OH, posi-
tive-ion mode, relative intensity): m/z (%) = 529.1 [M + Na]+ (100).
C22H34O11S (506.56): calcd. C 52.16, H 6.77, S 6.33; found C 52.29,
H 6.82, S 6.27.
2-O-(6-Deoxy-6-thioacetyl-β-D-glucopyranosyl)-1-O-hexanoyl-sn-
glycerol (7c): Reaction time 17 h, yield 67%; pink, gel-like com-
1
pound. [α]2D0 = –37.0 (CHCl3). H NMR (CDCl3): δ = 0.86 (t, J =
7.0 Hz, 3 H, CH3), 1.21–1.33 (m, 4 H, 2ϫCH2), 1.57 (m, 2 H,
CH2), 2.29 (m, 2 H, CH2), 2.34 (s, 3 H, –SCOCH3), 3.08 (dd, J6Јa,5Ј
= 8.0, J6Јa,6Јb = 14.8 Hz, 1 H, 6Јa-H), 3.30 (dd, J3Ј,4Ј = 9.1, J4Ј,5Ј
=
9.1 Hz, 1 H, 4Ј-H), 3.34–3.44 (m, 3 H, 2Ј-H, H5Ј-H, 6Јb-H), 3.51
(dd, J2Ј,3Ј = 9.0 Hz, 1 H, 3Ј-H), 3.56–3.72 (m, 2 H, 3a-H, 3b-H),
3.90 (m, 1 H, 2-H), 4.15–4.20 (m, 2 H, 1a-H, 1b-H), 4.38 (d, J1Ј,2Ј
= 7.8 Hz, 1 H, 1Ј-H) ppm. 13C NMR (CDCl3): δ = 13.83 (CH3),
22.24 (CH2), 24.51 (CH2), 30.48 (SCOCH3), 30.77 (C6Ј), 31.25
(CH2), 34.13 (CH2CO), 62.58 (C3), 63.01 (C1), 72.46 (C4Ј), 73.30
(C2Ј), 74.64 (C5Ј), 75.70 (C3Ј), 79.59 (C2), 102.75 (C1Ј), 173.96
(CO), 196.78 (SCO) ppm. ESI-MS (CH3OH, positive-ion mode,
relative intensity): m/z (%) = 433.1 (100) [M + Na]+. C17H30O9S
(410.48): C 49.74, H 7.37, S 7.81; found C 49.83, H 7.45, S 7.67.
General Procedure for the Synthesis of Thioacetates 7a–c: A com-
pound 5 (0.770 mmol) was dissolved in dry DMF (30 mL), and
potassium thioacetate (0.390 g, 3.41 mmol) was added. The mix-
ture was stirred under Ar at room temperature and the changing
of its color from blue to brown indicated progress of the reaction
that was monitored by TLC (CH2Cl2/CH3OH, 97:3 v/v). Owing to
co-elution of the starting and target compound, the reaction was
stopped when the TLC spot color had turned completely brown
from green (anisaldehyde-based reagent). DMF was then co-evapo-
General Procedure for the Synthesis of Sulfonates 1a–c: Potassium
monopersulfate triple salt (Oxone®, 0.830 g, 1.35 mmol) and potas-
sium acetate (1.30 g, 13.2 mmol) were added in that order to a solu-
tion of a compound 7 (0.450 mmol) in glacial acetic acid (15 mL).
The suspension was stirred at room temperature and the reaction
was monitored by TLC (CHCl3/CH3OH/H2O, 65:25:4). After dis-
appearing of the starting material, the solvent was evaporated un-
Eur. J. Org. Chem. 2009, 6019–6026
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6023

D. Colombo et al.
FULL PAPER
der vacuum. The obtained crude residue was purified directly by
flash column chromatography (from CHCl3/CH3OH, 80:20 v/v to
CHCl3/CH3OH/H2O, 65:25:4 v/v) to yield the pure sulfonate 1.
[M]. C15H27KO11S (454.53): calcd. C 39.64, H 5.99, S 7.05; found
C 39.72, H 6.10, S 6.98.
Configuration Assignment of Tosylates 5a–c: The converging syn-
thesis of the 2R ditosyl derivatives 8a–c was achieved by starting
from 5a–c and 6a–c as reported here.
1-O-Octadecanoyl-2-O-(β-D-sulfoquinovopyranosyl)-sn-glycerol Po-
tassium Salt (1a): Reaction time 7 h; yield 37 %; m.p. 220 °C
(amorphous solid). [α]2D0 = –6.5 (CHCl3/CH3OH/H2O, 65:25:4). H
1
General Procedure for the Synthesis of (2R)-Ditosylates 8a–c from
5a–c: Zinc bromide (0.135 g, 0.600 mmol) was added at –20 °C to
a solution of a compound 5 (0.150 mmol) in dry pyridine (5.0 mL).
After addition of tosyl chloride (0.143 g, 0.750 mmol) the reaction
mixture was stirred at –20 °C for 1 h and monitored by TLC
(CH2Cl2/CH3OH, 95:5 v/v). After quenching with CH3OH, the sol-
vent was evaporated under vacuum by repeated addition of toluene
and the obtained crude material was subjected to flash column
chromatography (CH2Cl2/CH3OH, 95:5 v/v) to yield the ditosyl de-
rivative 8 (about 70%).
NMR (CDCl3/CD3OD/D2O, 65:25:4, 320 K): δ = 0.84 (t, J = 7.1 Hz,
3 H, CH3), 1.17–1.34 (m, 28 H, 14ϫCH2), 1.56 (m, 2 H, CH2), 2.29
(m, 2 H, CH2), 3.00 (dd, J6Јa,5Ј = 8.6, J6Јa,6Јb = 14.6 Hz, 1 H, 6Јa-
H), 3.22 (dd, J3Ј,4Ј = 9.2, J4Ј,5Ј = 9.2 Hz, 1 H, 4Ј-H), 3.24 (dd, J1Ј,2Ј
= 7.9, J2Ј,3Ј = 9.2 Hz, 1 H, 2Ј-H), 3.32 (dd, J6Јb,5Ј = 2.4 Hz, 1 H, 6Јb-
H), 3.40 (dd, 1 H, 3Ј-H), 3.62 (dd, J2,3a = 6.0, J3a,3b = 12.0 Hz, 1 H,
3a-H), 3.68 (dd, J2,3b = 3.9 Hz, 1 H, 3b-H), 3.72 (ddd, 1 H, 5Ј-H),
3.94 (m, 1 H, 2-H), 4.15 (dd, J2,1a = 5.2, J1a,1b = 11.8 Hz, 1 H, 1a-
H), 4.18 (dd, J2,1b = 5.4 Hz, 1 H, 1b-H), 4.40 (d, 1 H, 1Ј-H) ppm.
13C NMR (CDCl3/CD3OD/D2O, 65:25:4, 320 K): δ = 14.19 (CH3),
22.96 (CH2), 25.19 (CH2), 29.41–30.24 (12 ϫCH2), 32.25 (CH2),
34.46 (CH2CO), 53.05 (C6Ј), 62.27 (C3), 63.84 (C1), 72.97 (C5Ј),
73.25 (C4Ј), 73.88 (C2Ј), 76.40 (C3Ј), 80.10 (C2), 103.45 (C1Ј), 175.08
(CO) ppm. ESI-MS (CH3OH, negative-ion mode, relative intensity):
m/z (%) = 583.3 [M]– (100). Calcd for C27H51O11S–, m/z 583.3 [M]–.
1-O-Octadecanoyl-3-O-tosyl-2-O-(6-O-tosyl-β-D-glucopyranosyl)-
sn-glycerol (8a): M.p. 208 °C (from Et2O). [α]2D0 = –2.2 (CHCl3). 1H
NMR ([D5]pyridine, 312 K): δ = 0.86 (t, J = 7.1 Hz, 3 H, CH3),
1.20–1.32 (m, 28 H, 14ϫCH2), 1.61 (m, 2 H, CH2), 2.23 (s, 3 H,
CH3), 2.24 (s, 3 H, CH3), 2.30 (m, 2 H, CH2), 3.71 (dd, J1Ј,2Ј = 7.8,
J2Ј,3Ј = 8.9 Hz, 1 H, 2Ј-H), 3.85 (dd, J3Ј,4Ј = 9.0, J4Ј,5Ј = 9.9 Hz, 1
H, 4Ј-H), 3.95 (ddd, J5Ј,6Јa = 6.1, J5Ј,6Јb = 1.7 Hz, 1 H, 5Ј-H), 4.03
(dd, 1 H, 3Ј-H), 4.40–4.46 (m, 2 H, 2-H, 1a-H), 4.51–4.61 (m, 3 H,
a-H, 3b-H, 1b-H), 4.62 (dd, J6Јa,6Јb = 10.5, J6Јa,5Ј = 6.1 Hz, 1 H,
6Јa-H), 4.86 (d, 1 H, 1Ј-H), 4.88 (dd, 1 H, 6Јb-H), 7.29 (m, 4 H,
Ph), 8.01 (d, J = 8.1 Hz, 2 H, Ph), 8.02 (d, J = 8.1 Hz, 2 H,
Ph) ppm. 13C NMR ([D5]pyridine, 312 K): δ = 13.75 (CH3), 20.82
(2ϫPhCH3), 22.42 (CH2), 24.63 (CH2), 28.80–29.80 (12ϫCH2),
31.62 (CH2), 33.67 (CH2CO), 62.16 (C1), 69.75 (C3), 70.30 (C4Ј,
C6Ј), 73.99 (C2Ј), 74.33 (C2), 74.54 (C5Ј), 77.37 (C3Ј), 104.26 (C1Ј),
127.83 (2 C, Ph), 128.03 (2 C, Ph), 129.87 (4 C, Ph), 133.03 and
133.42 (2 C, Ph), 144.66 and 144.76 (2 C, Ph), 172.69 (CO) ppm.
ESI-MS (CH3OH, positive-ion mode, relative intensity): m/z (%) =
851.2 [M + Na]+ (100). C41H64O13S2 (829.07): calcd. C 59.40, H
7.78, S 7.74; found C 59.51, H 7.85, S 7.68.
C
27H51KO11S (622.85): calcd. C 52.07, H 8.25, S 5.15; found C
52.24, H 8.19, S 5.21.
1-O-Dodecanoyl-2-O-(β-D-sulfoquinvopyranosyl)-sn-glycerol Potas-
sium Salt (1b): Reaction time 7 h; yield 35%; m.p. 180 °C (amorph-
ous solid). [α]2D0 = –7.0 (CHCl3/CH3OH/H2O, 65:25:4). 1H NMR
(CDCl3/CD3OD/D2O, 65:25:4, 320 K): δ = 0.84 (t, J = 7.1 Hz, 3
H, CH3), 1.18–1.31 (m, 16 H, 8ϫCH2), 1.56 (m, 2 H, CH2), 2.29
(m, 2 H, CH2), 3.00 (dd, J6Јa,5Ј = 8.6, J6Јa,6Јb = 14.6 Hz, 1 H, 6Јa-
H), 3.21 (dd, J3Ј,4Ј = 8.9, J4Ј,5Ј = 8.9 Hz, 1 H, 4Ј-H), 3.23 (dd, J1Ј,2Ј
= 7.9, J2Ј,3Ј = 9.1 Hz, 1 H, 2Ј-H), 3.32 (dd, J6Јb,5Ј = 2.3 Hz, 1 H,
6Јb-H), 3.39 (dd, 1 H, 3Ј-H), 3.62 (dd, J2,3a = 6.0, J3a,3b = 12.1 Hz,
1 H, 3a-H), 3.68 (dd, J2,3b = 3.9 Hz, 1 H, 3b-H), 3.72 (ddd, 1 H,
5Ј-H), 3.94 (m, 1 H, 2-H), 4.15 (dd, J2,1a = 5.4, J1a,1b = 11.8 Hz, 1
H, 1a-H), 4.18 (dd, J2,1b = 5.1 Hz, 1 H, 1b-H), 4.41 (d, 1 H, 1Ј-
H) ppm. 13C NMR (CDCl3/CD3OD/D2O 65:25:4, 320 K): δ =
14.19 (CH3), 22.96 (CH2), 25.20 (CH2), 29.52 (CH2), 29.63 (2CH2),
29.83 (CH2), 29.94 (2ϫCH2), 32.24 (CH2), 34.47 (CH2CO), 53.11
(C6Ј), 62.28 (C3), 63.89 (C1), 72.99 (C5Ј), 73.32 (C4Ј), 73.92 (C2Ј),
76.45 (C3Ј), 80.02 (C2), 103.43 (C1Ј), 175.14 (CO) ppm. ESI-MS
(CH3OH, negative-ion mode, relative intensity): m/z (%) = 499.2
[M]– (100). Calcd for C21H39O11S–, m/z 499.2 [M]. C21H39KO11S
(538.69): calcd. C 46.82, H 7.30, S 5.95; found C 46.98, H 7.41, S
5.84.
1-O-Dodecanoyl-3-O-tosyl-2-O-(6-O-tosyl-β-D-glucopyranosyl)-sn-
glycerol (8b): M.p. 196 °C (from Et2O). [α]2D0 = –1.8 (CHCl3). 1H
NMR ([D5]pyridine, 312 K): δ = 0.86 (t, J = 7.1 Hz, 3 H, CH3),
1.18–1.30 (m, 16 H, 8ϫCH2), 1.61 (m, 2 H, CH2), 2.22 (s, 3 H,
CH3), 2.23 (s, 3 H, CH3), 2.30 (m, 2 H, CH2), 3.71 (dd, J1Ј,2Ј = 7.8,
J2Ј,3Ј = 8.9 Hz, 1 H, 2Ј-H), 3.86 (dd, J3Ј,4Ј = 9.0, J4Ј,5Ј = 9.9 Hz, 1
H, 4Ј-H), 3.95 (ddd, J5Ј,6Јa = 6.1, J5Ј,6Јb = 1.8 Hz, 1 H, 5Ј-H), 4.04
(dd, 1 H, 3Ј-H), 4.41–4.46 (m, 2 H, 2-H, 1a-H), 4.52–4.62 (m, 3 H,
4a-H, 3b-H, 1b-H), 4.62 (dd, J6Јa,6Јb = 10.5, J6Јa,5Ј = 6.1 Hz, 1 H,
6Јa-H), 4.86 (d, 1 H, 1Ј-H), 4.89 (dd, 1 H, 6Јb-H), 7.29 (m, 4 H,
Ph), 8.01 (d, J = 8.1 Hz, 2 H, Ph), 8.02 (d, J = 8.1 Hz, 2 H,
Ph) ppm. 13C NMR ([D5]pyridine, 312 K): δ = 13.75 (CH3), 20.81
(2 ϫ PhCH3), 22.42 (CH2), 24.63 (CH2), 28.90 (CH2), 29.07
(2 ϫ CH2), 29.25 (CH2), 29.36 (2 ϫ CH2), 31.61 (CH2), 33.67
(CH2CO), 62.16 (C1), 69.76 (C3), 70.30 (C4Ј, C6Ј), 73.99 (C2Ј),
74.33 (C2), 74.54 (C5Ј), 77.37 (C3Ј), 104.26 (C1Ј), 127.83 (2 C, Ph),
128.04 (2 C, Ph), 129.88 (4 C, Ph), 133.04 and 133.39 (2 C, Ph),
144.66 and 144.77 (2 C, Ph), 172.69 (CO) ppm. ESI-MS (CH3OH,
positive-ion mode, relative intensity): m/z (%) = 767.2 (100) [M +
Na]+. C35H52O13S2 (744.91): calcd. C 56.43, H 7.04, S 8.61; found
C 56.27, H 7.12, S 8.50.
1-O-Hexanoyl-2-O-(β-D-sulfoquinvopyranosyl)-sn-glycerol Potas-
sium Salt (1c): Reaction time 7 h; yield 45%; m.p. 120 °C (amorph-
ous solid). [α]2D0 = –6.9 (CHCl3/CH3OH/H2O 65:25:4). 1H NMR
(CDCl3/CD3OD/D2O 65:25:4, 320 K): δ = 0.85 (t, J = 7.0 Hz, 3 H,
CH3), 1.21–1.33 (m, 4 H, 2ϫCH2), 1.57 (m, 2 H, CH2), 2.30 (m,
2 H, CH2), 3.00 (dd, J6Јa,5Ј = 8.7, J6Јa,6Јb = 14.6 Hz, 1 H, 6Јa-H),
3.22 (dd, J3Ј,4Ј = 9.0, J4Ј,5Ј = 9.0 Hz, 1 H, 4Ј-H), 3.23 (dd, J1Ј,2Ј
=
7.8, J2Ј,3Ј = 9.1 Hz, 1 H, 2Ј-H), 3.32 (dd, J6Јb,5Ј = 2.3 Hz, 1 H, 6Јb-
H), 3.40 (dd, 1 H, 3Ј-H), 3.63 (dd, J2,3a = 6.0, J3a,3b = 12.1 Hz, 1
H, 3a-H), 3.68 (dd, J2,3b = 3.7 Hz, 1 H, 3b-H), 3.72 (ddd, 1 H, 5Ј-
H), 3.94 (m, 1 H, 2-H), 4.15 (dd, J2,1a = 5.4, J1a,1b = 11.7 Hz, 1 H,
1a-H), 4.19 (dd, J2,1b = 5.1 Hz, 1 H, 1b-H), 4.41 (d, 1 H, 1Ј-
H) ppm. 13C NMR (CDCl3/CD3OD/D2O 65:25:4, 320 K): δ = 1-O-Hexanoyl-3-O-tosyl-2-O-(6-O-tosyl-β-
D-glucopyranosyl)-sn-
13.92 (CH3), 22.54 (CH2), 24.81 (CH2), 31.59 (CH2), 34.41 glycerol (8c): M.p. 180 °C (from Et2O). [α]2D0 = –1.2 (CHCl3). 1H
(CH2CO), 52.99 (C6Ј), 62.20 (C3), 63.87 (C1), 73.04 (C5Ј), 73.25 NMR ([D5]pyridine, 312 K): δ = 0.78 (t, J = 7.1 Hz, 3 H, CH3),
(C4Ј), 73.87 (C2Ј), 76.42 (C3Ј), 80.05 (C2), 103.45 (C1Ј), 175.14 1.14–1.21 (m, 4 H, 2ϫCH2), 1.55 (m, 2 H, CH2), 2.22 (s, 6 H,
(CO) ppm. ESI-MS (CH3OH, negative-ion mode, relative inten-
2ϫCH3), 2.25 (m, 2 H, CH2), 3.71 (dd, J1Ј,2Ј = 7.8, J2Ј,3Ј = 8.9 Hz,
1 H, 2Ј-H), 3.85 (dd, J3Ј,4Ј = 8.9, J4Ј,5Ј = 9.9 Hz, 1 H, 4Ј-H), 3.95
sity): m/z (%) = 415.1 [M]– (100). Calcd for C15H27O11S–, m/z 415.1
6024
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Eur. J. Org. Chem. 2009, 6019–6026

2-O-(β--Sulfoquinovosyl)-monoacylglycerols
(ddd, J5Ј,6Јa = 6.0, J5Ј,6Јb = 1.6 Hz, 1 H, 5Ј-H), 4.04 (dd, 1 H, 3Ј-
H), 4.39–4.45 (m, 2 H, 2-H, 1a-H), 4.50–4.61 (m, 3 H, 3a-H, 3b-
1.63 (m, 2 H, CH2), 2.21 (s, 3 H, CH3), 2.23 (s, 3 H, CH3), 2.32
(m, 2 H, CH2), 3.84 (dd, J1Ј,2Ј = 7.8, J2Ј,3Ј = 8.6 Hz, 1 H, 2Ј-H),
H, 1b-H), 4.62 (dd, J6Јa,6Јb = 10.5, J6Јa,5Ј = 6.2 Hz, 1 H, 6Јa-H), 3.90 (dd, J3Ј,4Ј = 9.0, J4Ј,5Ј = 9.6 Hz, 1 H, 4Ј-H), 3.93 (ddd, J5Ј,6Јa
4.86 (d, 1 H, 1Ј-H), 4.89 (dd, 1 H, 6Јb-H), 7.28 (d, J = 8.1 Hz, 4 = 5.6, J5Ј,6ЈbϽ1.0 Hz, 1 H, 5Ј-H), 4.04 (dd, 1 H, 3Ј-H), 4.46 (m, 1
H, Ph), 8.00 (d, J = 8.1 Hz, 2 H, Ph), 8.02 (d, J = 8.1 Hz, 2 H,
Ph) ppm. 13C NMR ([D5]pyridine, 312 K): δ = 13.46 (CH3), 20.80
(2ϫPhCH3), 21.97 (CH2), 24.20 (CH2), 30.90 (CH2), 33.56
H, 3a-H), 4.50 (m, 1 H, 2-H), 4.52 (m, 1 H, 1a-H), 4.57 (m, 1 H,
1b-H), 4.59 (m, 1 H, 3b-H), 4.65 (dd, J6Јa,6Јb = 10.5 Hz, 1 H, 6Јa-
H), 4.87 (d, 1 H, 1Ј-H), 4.89 (dd, 1 H, 6Јb-H), 7.25 (d, J = 8.0 Hz,
(CH2CO), 62.14 (C1), 69.73 (C3), 70.24 (C4Ј or C6Ј), 70.28 (C4Ј 2 H, Ph), 7.28 (d, J = 8.0 Hz, 2 H, Ph), 8.01 (m, 4 H, Ph) ppm.
or C6Ј), 73.95 (C2Ј), 74.29 (C2), 74.50 (C5Ј), 77.33 (C3Ј), 104.22 13C NMR ([D5]pyridine, 312 K): δ = 13.76 (CH3), 20.82
(C1Ј), 127.80 (2 C, Ph), 128.00 (2 C, Ph), 129.86 (4 C, Ph), 132.98
and 133.36 (2 C, Ph), 144.66 and 144.76 (2 C, Ph), 172.63
(CO) ppm. ESI-MS (CH3OH, positive-ion mode, relative intensity):
m/z (%) = 683.1 (100) [M + Na]+. C29H40O13S2 (660.2): calcd. C
52.71, H 6.10, S 9.71; found C 52.77, H 6.19, S 9.63.
(2ϫPhCH3), 22.43 (CH2), 24.69 (CH2), 28.86–29.70 (12ϫCH2),
31.63 (CH2), 33.68 (CH2CO), 63.08 (C3), 68.75 (C1), 70.33 (C4Ј
and C6Ј), 74.10 (C2Ј), 74.33 (C2), 74.58 (C5Ј), 77.49 (C3Ј), 103.97
(C1Ј), 127.83 (2 C, Ph), 127.93 (2 C, Ph), 129.86 (4 C, Ph), 133.01
(2 C, Ph), 133.40 (2 C, Ph), 144.60 and 144.76 (2 C, Ph), 172.65
(CO) ppm. ESI-MS (CH3OH, positive-ion mode, relative intensity):
m/z (%) = 851.4 (100) [M + Na]+. C41H64O13S2 (829.07): calcd. C
59.40, H 7.78, S 7.74; found C 59.28, H 7.89, S 7.61.
General Procedure for the Synthesis of 2R Ditosylates 8a–c from
6a–c: Zinc bromide (0.126 g, 0.560 mmol) was added at –20 °C to
a solution of a compound 6 (0.140 mmol) in dry pyridine (3.0 mL).
After addition of tosyl chloride (0.133 g, 0.700 mmol) the reaction
mixture was stirred at –20 °C for 1 h with monitoring by TLC
(CH2Cl2/CH3OH, 95:5 v/v). After conventional workup, com-
pounds identical (NMR, MS, m.p.) to the previously obtained 8a–
c were obtained in about 70% yields.
Short-Term In-Vitro Bioassay for Antitumor Promoters: Inhibition
was tested by a short-term in-vitro assay for EBV activation in Raji
cells (originally obtained from Prof. G. Klein, Karolinska Institute,
Stockholm, Sweden) cultivated in RPMI 1640 medium containing
fetal calf serum (10%) and induced with TPA as described pre-
viously.[6,14] Raji cells (1ϫ106 mL–1) were incubated at 37 °C for
48 h in a medium (1 mL) containing n-butyric acid (4 m), TPA in
DMSO (32 pmol), and a known amount of the test compound in
DMSO. The cells were stained with high-titer EBV-positive sera
from nasopharyngeal carcinoma patients and fluorescein-isothio-
cyanate-labeled antihuman IgG. After staining, they were detected
by a conventional indirect immunofluorescence technique. The as-
says were performed in triplicate for each compound, with at least
500 cells being counted. The average EBV-EA inhibitory activities
of the test compounds were determined by comparison with con-
trol experiments (100%) with butyric acid (4 m) and TPA (32 p),
in which EBV-EA induction was typically around 30%. The viabil-
ity of the cells was assayed against treated cells by the Trypan Blue
staining method. For an accurate determination of cytotoxicity, the
cell viability was required to be more than 60% 2 d after treatment
with the compounds.
Chemoenzymatic Synthesis of the (2S)-Ditosylate 10a: The tosyl de-
rivative 4 (0.080 g, 0.196 mmol) was dissolved in pyridine (1.0 mL),
and trifluoroethyl octadecanoate (0.360 g, 0.982 mmol) and LCA
(0.250 g) were added in that order. The suspension was stirred at
45 °C and monitored by TLC (CH2Cl2/CH3OH, 90:10 v/v). The
reaction was stopped after 28 h by filtering off of the enzyme,
which was washed with pyridine. After removal of the solvent un-
der vacuum, the crude product was subjected to flash column
chromatography (CH2Cl2/CH3OH, 95:5 v/v), yielding the 1-O-
monoester 5a (0.059 g, 0.087 mmol) and 3-O-octadecanoyl-2-O-(6-
O-tosyl-β--glucopyranosyl)-sn-glycerol (9a, 0.066 g, 0.098 mmol)
in that order; m.p. 98 °C (amorphous solid). [α]2D0 = –10.9 (CHCl3).
1H NMR ([D5]pyridine, 312 K): δ = 0.86 (t, J = 7.0 Hz, 3 H, CH3),
1.19–1.38 (m, 28 H, 14ϫCH2), 1.68 (m, 2 H, CH2), 2.22 (s, 3 H,
CH3), 2.42 (m, 2 H, CH2), 3.91 (dd, J1Ј,2Ј = 7.8, J2Ј,3Ј = 9.0 Hz, 1
H, 2Ј-H), 3.93 (dd, J3Ј,4Ј = 9.0, J4Ј,5Ј = 9.8 Hz, 1 H, 4Ј-H), 3.99
(ddd, J5Ј,6Јa = 5.9, J5Ј,6Јb = 1.5 Hz, 1 H, 5Ј-H), 4.05–4.13 (m, 3 H,
1a-H, 1b-H, 3Ј-H), 4.41 (m, 1 H, 2-H), 4.60 (dd, J3a,2 = 4.9, J3a,3b
= 11.4 Hz, 1 H, 3a-H), 4.68 (dd, J3b,2 = 5.4 Hz, 1 H, 3b-H), 4.70
(dd, J6Јa,6Јb = 10.4 Hz, 1 H, 6Јa-H), 4.93 (dd, 1 H, 6Јb-H), 5.04 (d,
1 H, 1Ј-H), 7.27 (d, J = 8.1 Hz, 2 H, Ph), 8.02 (d, 2 H, Ph) ppm.
13C NMR ([D5]pyridine, 312 K): δ = 13.76 (CH3), 20.82 (PhCH3),
22.44 (CH2), 24.82 (CH2), 28.90–29.69 (12ϫCH2), 31.64 (CH2),
33.91 (CH2CO), 61.56 (C1), 64.06 (C3), 70.46 (C4Ј and C6Ј), 74.62
Supporting Information (see also the footnote on the first page of
this article): 13C NMR spectra of target compounds 1a–c.
Acknowledgments
This study was supported in part by the University of Milan (Italy)
(C2Ј and C5Ј), 77.64 (C3Ј), 78.95 (C2), 104.23 (C1Ј), 127.86 and and in part by Grants-in-Aid from the Ministry of Education, Sci-
129.82 (4 C, Ph), 133.50 and 145.54 (2 C, Ph), 173.09 (CO) ppm.
ESI-MS (CH3OH, positive-ion mode, relative intensity): m/z (%) =
697.3 (100) [M + Na]+. C34H58O11S (674.88): calcd. C 60.51, H
8.66, S 4.75; found C 60.73, H 8.59, S 4.81.
ence and Culture (Japan) and the Ministry of Health and Welfare
(Japan). The authors thank Professor Lucio Toma for helpful dis-
cussions.
Zinc bromide (0.067 g, 0.297 mmol) was added at –20 °C to a solu-
tion of compound 9a (0.050 g, 0.074 mmol) in dry pyridine
(3.0 mL). After addition of tosyl chloride (0.070 g, 0.367 mmol) the
reaction mixture was stirred at –20 °C for 3 h and monitored by
TLC (CH2Cl2/CH3OH, 95:5 v/v). The reaction was stopped and
worked up as described previously, yielding the ditosyl derivative
3-O-octadecanoyl-1-O-tosyl-2-O-(6-O-tosyl-β--glucopyranosyl)-
sn-glycerol (10a, 0.050 g, 0.060 mmol) after flash column
chromatography (CH2Cl2/CH3OH, 95:5 v/v); m.p. 190 °C (amorph-
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Eur. J. Org. Chem. 2009, 6019–6026
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Received: August 18, 2009
Published Online: October 8, 2009
6026
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 6019–6026