Chemoenzymatic synthesis and antitumor promoting activity of 6'- and 3-esters of 2-O-β-D-glucosylglycerol

Article abstract of DOI:10.1016/S0968-0896(99)00137-6

Through a simple chemoenzymatic approach 6'- and 3-esters of 2-O-β-D-glucosylglycerol 1, with short-medium length fatty acid acyl chains, were prepared. The study of their in vitro antitumor promoting effect on Epstein-Barr virus early antigen (EBV-EA) ac

Full text of DOI:10.1016/S0968-0896(99)00137-6

Bioorganic & Medicinal Chemistry 7 (1999) 1867±1871
Chemoenzymatic Synthesis and Antitumor Promoting Activity
of 60- and 3-Esters of 2-O-ꢀ-D-Glucosylglycerol
Diego Colombo, a,* Federica Compostella, a Fiamma Ronchetti, a Antonio Scala, a
Lucio Toma, b Harukuni Tokuda c and Hoyoku Nishino c
a
Á
Dipartimento di Chimica e Biochimica Medica, Universita di Milano, Via Saldini 50, 20133 Milano, Italy
b
Á
Dipartimento di Chimica Organica, Universita di Pavia, Via Taramelli 10, 27100 Pavia, Italy
cDepartment of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-084, Japan
Received 18 December 1998; accepted 6 March 1999
AbstractÐThrough a simple chemoenzymatic approach 60- and 3-esters of 2-O-b-d-glucosylglycerol 1, with short-medium length
fatty acid acyl chains, were prepared. The study of their in vitro antitumor promoting e€ect on Epstein±Barr virus early antigen
(EBV-EA) activation, in comparison with that of the 1-esters previously described, con®rms the signi®cant activity of such mono-
acylated glycoglycerolipid analogues and establishes for the glucose series that the 1-substitution and the hexanoyl chain are the
proper structural features for the maximum activity. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
synthesis of short-medium length (from C4 to C10) 60-
and 3-O-acyl-2-O-b-d-glucosylglycerols 3a±d and 4a±d
and on the dependence of their antitumor promoting
potential on the acylation site and on the chain length.
Recently tumor inhibitory activity of glycosylglycerols
and glycoglycerolipids has been demonstrated on the
basis of their in vitro and in vivo antitumor promoting
e€ect on Epstein±Barr virus early antigen (EBV-EA)
activation induced by the tumor promoter 12-O-tetra-
decanoylphorbol-13-acetate (TPA).1±3 Our e€orts in
this ®eld have been directed toward the synthesis of
di€erent gluco-4,5 and galactosylglycerols6,7 in order to
study the relationship between the molecular structure
of glycoglycerolipids and their activity. In particular, we
could easily prepare5 highly enriched 1-O-acyl deriva-
tives of 2-O-b-d-glucosylglycerol (1), such as 2, con-
taining small amounts of their 60-O- and 3-O-acyl
isomers, through a direct lipase catalyzed monoacyla-
tion of the substrate mediated by Pseudomonas cepacia
lipase (LPS). These compounds were then assayed as
cancer chemopreventive agents and resulted highly
active especially for short-medium length fatty acid acyl
chains.3
Results and Discussion
Chemoenzymatic synthesis of esters 3 and 4
However, the biological activity might be related not to
the 1-O-acyl derivatives 2, but to the small amounts of
the isomers 3 and 4. Therefore, we report here on the
1,3-Di-O-benzyl-2-O-b-d-glucopyranosyl-sn-glycerol (5)
was chosen as the key compound for the preparation of
both the 60- and 3-O-acylglucosylglycerols 3 and 4.
Key words: Glucosylglycerols; lipases; antitumor promoting activity;
Epstein±Barr virus early antigen.
* Corresponding author. Tel.: +39-02-7064-5230; fax: +39-02-236-
1407; e-mail: diego.colombo@unimi.it
To get the 60-esters 3 (Scheme 1), compound 5, obtained
from the known8,9 1,3-di-O-benzyl-2-O-(2,3,4,6-tetra-
O-acetyl-b-d-glucopyranosyl)-sn-glycerol, was directly
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00137-6

1868
D. Colombo et al. / Bioorg. Med. Chem. 7 (1999) 1867±1871
enzymatic reaction a€orded the 3-O-acyl-2-O-(2,3,4,6-
tetra-O-chloroacetyl-b-d-glucopyranosyl)-sn-glycerols (9)
in good diastereoselectivity and yields; the desired 3-O-
acyl-2-O-b-d-glucosylglycerols (4) were ®nally obtained
pure by mild removing of the MCA groups of 9 by hy-
drazine±acetate treatment12 followed by crystallization.
The con®guration at C-2 of compounds 4 was deter-
mined by chemical correlation to commercial 1,2- and
2,3-O-isopropylidene-sn-glycerol. Compounds 4 were
hydrolyzed with b-glucosidase from almonds; the
monoacyglycerols present in the reaction mixtures were
transformed into the isopropylidene derivatives and
analyzed by chiral GLC in comparison with authentic
samples as described in ref 7 for the corresponding 3-
galactosylesters. In this way the con®guration of 3-O-
acyl-2-O-b-d-glucopyranosyl-sn-glycerols was assigned
to the monoesters 4.
Scheme 1. Reagents: (i) MeOH, MeONa; (ii) LPS, CF3CH2OCO
(CH2)nCH3, THF; (iii) H2, Pd/C.
submitted to transesteri®cation catalyzed by LPS in the
presence of the appropriate tri¯uoroethylester in tetra-
hydrofuran as the solvent. The reactions were com-
pletely regioselective and a€orded, through the expected
acylation of the primary hydroxyl group,10 the 1,3-di-O-
benzyl-2-O-(6-O-acyl-b-d-glucopyranosyl)-sn-glycerols 6
in good yields. Catalytic hydrogenolysis of 6 with palla-
dium on activated carbon in methanol ®nally yielded
almost quantitatively pure 2-O-(6-O-acyl-b-d-glucopyr-
anosyl)-sn-glycerols 3.
Inhibitory e€ects of 3 and 4 on EBV-EA
Compounds 3a±d and 4a±d were assayed for their
antitumor promoting activities using a short-term in
vitro assay for Epstein±Barr virus early antigen acti-
vation in Raji cells induced by 12-O-tetradecanoyl-
phorbol-13-acetate (TPA).2 Their inhibitory e€ects on
activation of the early antigen (% to control) and the
viabilities of Raji cells (% viability) are shown in Table
1, in comparison with those of 1-esters 2a±d, previously
described by us.3
In order to obtain the 3-esters 4, the hydroxyl groups of
the glucosyl moiety of 5 were temporary protected as
chloroacetates (MCA); thus, after removal of the benzyl
groups, the glycerol hydroxymethyls should be available
for the selective acylation catalyzed by lipase from
Candida antarctica (LCA) which is known to occur on
such substrates with opposite diastereoselectivity with
respect to LPS.4
As reported in Table 1 all the tested compounds showed
signi®cant and comparable activity even if the already
studied 1-esters 23 resulted the most active. In particular
the antitumor promoting activity followed the order: 1-
esters >60-esters >3-esters for all the concentrations
and all the acyl chain length employed, and for each
isomeric series the hexanoyl derivatives 2b, 3b and 4b
resulted the most potent compounds, strengthening
previous ®ndings.3 These results establish that the
activity previously ascertained for compounds 2 is
actually due to them and not to the small amounts of
compounds 3 and 4 they contain as byproducts.
So 5 was converted (Scheme 2) into 1,3-di-O-benzyl-2-
O-(2,3,4,6-tetra-O-chloroacetyl-b-d-glucopyranosyl)-sn-
glycerol (7) by chloroacetic anhydride treatment in a
dichloromethane±pyridine mixture at 0ꢀC.11 Catalytic
hydrogenolysis of 7 yielded 2-O-(2,3,4,6-tetra-O-chloro-
acetyl-b-d-glucopyranosyl)-sn-glycerol (8), which was
submitted to LCA transesteri®cation with the appro-
priate tri¯uoroethyl esters in tetrahydrofuran. The
In conclusion, this study has allowed to prepare other
series of interestingly active glucosylglycerols, once
more synthetic analogues more potent than the natural
compounds,3 and establishes the hexanoyl chain as the
proper length for the best activity in the series. The role
of the acyl chain, which appear not strictly related to a
positional requirement, needs further elucidation.
Experimental
General procedures
1H NMR spectra were recorded with a Bruker AM-500
spectrometer on CDCl3 solutions at 303 K, unless
otherwise stated; chemical shifts are reported as d (ppm)
relative to tetramethylsilane as internal standard. Mass
experiments were performed through chemical ionisa-
tion mass spectrometry (CI±MS) as described in ref 13.
Scheme 2. Reagents: (i) (ClCH2CO)2O, CH2Cl2/Py, 0ꢀC; (ii) H2, Pd/
C; (iii) LCA, CF3CH2OCO(CH2)nCH3, THF; (iv) NH2NH2.AcOH,
AcOEt/MeOH; MCA=COCH2Cl.

D. Colombo et al. / Bioorg. Med. Chem. 7 (1999) 1867±1871
1869
Table 1. Percentages of EBV-EA induction in the presence of 2, 3 and 4 with respect to positive control (100%)
Compound
Relative concentration (mol ration/TPA)a
1000
500
100
10
% to positive control‹SE (n=3)
2a3
2b3
2c3
2d3
3a
3b
3c
3d
4a
0.0b‹0.1 (60)c
0.0‹0 (70)
0.0‹0 (70)
0.0‹0 (60)
0.0‹0 (70)
0.0‹0 (70)
0.0‹0 (70)
0.0‹0 (70)
0.0‹0.5 (70)
0.0‹0 (70)
0.0‹0 (70)
0.0‹0.7 (70)
15.7‹0.4
10.6‹0.6
16.9‹0.7
18.4‹1.0
27.9‹0.8
19.3‹0.3
21.1‹0.4
27.3‹0.6
31.6‹0.5
25.1‹0.6
27.7‹0.4
36.2‹0.6
37.2‹1.5
30.9‹1.8
55.0‹1.9
56.5‹2.5
51.3‹1.3
37.6‹1.5
57.2‹1.4
62.8‹2.3
56.2‹1.4
42.3‹1.1
69.5‹1.3
76.3‹1.3
79.4‹2.0
68.2‹2.5
89.2‹2.2
88.2‹1.9
88.4‹1.0
78.1‹1.8
90.6‹1.1
92.1‹1.2
93.6‹0.2
82.7‹1.1
92.1‹0.3
95.8‹0.3
4b
4c
4d
a
TPA (32 pmol/mL).
Values represent relative percentages to the positive control value (at least 500 cells were counted).
Values in parentheses are viability percentages of Raji cells.
b
c
Melting points were recorded on a Buchi 510 capillary
melting point apparatus and were uncorrected. Optical
rotations were determined on a Perkin±Elmer 241
polarimeter in methanol solutions (c ˆ 1:0) in a 1 dm
cell at 20ꢀC unless otherwise stated. Analytical thin
layer chromatography (TLC) was carried out on Merck
60 F254 silica gel plates (0.25 mm thickness) and the
spots were detected by spraying with 50% aqueous
H2SO4 or with anisaldehyde based reagent and heating
at 110ꢀC. Flash chromatography was performed with
Merck 60 silica gel (230±400 mesh). P. cepacia lipase
(lipase PS, LPS, speci®c activity 30.5 triacetin units/mg
solid), a generous gift from Amano Pharmaceutical Co
(Mitsubishi Italia), was supported on Celite;4 C. ant-
arctica lipase SP 435 L, immobilized on a macroporous
acrylic resin, (Novozym1 435, LCA, speci®c activity 9.5
PL units/mg solid), was a generous gift from Novo
Nordisk A/S; b-glucosidase from almonds (speci®c
activity about 6 salicin units/mg solid) was purchased
from Fluka. LPS and LCA were kept under vacuum
overnight prior to use. The tri¯uoroethyl esters were
synthesized according to ref 5. Evaporation under
reduced pressure was always e€ected with the bath
temperature kept below 40ꢀC. Tetrahydrofuran was
distilled over sodium-benzophenone, and pyridine over
calcium hydride prior to use. The elemental analyses of
the new compounds were consistent with the theoretical
ones.
3.51±3.62 (m, 5H, H2-1, H2-3 and H-40), 3.71 (dd, 1H,
J6 a,6 b=12.0 Hz, H-60a), 3.74 (dd, 1H, H-60b), 4.0 (m,
1H, H-2), 4.43 (d, 1H, H-10), 4.44±4.50 (m, 4H, 2
CH2Ph), 7.20±7.35 (m, 10H, Ph).
0
0
General procedure for the chemoenzymatic synthesis of
monoesters 3
1,3-Di-O-benzyl-2-O-(6-O-acyl-ꢀ-D-glucopyranosyl)-sn-
glycerols (6). 1,3-Di-O-benzyl-2-O-b-d-glucopyranosyl-
sn-glycerol (5) (0.50 g, 1.15 mmol) was dissolved in tet-
rahydrofuran (6 mL); the appropriate tri¯uoroethyl
ester (3.45 mmol) and LPS (2.50 g) were added in the
order and the suspension was stirred at 45ꢀC. The reac-
tion was monitored by TLC and stopped after 24 h by
®ltering o€ the enzyme which was washed with tetra-
hydrofuran. The solvent was removed under reduced
pressure and the crude residue was submitted to silica
gel ¯ash chromatography (ethyl acetate:petroleum
ether, 80:20, v/v). In this way 60-O-acylderivatives 6
1
were obtained. For the H NMR signals see Table 2.
1,3-Di-O-benzyl-2-O-(6-O-butanoyl-ꢀ-D-glucopyranosyl)-
sn-glycerol (6a). (83% yield): oil, [a]d 14.3 (chloro-
form).
1,3-Di-O-benzyl-2-O-(6-O-hexanoyl-ꢀ-D-glucopyranosyl)-
sn-glycerol (6b). (80% yield): oil, [a]d 14.8 (chloro-
form).
1,3-Di-O-benzyl-2-O-ꢀ-D-glucopyranosyl-sn-glycerol (5).
50 mL of 0.7 M sodium metoxide in methanol were
added to a solution of the known8,9 1,3-di-O-benzyl-2-
O-(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)-sn-glycerol
(12 g, 20 mmol) in methanol (18 mL). After 2 h at room
temperature the reaction mixture was neutralized with
Dowex 50Â8 H+ and ®ltered. The solvent was removed
under vacuum and ¯ash chromatography (ethyl acet-
ate:methanol, 90:10, v/v) of the crude product a€orded
pure 5 (87% yield): oil; [a]d 5.0 (chloroform); 1H
1,3-Di-O-benzyl-2-O-(6-O-octanoyl-ꢀ-D-glucopyranosyl)-
sn-glycerol (6c). (81% yield): oil, [a]d 14.4 (chloro-
form).
1,3-Di-O-benzyl-2-O-(6-O-decanoyl-ꢀ-D-glucopyranosyl)-
sn-glycerol (6d). (80% yield): oil, [a]d 13.8 (chloro-
form).
2-O-(6-O-Acyl-ꢀ-D-glucopyranosyl)-sn-glycerols (3). Com-
pound 6 (0.40 mmol) was dissolved in methanol (18 mL)
and 10% palladium on activated carbon (0.12 g)
was added. This mixture was shaken under hydrogen
0
0
0
0
NMR: d 3.21 (ddd, 1H, J5 ,4 =9.0 Hz, J5 ,6 a=3.0 Hz,
J5 ,6 b=4.0 Hz, H-50), 3.40 (dd, 1H, J2 ,1 =8.0 Hz,
0
0
0
0
0
0
0 0
J2 ,3 =9.0 Hz, H-2 ), 3.49 (dd, 1H, J3 , 4 =9.0 Hz, H-3 ),
0
0

1870
D. Colombo et al. / Bioorg. Med. Chem. 7 (1999) 1867±1871
1
Table 2. Signi®cant H NMR signals of compounds 3a, 4a, 6 and 9
Chemical shifts, ꢀb
H-10
H-60a
H-60b
H-2
H-1a
H-1b
H-3a
H-3b
3a
3b
3c
3d
5.11
5.12
5.12
5.13
5.13
5.15
5.15
5.15
4.44
4.44
4.44
4.45
4.78
4.78
4.78
4.78
4.70
4.71
4.73
4.73
4.36
4.36
4.36
4.37
4.25
4.24
4.24
4.24
4.29
4.29
4.29
4.29
4.96
4.98
4.98
4.99
4.50
4.51
4.52
4.51
4.42
4.42
4.42
4.43
4.35
4.35
4.34
4.34
4.39
4.38
4.38
4.38
4.46
4.48
4.50
4.50
4.05
4.05
4.05
4.05
3.90
3.90
3.90
3.89
4.11
4.15
4.15
Ð
Ð
Ð
Ð
4.26
4.24
4.25
4.25
4.14
4a
4.07±4.15
4.08±4.16
4.09±4.17
4.09±4.17
3.50
3.50
3.50
3.49
4.60
4.62
4.63
4.64
4.66
4.69
4.70
4.70
4bc
4cd
4d
6a
6b
6c
Ð
Ð
Ð
Ð
3.64
3.66
3.65
3.65
6d
9ae
9be
9ce
9de
3.62
3.62
3.61
3.61
3.68
3.67
3.67
3.67
4.09
4.08
4.08
4.08
4.27
4.28
4.27
4.27
a
Pyridine-d5 solutions.
When measurable, the coupling constants in Hz results: J1 ,2 =8.0, J6 a,6 b=12.0; 3: J6 b,5 =0.5, J6 a,5 =5.5; 4: J3a,3b=12.0, J6 b,5 =2.0,
b
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
J6 a,5 =5.0, J3b,2=5.5, J3a,2=5.5; 6: J6 b,5 =4.0, J6 a,5 =2.0; 9: J1a,1b=12.0, J3a,3b=12.0, J6 b,5 =5.0, J6 a,5 =2.0, J1b,2=3.5,
J1a,2=6.0, J3b,2=5.0, J3a,2=6.0.
c
Signi®cant 1H NMR signals for the 1-O-isomer 2b: 4.66 (dd, 1H, J1a,1b=12.0 Hz, J1a,2=4.5 Hz, H-1a), 4.69 (dd, 1H, J1b,2=5.5 Hz, H-1b), 5.10
0
0
0
(d, 1H, J1 ,2 =8.0 Hz, H-1 ).
d
Signi®cant 1H NMR signals for the 1-O-isomer 2c: 4.67 (dd, 1H, J1a,1b=12.0 Hz, J1a,2=4.5 Hz, H-1a), 4.70 (dd, 1H, J1b,2=5.5 Hz, H-1b), 5.10
0
0
0
(d, 1H, J1 ,2 =8.0 Hz, H-1 ).
e
0
0
Signi®cant 1H0 NMR signals for the 1-O-isomers 09; a: 4.71 (d, 1H, J1 ,2 =8.0 Hz, H-1 ), b: 4.71 (d, 1H, J1 ,2 =8.0 Hz, H-1 ), c: 4.71 (d, 1H,
0
0
0
0
0
J1 ,2 =8.0 Hz, H-1 ), d: 4.71 (d, 1H J1 ,2 =8.0 Hz, H-1 ).
0
0
0
atmosphere for 1 h and then ®ltered through a Celite
bed a€ording quantitatively the debenzylated com-
pounds 3. For the H NMR signals see Table 2.
yielded pure 1,3-di-O-benzyl-2-O-(2,3,4,6-tetra-O-chloro-
acetyl-b-d-glucopyranosyl)-sn-glycerol (7), (90% yield):
1
1
oil; [a]d +0.6 (chloroform); H NMR: d 3.46±3.65 (m,
0
0
4H, H2-1 and H2-3), 3.70 (ddd, 1H, J5 ,4 =10.0,
0
J5 ,6 a=3.0, J5 ,6 b=4.0 Hz, H-50), 3.82, 3.96 and 4.04
(3s, 6H, 3 CH2Cl), 3.97±4.00 (m, 2H, CH2Cl), 4.02 (m,
0
0
0
2-O-(6-O-Butanoyl-ꢀ-D-glucopyranosyl)-sn-glycerol (3a).
(98% yield), mp 76±78ꢀC (ethanol), [a]d 4.0, MS m/z
342 [M+NH4]+.
1H, H-2), 4.23 (dd, 1H, J6 a,6 b=12.0 Hz, H-60a), 4.27
(dd, 1H, H-60b), 4.46±4.52 (m, 4H, 2 CH2Ph), 4.84 (d,
0
0
0
0
0
0
0
2-O-(6-O-Hexanoyl-ꢀ-D-glucopyranosyl)-sn-glycerol (3b).
(97% yield), mp 103±105ꢀC (ethanol), [a]d 8.0, MS
m/z 370 [M+NH4]+.
1H, J1 ,2 =8.0 Hz, H-1 ), 5.03 (dd, 1H, J2 ,3 =10.0 Hz,
0
H-20), 5.13 (dd, 1H, J4 ,3 =10.0 Hz, H-4 ), 5.29 (dd,
1H, H-30), 7.20±7.35 (m, 10H, Ph); MS m/z 758
[M+NH4]+.
0
0
2-O-(6-O-Octanoyl-ꢀ-D-glucopyranosyl)-sn-glycerol (3c).
(98% yield), mp 109±111ꢀC (ethanol), [a]d 6.0, MS
m/z 398 [M+NH4]+.
2-O-(2,3,4,6-Tetra-O-chloroacetyl-ꢀ-D-glucopyranosyl)-
sn-glycerol (8). Compound 7 (3.00 g, 4.05 mmol) was
submitted to hydrogenolysis as described above a€ord-
ing 2-O-(2,3,4,6-tetra-O-chloroacetyl-b-d-glucopyrano-
syl)-sn-glycerol (8), (88% yield): oil; [a]d +2.4
2-O-(6-O-Decanoyl-ꢀ-D-glucopyranosyl)-sn-glycerol (3d).
(97% yield), mp 113±114ꢀC (ethanol), [a]d 4.0, MS
m/z 426 [M+NH4]+.
1
(chloroform); H NMR: d 3.58±3.68 (m, 4H, H2-1 and
0
0
H2-3), 3.78 (m, 1H, H-2), 3.88 (ddd, 1H, J5 ,4 =10.0,
0
J5 ,6 a=6.0, J5 ,6 b=3.0 Hz, H-50), 3.97 and 4.12 (2s, 4H,
2 CH2Cl), 3.99±4.05 (m, 4H, 2 CH2Cl), 4.30 (dd, 1H,
0
0
0
General procedure for the chemoenzymatic synthesis of
monoesters 4
J6 a,6 b=12.0 Hz, H-60a), 4.37 (dd, 1H, J6 b), 4.77 (d,
0
0
0
0
0
0
0
0
1,3-Di-O-benzyl-2-O-(2,3,4,6-tetra-O-chloroacetyl-ꢀ-D-
glucopyranosyl)-sn-glycerol (7). Chloroacetic anhydride
(3.92 g, 22.92 mmol) was added at 0ꢀC to a solution of 5
(2.00 g, 4.61 mmol) in dichloromethane (40 mL) and
pyridine (5.4 mL). After 15 min the reaction mixture
was diluted with dichloromethane (160 mL) and washed
with 1 M HCl (80 mL), water (80 mL), 10% NaHCO3
(80 mL), and two volumes of water (80 mL). Organic
layer was dried over Na2SO4 and the solvent removed
under reduced pressure. Flash chromatography (ethyl
acetate:petroleum ether, 30:70, v/v) of the crude product
1H, J1 ,2 =8.0 Hz, H-1 ), 5.10 (dd, 1H,0 J2 ,3 =10.0 Hz,
H-20), 5.13 (dd, 1H, J4 ,3 =10.0 Hz, H-4 ), 5.36 (dd, 1H,
0
0
H-30).
3-O-Acyl-2-O-(2,3,4,6-tetra-O-chloroacetyl-ꢀ-D-gluco-
pyranosyl)-sn-glycerols (9). 2-O-(2,3,4,6-Tetra-O-chlor-
oacetyl-b-d-glucopyranosyl)-sn-glycerol (8) (0.50 g, 0.89
mmol) was dissolved in 10 mL of tetrahydrofuran; the
appropriate tri¯uoroethyl ester (2.67 mmol) and LCA
(1.50 g) were added in the order and the suspension was
stirred at 45ꢀC for 8 h. The reaction was treated as

D. Colombo et al. / Bioorg. Med. Chem. 7 (1999) 1867±1871
1871
reported for the LPS transesteri®cation procedure and
Short term in vitro bioassay for antitumor promoters
after ¯ash chromatography (ethyl acetate:petroleum
ether from 50:50 to 70:30, v/v) 3-O-acyl-2-O-(2,3,4,6-
tetra-O-chloroacetyl-b-d-glucopyranosyl)-sn-glycerols 9,
contaminated by about 10% of the corresponding 1-O-
The inhibitions were assayed using a short term in vitro
assay for Epstein±Barr virus activation in Raji cells
induced by 12-O-tetradecanoylphorbol-13-acetate (TPA)
as a primary screening test for antitumor promoters.2
For each compound assays were performed in triplicate.
No sample exhibited signi®cant toxicity against Raji
cells. The results are reported in Table 1.
1
diastereoisomers detected by H NMR, were obtained
1
in 60±65% yield. For the H NMR signals see Table 2.
3-O-Acyl-2-O-ꢀ-D-glucopyranosyl-sn-glycerols. (4). To a
solution of 9 (0.50 mmol) in a 1:1 ethyl acetate:
methanol mixture (9.5 mL) hydrazine acetate (0.69 g,
7.50 mmol) was added. After 6 h at room temperature
the solvent was removed under reduced pressure and the
obtained residue submitted to ¯ash chromatography
(dichloromethane:methanol, 80:20, v/v). Final crystal-
lization allowed to remove the 1-isomer yielding pure 4.
Acknowledgements
This study was supported in part by MURST
(Rome, Italy) and University of Milan (Italy) and in
part by Grants-in-Aid from the Ministry of Education,
Science and Culture, and Ministry of Health and Wel-
fare (Japan).
1
For the H NMR signals see Table 2.
3-O-Butanoyl-2-O-ꢀ-D-glucopyranosyl-sn-glycerol (4a).
(60% yield), mp 138±140ꢀC (ethanol-diisopropylether),
[a]d 8.6, MS m/z 342 [M+NH4]+.
References
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3-O-Hexanoyl-2-O-ꢀ-D-glucopyranosyl-sn-glycerol (4b).
(67% yield), mp 135±137ꢀC (ethanol), [a]d 8.4, MS
m/z 370 [M+NH4]+.
3-O-Octanoyl-2-O-ꢀ-D-glucopyranosyl-sn-glycerol (4c).
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Standard samples. Standard 3-O-acyl-1,2-O-isopropyl-
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water (0.5 mL) and 5 mg of b-glucosidase from almonds
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samples were analyzed on a chiral GLC-capillary col-
umn (dimethylpentyl-b-cyclodextrin, 25 m, ID 0.25 mm,
from MEGA-Italy) in the conditions described in ref 7.