New 6-amino-6-deoxy-glycoglycerolipids derived from 2-O-β-d- glucopyranosylglycerol: Insights into the structure-activity relationship of glycoglycerolipids as anti-tumor promoters

Article abstract of DOI:10.1016/j.carres.2013.03.007

As part of a project aimed at obtaining compounds capable of inhibiting tumor promotion, new 6-amino-6-deoxyglycoglycerolipids (AGGLs) derived from 2-O-β-d-glucopyranosyl-sn-glycerol were synthesized and tested for their anti-tumor-promoting activity using a short-term in vitro assay of the inhibition of Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). The corresponding 6-amino-6-deoxy-β-d-octylglucosides were also prepared as simplified aminoglycolipid models and tested. Comparison with the activity of a series of previously studied glycoglycerolipids showed that replacing the 6-oxygen of the glucose moiety by a nitrogen atom greatly reduced the in vitro activity of the compounds. A two-stage mouse skin carcinogenesis test of two representative aminoglycoglycerolipids confirmed their reduced activity also in this in vivo model.

Full text of DOI:10.1016/j.carres.2013.03.007

Accepted Manuscript
New 6-amino-6-deoxy-glycoglycerolipids derived from 2-O-β-D-glucopyrano‐
sylglycerol: insights into the structure-activity relationship of glycoglyceroli‐
pids as anti-tumor promoters
Diego Colombo, Clarissa Gagliardi, Maria Vetro, Fiamma Ronchetti, Midori
Takasaki, Takao Konoshima, Nobutaka Suzuki, Harukuni Tokuda
PII:
S0008-6215(13)00086-4
DOI:
http://dx.doi.org/10.1016/j.carres.2013.03.007
CAR 6418
Reference:
To appear in:
Carbohydrate Research
Received Date:
Revised Date:
Accepted Date:
18 January 2013
5 March 2013
8 March 2013
Please cite this article as: Colombo, D., Gagliardi, C., Vetro, M., Ronchetti, F., Takasaki, M., Konoshima, T., Suzuki,
N., Tokuda, H., New 6-amino-6-deoxy-glycoglycerolipids derived from 2-O-β-D-glucopyranosylglycerol: insights
into the structure-activity relationship of glycoglycerolipids as anti-tumor promoters, Carbohydrate Research
(2013), doi: http://dx.doi.org/10.1016/j.carres.2013.03.007
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New 6-amino-6-deoxy-glycoglycerolipids derived from 2-O--D-glucopyranosylglycerol:
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insights into the structure-activity relationship of glycoglycerolipids as anti-tumor promoters
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Diego Colomboa,*, Clarissa Gagliardia, Maria Vetroa, Fiamma Ronchettia, Midori Takasakib, Takao
Konoshimab, Nobutaka Suzukic, Harukuni Tokudac
aDepartment of Medical Biotechnology and Translational Medicine, University of Milano, Via
Saldini 50, 20133 Milano, Italy. bDepartment of Pharmacology, Chiba Institute of Science, Chiba,
JAPAN, cDepartment of Complementary and Alternative Medicine, Clinical R&D, Graduate School
of Medical Science, Kanazawa University 13-1 Takara-machi Kanazawa JAPAN 920-8640.
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1

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Abstract
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As part of a project aimed at obtaining compounds capable of inhibiting tumor promotion, new 6-
amino-6-deoxyglycoglycerolipids (AGGLs) derived from 2-O--D-glucopyranosyl-sn-glycerol
were synthesised and tested for their anti-tumor-promoting activity using a short-term in vitro assay
of the inhibition of Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-
tetradecanoylphorbol-13-acetate (TPA). The corresponding 6-amino-6-deoxy--D-octylglucosides
were also prepared as simplified aminoglycolipid models and tested. Comparison with the activity
of a series of previously studied glycoglycerolipids showed that replacing the 6-oxygen of the
glucose moiety by a nitrogen atom greatly reduced the in vitro activity of the compounds. A two-
stage mouse skin carcinogenesis test of two representative aminoglycoglycerolipids confirmed their
reduced activity also in this in vivo model.
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Key words: Aminoglycoglycerolipids; cancer chemoprevention; EBV-EA; AGGL
a
* Corresponding author. Department of Medical Biotechnology and Translational Medicine,
University of Milan, Via Saldini 50, 20133 Milano, Italy. Tel: +390250316039 Fax:
+390250316036 Email: diego.colombo@unimi.it
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1. Introduction
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Glycoglycerolipids, such as mono- and digalactosyldiacylglycerols (MGDG, Figure 1, and DGDG)
and the anionic sulfoquinovosylacylglycerols (SQAG, Figure 1), are widespread in nature
especially in plants and bacteria as they are the main lipid components of the thylakoid membranes
of chloroplasts and cyanobacteria, in which it is thought they play an important role in a variety of
cell functions.1 Their general structure consists of a carbohydrate moiety that is beta-(MGDG and
DGDG) or alpha-linked (SQAG) to the sn-3 position of glycerol, which is acylated at the residual
hydroxyls by fatty acids of different lengths and degrees of unsaturation.
Given the remarkable biological activities of some MGDGs2 and particularly their ability to inhibit
in vitro and in vivo tumor-promoting-activity3,4 induced by some tumor promoters,5 we have
previously prepared a series of analogues based on the easily accessible 2-O--D-glucosylglycerol
skeleton6 in order to study their structure and anti-tumor-promoting activity relationships, obtaining
in some cases more potent compounds than the natural ones.7 More recently, we have also prepared
2-O-β-D-sulfoquinovosylacylglycerol analogues of SQAG, and compared their activity with that of
the earlier compounds.8,9 Interestingly, these compounds were significantly less active suggesting
that the negative charge of the sulfonate group had a depressing effect on the activity.
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Our continuing interest in the development of novel anti-tumor promoters has now led us to study
the activity of nitrogen-containing glycoglycerolipids and, once again, nature inspired us for new
compounds as glycoglycerolipids bearing an amino-group at the C-6 of the sugar are reported,
namely 6-amino-6-deoxyglycoglycerolipids (AGGLs, Figure 1) although much rarer in nature than
MGDGs and SQDGs (to the best of our knowledge only a few recent papers have described their
isolation from algae10-12 or plants13,14). All the reported compounds share a common structure in
which 6-amino-6-deoxyglucose is alpha-linked to the sn-3 position of glycerol (the remaining
glycerol hydroxyls are esterified with saturated10-12,14 or unsaturated13 fatty acids, and the amino
group can be free10,12-14 or acylated),11,13 and they have anti-bacterial,13 anti-tumor,10,13 anti-stress,14
free-radical scavenging,12 and human Myt1 kinase inhibiting activities.11 Remarkably, the Myt1
kinase inhibitory effect of 1,2-di-O-palmitoyl-3-O-(N-palmitoyl-6-amino-6-deoxy--D–
glucopyranosyl)-sn-glycerol has independently prompted two different research groups to
synthesise it because of its potential use in cancer studies and therapy,15,16 and some analogues have
also been prepared.17
3

SO3H
OH
OH
R
O
O
1
2
3
O
HO
HO
R
O
O
O
HO
2
R'
OH
OH
3
1
O
O
4
5
2
R'
MGDG
SQAG
3
1
6
NHR''
7
8
9
O
R and R' = acyl chains
R'' = H or acyl chain
HO
HO
R
O
OH
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O
O
2
R'
AGGL
3
1
Figure 1 . Structures of natural monogalactosyldiacylglycerols (MGDG),
sulfoquinovosylacylglycerols (SQAG) and 6-amino-6-deoxyglycoglycerolipids (AGGL).
In this paper we describe the synthesis and anti-tumor-promoting activity of new 6-amino-6-
deoxyglycoglycerolipids derived from 2-O--D-glucopyranosyl-sn-glycerol carrying acyl chains of
different lengths (hexanoyl and octadecanoyl) on the glycerol and sugar moieties. We prepared 2-
O-(N-acyl-6-amino-6-deoxy--D-glucopyranosyl)-sn-glycerols 1a-b, the corresponding 1-O-esters
2a-b, octyl 6-amino-6-deoxy--D-glucopyranoside 3 and the related hexanoyl amide 4 (see Figure
2) as simplified aminoglycolipid models, and their anti-tumor-promoting activity was tested using a
short-term in vitro assay of the inhibition of Epstein-Barr virus early antigen (EBV-EA) activation
induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).7 The most active
glycoglycerolipids 1a and 2a were also tested using an in vivo two-stage mouse skin carcinogenesis
test, and the biological data were compared with those of the known compounds 5-10.9,18,19
R
NHR
O
1
3
O
OR'
OH
HO
HO
HO
HO
O
O(CH2)7CH3
2
OH
OH
1: R= NHCO(CH2) CH3; R'= H
3: R= H
4: R= CO(CH2)4CH3
n
2: R= NHCO(CH2) CH3; R'= CO(CH2) CH3
n
n
a: n= 4; b: n= 16
5: R= OCO(CH2)4CH3; R'= H
OH
OH
O
6: R= OCO(CH2)4CH3; R'= CO(CH2)4CH3
7: R= OH; R'= CO(CH2)4CH3
O(CH2)8CH3
HO
OH
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8: R= OH; R'= CO(CH2)16CH3
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9: R= SO3 ; R'= CO(CH2)4CH3
Figure 2. Structures of the studied compounds 1-4 and the
reference compounds 5-10.
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2. Results and discussion
2.1. Chemistry
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2.1.1. Synthesis of compounds 1a,b and 2a,b
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Compounds 1a,b and 2a,b were efficiently obtained by means of the synthetic pathway shown in
Scheme 1, which uses a lipase catalysed acylation to recover the 1-O-acylderivatives 2a,b directly
from 1a,b. Treatment of the known tosylate 109 with sodium azide in DMF, followed by the
Staudinger reduction of the obtained azide 11 allowed the introduction of the amino function on C6
of the glucose moiety, thus affording the amino derivative 12, which was acylated to the amides
13a,b by means of the proper acyl chloride in pyridine. Debenzylation to the target compounds 1a,b
was finally obtained by means of Pd/C catalysed hydrogenolysis. Exploiting the known selectivity
of lipase from Pseudomonas cepacia (LPS) for 2-O--D-glucopyranosylglycerol in lipase-mediated
transesterification reactions in organic solvent,6 we directly acylated the sn-1 positions of 1a,b
simply by treating them with the desired trifluoroethylester in pyridine (in the presence of LPS as
catalyst), thus obtaining the N,O-diacylderivatives 2a,b with good conversion yields (see
Experimental).
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OTs
N3
O
O
OBn
OBn
OBn
OBn
HO
HO
HO
HO
a
O
O
OH
OH
b
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11
NHCOR
NH2
O
O
OBn
OBn
OBn
OBn
HO
HO
HO
HO
c
O
O
OH
OH
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13a R=C5H11
13b R=C17H35
d
e
1a, 1b
2a, 2b
Scheme 1. a) NaN3, DMF; b) PPh3 polymer-bound, THF/H2O (90/10); c) RCOCl, Py; d) H2, Pd/C,
MeOH; e) LPS, Py, CF3CH2OCOR.
2.1.2. Configuration assignment of compounds 2a,b
As the procedure used to obtain compounds 2a and 2b was the same except for the different acyl
carriers used in the final enzymatic step of the synthesis (step e, Scheme 1), only the configuration
of compound 2a was assigned (see Scheme 2). As can be seen, 2a was correlated to the known 1-O-
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hexanoyl-2-O-(6-O-tosyl--D-glucopyranosyl)-sn-glycerol 149 by way of the trityl derivative 18,
which was directly obtained from 2a by selectively tritylating the glycerol primary hydroxyl with
trityl chloride in pyridine (step e). The same compound 18 was obtained in four steps starting from
tosylate 14, which was first converted into the corresponding azide 15 by means of sodium azide
treatment in DMF. Subsequent tritylation afforded the trityl derivative 16 and a Staudinger
reduction yielded the amine 17, which was finally transformed (step d) to the target 3-O-trityl amide
18 that was identical to that directly obtained from 2a.
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N3
OTs
O
O
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OCOC5H11
OH
OCOC5H11
HO
HO
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O
H
HO
HO
3
OH
OH
OH
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NHCOC5H11
O
O
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OCOC5H11
OCPh3
OCOC5H11
HO
HO
HO
HO
O
O
H
3
OH
OH
OH
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NH2
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OCOC5H11
OCPh3
OCOC5H11
H
HO
HO
HO
HO
d
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O
3
OH
OH
OCPh3
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Scheme 2. a) NaN3, DMF; b) CClPh3, Py 100°C; c) PPh3 polymer-bound, THF/H2O (90/10); d)
C5H11COCl, Py ; e) CClPh3 ,Py 100°C
2.1.3. Synthesis of compounds 3 and 4
In addition to the 6’-amides 1a,b and 2a,b, we thought it necessary to obtain the related 6’-amino
compounds in order to compare the influence on biological activity of the free vs acylated amino
group at position 6 of the sugar. We therefore attempted to obtain analogues carrying the free amino
group at C6 of the glucose moiety using an alternative route that should have allowed us to obtain
both the 6’-NH2 and the N-acylated derivatives as shown in Scheme 3.
1-O-Hexanoyl or octadecanoyl-2-O-(6-O-tosyl--D-glucopyranosyl)-sn-glycerols 14 or 199 easily
provided the corresponding azides 15 and 20 (see experimental) but, unfortunately, the Staudinger
reduction promoted the migration of the acyl chain from the 1-O to 3-O position of sn-glycerol.
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Complex mixtures of the amino derivatives 21, 22 or 23, 24 were obtained after column
chromatography, as shown by their TLC (CH2Cl2:MeOH 8:2, NH3 2%) and 1H-NMR analyses in
deuterated methanol at room temperature (see Figure 3 for the hexanoyl derivatives). In particular,
the migration and formation of the fully deacylated 6-amino-6-deoxy-2-O--D-glucopyranoside 25
were clearly observed by collecting 1H-NMR spectra at different times (Figure 3).
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OTs
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OR
HO
O
HO
OH
OH
14 : R = COC5H11
19 : R = COC17H35
NH2
a
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O
OR
H
HO
HO
2
3
N3
O
OH
OR'
O
OR
OH
HO
HO
b
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22 : R = COC17H35 ; R'= H
23 : R = H ; R'= COC5H11
24 : R = H; R'= COC17H35
25 : R = H; R'= H
O
OH
15 : R = COC5H11
20 : R = COC17H35
Scheme 3. a) NaN3, DMF; b) PPh3 polymer-bound, THF/H2O (90/10)
Figure 3. 1H-NMR resonances of glycerol H-2 of compounds 21, 23 and 25 in deuterated
methanol at room temperature. The spectra were collected at 0, 44 and 186 hours.
Given the observed intrinsic instability of the 1-O-acyl-6’-aminoglucosylglycerols, we did not
explore different means of synthesis (e.g. by removing the trityl group from 17), but decided to test
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the role of the 6’-NH2 in octyl 6-amino-6-deoxy--D-glucopyranoside (3) as a simple model that has
both a lipophilic aglycone and the 6-amino group on the hydrophilic sugar head. The synthesis
(Scheme 4) started from the sodium azide treatment of octyl 2,3,4-tri-O-acetyl-6-O-tosyl-β-D-
glucopyranoside (26) (obtained from the one-pot tosylation/acetylation20 of commercially available
octyl β-D-glucopyranoside, see experimental), which afforded octyl 2,3,4-tri-O-acetyl-6-azido-6-
deoxy--D-glucopyranoside 27. Deacetylation by means of the Zemplén reaction yielded the azide
28, which was finally reduced by means of a Staudinger procedure to the amine 3 (direct reduction
of 27 yielded the acetyl migration compound octyl 2,3-di-O-acetyl-N-acetyl-6-amino-6-deoxy--D-
glucopyranoside, data not shown). Octyl N-hexanoyl-6-amino-6-deoxy--D-glucopyranoside (4)
was also prepared by treating 3 with hexanoyl chloride in pyridine in order to obtain the reference
N-acylated derivative (Scheme 4).
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N3
OTs
O
O
AcO
AcO
AcO
AcO
a
OC8H17
OC8H17
OAc
OAc
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27
NH2
O
HO
b
OC8H17
HO
OH
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d
c
N3
NHCOC5H11
O
O
HO
HO
HO
OC8H17
OC8H17
HO
OH
OH
28
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Scheme 4. a) NaN3, DMF; b) MeONa, MeOH; c) PPh3 polymer-bound, THF/H2O (90/10); d)
C5H11COCl, Py.
2.2. Biological evaluation
2.2.1. EBV-EA assay for antitumor promoters
It is known that Epstein-Barr virus (EBV) is activated by tumor promoters to produce viral early
antigens (EAs), and evaluating its inhibition is often used as a primary screening for in vitro anti-
tumor-promoting activities.4 The inhibitory effects of AGGLs 1a,b and 2a,b and 6-aminoglucosides
3 and 4 were assayed using a short-term in vitro assay of the EBV-EA activation induced in Raji
cells by the tumor promoter TPA (as described in Refs. 3 and 21). Table 1 shows the in vitro
inhibitory activity of compounds 1-4 and, for purposes of comparison, the effects of the previously
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studied 2-O-(6-O-hexanoyl-β-D-glucopyranosyl)-sn-glycerol (5),18 1-O-hexanoyl-2-O-(6-O-
hexanoyl-β-D-glucopyranosyl)-sn-glycerol (6),18 1-O-hexanoyl-2-O-(β-D-glucopyranosyl)-sn-
glycerol (7),18 1-O-octadecanoyl-2-O-(β-D-glucopyranosyl)-sn-glycerol (8)9, 1-O-hexanoyl-2-O-(β-
D-sulfoquinovopyranosyl)-sn-glycerol (9),9 and nonyl -D-galactopyranoside (10)19 are also
reported. Compounds 1-4 were only weak cytotoxic against Raji cells (50% viability at 1000 mol
ratio/TPA, Table 1) and, in general, all of the compounds were poorly active as indicated by their
inhibitory percentages in comparison with control values (400-570 IC50, mol ratio/TPA, Table 1).
However, comparison of the short acyl chain derivatives, hexanoyl-amides 1a and 2a (IC50 400 and
465, Table 1), with the highly active hexanoates 5 and 6 (IC50 51.8 and 37.3, Table 1, Ref. 18)
revealed a remarkable effect. Substituting the 6’-ester oxygen with a nitrogen atom greatly
decreased the activity, as can be seen by comparing the corresponding IC50 values shown in Table 1
(51.8 vs 400 for 5 vs 1a and 37.3 vs 465 for 6 vs 2a). The highly negative effect induced by the
nitrogen atom was similar to that observed in the case of the SQAG hexanoate 9,9 in which an
anionic sulfonate head substitutes the primary hydroxyl of glucose (IC50 29.3 vs 417 for 7 vs 9,
Table 1). However, the same strong depressing effect was not observed when the long chain
octadecanoyl-amide 1b (IC50 502, Table 1) was compared with the slightly active 1-O-
octadecanoyl-2-O-(β-D-glucopyranosyl)-sn-glycerol (8) (IC50 550, Table 1, Ref. 9), thus confirming
that, as already reported,9 the effects of two negative groups such as a long acyl chain22 and a
nitrogen atom are not additive.
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The activity of octylglucosides 3 and 4 (IC50 489 and 474 respectively, Table 1) also seemed to be
much less than that of compounds 5 and 6 (IC50 51.8 and 37.3, Table 1, Ref. 18), although in
principle the reduction could be due to both the presence of the nitrogen atom and the lack of the
glycerol backbone, as previously observed in the case of nonyl -D-galactopyranoside (10) (IC50
410, Table 1, Ref. 19) (the nature of the glycosyl moiety, e.g galactose vs glucose, plays a
secondary role in the EBV inhibition of glycoglycerolipids)22. Nevertheless, the data relating to
these simple glycosides allowed us to compare the effect of the amino vs hydroxyl group: the
presence of the amino group in 3 reduced its activity in comparison with 10 (IC50 489 vs 410, Table
1). Furthermore, the fact that there was no difference in the inhibitory effects of 6-aminoglucoside 3
and its amide 4 (IC50 489 vs 474, Table 1) suggests that nitrogen (acylated or not) also plays a role
in the lower level of activity of glycoglycerolipids.
9

Table 1
1
2
3
Inhibitory effects of 1a-b, 2a-b, 3, 4 and 5-9 on TPA-induced EBV-EA activation.
4
5
6
Concentration (mol ratio/TPA)
7
8
9
1000
500
100
10
10
11
12
13
14
15
16
17
18
19
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21
22
23
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65
a
IC50
% to control ± SD (n = 3)
1a
2a
1b
2b
3
11.5±1.0(50)
14.3±1.2(50)
19.8±1.6(50)
23.5±1.9(50)
17.3±1.5(50)
15.2±1.3(50)
0.0±0.0(70)
0.0±0.0(70)
0.0±0.0(70)
19.6±0.7(60)
14.3±1.2(50)
12.4±0.3(70)
53.7±2.0(60)
55.2±2.1(60)
58.4±2.0(60)
62.5±2.3(60)
57.4±2.3(60)
56.3±2.0(60)
80.6±1.5(80)
100±0.3(80)
100±0.3(80)
100±0.5(80)
100±0.3(80)
100±0.5(80)
100±0.5(80)
400
465
502
570
489
474
51.8
37.3
29.3
550
417
410
81.3±1.7(80)
84.1±1.9(80)
87.0±1.9(80)
83.9±1.8(80)
83.4±1.6(80)
4
5b
6b
7b
8c
9c
10d
19.3±0.3(>80) 37.6±1.5(>80) 78.1±1.8(>80)
15.1±0.5 (80)
10.6±0.6 (70)
57.4±2.2(70)
53.6±2.0(70)
34.6±1.2(80)
30.9±1.8(80)
90.6±0.5(80)
80.8±1.9(80)
71.5±1.9(80)
68.2±2.5(80)
100±0.0(80)
100±0.5(80)
50.6±1.7(>80) 80.5±2.3(>80) 100±0.3(>80)
a
Values are EBV-EA activation (%) in the presence of the test compound relative to the
control (100%). Activation was attained by treatment with TPA (32 pmol/mL). IC50
represents the mol ratio of compound, relative to TPA, required to inhibit 50% of the
positive control activated with TPA (32 pmol/mL). bSee ref.18. cSee ref.9. dSee ref.19.
10

2.2.2. Two-stage mouse skin carcinogenesis test
1
2
Compounds 1a and 2a, as representatives of N-containing glycoglycerolipids, were also submitted
to an in vivo two-stage mouse skin carcinogenesis test of mouse skin papillomas using DMBA as an
initiator and TPA as a promoter. After 20 weeks of promotion, there was no statistically significant
difference in body weight between the control and treatment groups. Activity was estimated on the
basis of incidence (percentage of mice bearing papillomas, Fig. 4A) and multiplicity (average
number of papillomas per mouse, Fig. 4B). After ten weeks of promotion, 80% of the mice in the
control group (TPA treatment alone) bore papillomas (Fig. 4A) and, after 10 and 20 weeks, the
number of papillomas per mouse was respectively two and eight (Fig. 4B). Both of the tested
compounds poorly inhibited tumor promotion, and did not reduce the percentage of mice bearing
papillomas after 20 weeks of treatment, but only made the process slightly slower. After ten weeks
of promotion 40% of the mice treated with 1a bore papillomas, and 50% of those treated with 2a;
after 20 weeks of promotion, all of the mice in both groups were affected (Fig. 4A). The decrease in
the number of papillomas per mouse was also poor: after ten weeks of promotion, the affected mice
treated with 1a had 1.6 papillomas (76% with respect to the control group) and those treated with
1b had 1.7 (81%), (Fig. 4B); the corresponding figures after 20 weeks were 5.8 (73%) and 6.2
(78%) (Fig. 4B). In comparison, mice treated with 1-O-hexanoyl-2-O--D-glucopyranosyl-sn-
glycerol (7) (a similar glycoglycerolipid whose in vivo activity was tested)23 for 20 weeks showed
an incidence of 80% and a multiplicity of 4.0 papillomas per mouse (44% of the number in the
control group) (Figs. 4A and 4B). Overall, the results of the in vivo experiments are in line with the
in vitro findings, and confirmed that the protective effect of 1a and 2a against TPA tumor-
promoting activity was markedly reduced.
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4
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11

1
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4
A
100
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10
0
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0
5
10
15
20
Weeks of promotion
B
10
8
6
4
2
0
0
5
10
15
20
Weeks of promotion
Figure 4. Inhibitory effects of compounds 1a and 2a (85 nmol) on DMBA-TPA mouse skin
carcinogenesis. All mice were initiated with DMBA (390 nmol) and promoted with TPA (1.7 nmol)
twice a week starting one week after initiation. A: percentage of mice with papillomas (papilloma
incidence); B: averaged number of papillomas per mouse (papilloma multiplicity). (, TPA alone;
∆, TPA + 1a; , TPA + 2a,  TPA + 7 ). At 20 weeks of promotion the averaged number of
papillomas per mouse was reduced, with respect to the control group (7.9 ± 1.8), to 5.8 ± 1.1 for 1a
(P < 0.1), 6.2 ± 1.3 for 2a (P <0.1 ) and 4.0 ± 0.9 (P <0.001 ) for 7 (control group 9.1 ± 1.6) 23.
12

3. Conclusion
1
2
It is not yet known what molecular target is responsible for the anti-tumor-promoting activity of
glycolglycerolipids in the EBV-EA assay. However, as some are able to inhibit PKC translocation
to the plasma membrane in TPA-treated fibroblasts,24 it is possible that this kinase (the most
important target of phorbol esters and the main signalling molecule involved in their carcinogenic
effects)25,26 could be targeted by this class of compounds. As a part of a project aimed at modifying
bioactive glycoglycerolipids in order to obtain compounds capable of inhibiting tumor promotion,
in this work the AGGL 1a-b and 2a-b, based on 2-O--D-glucopyranosylglycerol, were efficiently
prepared and their ability to inhibit TPA induced tumor-promotion was studied. Although they were
less active than previously studied MGDG analogues, they provided further interesting information
concerning the structure/anti-tumor-promoting activity of glycoglycerolipids. In particular, the in
vitro and in vivo biological assays showed that the N-hexanoyl-6’-amino derivatives 1a and 2a were
much less active than the corresponding potent 6’-O-acylated glucoglycerolipids 5 and 6, even
though the only structural difference was the replacement of an oxygen atom by a nitrogen atom. As
in the case of the anionic 6’-sulfonate 9, which was scarcely active in comparison with 5 and 6,9 the
reduced activity of the newly prepared 1a and 2a was clearly related to the manipulation of the 6-
position of the sugar, which therefore seems to play a crucial role in the anti-tumor promoting
activity of glycoglycerolipids. Finally, 2-O--D-glucopyranosylglycerol-based AGGLs represent
new and easily accessible analogues of rare natural bioactive AGGLs, thus offering new tools for
the study of AGGL-related biological activities.
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4
5
6
7
8
9
10
11
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4. Experimental
4.1. Chemical procedures
4.1.1. Materials
Pseudomonas cepacia lipase (LPS, lipase PS, specific activity 30.5 triacetin units/mg solid), from
Amano Pharmaceutical Co (Mitsubishi Italia), was supported on celite.6 All reagents were bought at
highest commercial quality and used without further purification except where noted. Air- and
moisture-sensitive liquids and solutions were transferred via oven-dried syringe or stainless steel
cannula through septa. Evaporation under reduced pressure was always effected with a bath
temperature below 45°C. Dry solvents and liquid reagents were distilled prior to use. Pyridine (Py)
and dichloromethane were distilled from calcium hydride; dimethylformamide (DMF) was dried on
13

activated 4 Å molecular sieves. The acyl carriers, 2,2,2-trifluoroethyl esters, were synthesized
according to reference 27. 1,3-Di-O-benzyl-2-O-(6-O-tosyl--D-glucopyranosyl)-sn-glycerol 10, 1-
O-hexanoyl-2-O-(6-O-tosyl--D-glucopyranosyl)-sn-glycerol 14 and 1-O-octadecanoyl-2-O-(6-O-
tosyl--D-glucopyranosyl)-sn-glycerol 19 were synthesized according to a literature procedure.9
Optical rotations were measured with a Perkin-Elmer 241 polarimeter. Melting points were
recorded on a Büchi 510 capillary melting point apparatus and were uncorrected. The structures of
all the new synthesized compounds were confirmed through full 1H and 13C NMR characterization
and mass spectroscopy. 1H NMR analysis were performed at 500 MHz with a Bruker FT-NMR
AVANCE™ DRX500 spectrometer using a 5 mm z-PFG (pulsed field gradient) broadband reverse
probe at 298 K, and 13C NMR spectra at 125.76 MHz were done of all the new compounds. The
signals were unambiguously assigned by 2D COSY and HSQC experiments (standard Bruker pulse
program). Chemical shifts are reported as  (ppm) relative to residual CHCl3 , CH3OH or Py fixed
at 7.24, 3.30 and 7.19 (higher field line) ppm, respectively, for 1H NMR spectra and relative CDCl3
fixed at 77.0 (central line), CD3OD at 47.0 (central line) or Pyd5 123.0 (higher field line) ppm for
13C NMR spectra; scalar coupling constants are reported in hertz. Mass spectra were recorded in
negative or positive-ion electrospray (ESI) mode on a Thermo Quest Finnigan LCQTM DECA ion
trap mass spectrometer; the mass spectrometer was equipped with a Finningan ESI interface;
sample solutions were injected with a ionization spray voltage of 4.5 kV or 5.0 kV (positive 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 by Finnigan
Xcalibur software system. All reactions were monitored by TLC on silica gel 60 F-254 plates
(Merck), spots being developed with an anysaldehyde-based reagent, and subsequently heated at
110 °C. Flash column chromatography was performed on silica gel 60 (230-400 mesh, Merck).
TLC, NMR and MS analysis confirmed purity of all synthesized compounds (see supplementary
material for 13C and 1H NMR spectra). High resolution mass spectra (HRMS) of the target
compounds (1a-b, 2a-b, 3 and 4) and of tritylated 18 (the key compound for the configuration
assignment) were also recorded at C.I.G.A. (Centro Interdipartimentale Grandi Attrezzature,
University of Milan) on a Fourier Transform Ion Ciclotron Resonance (FT-ICR) mass spectrometer
APEX II & Xmass software (Bruker Daltonics) – 4.7 T magnet (Magnex), in positive-ion
electrospray (ESI) mode. Samples were injected as <1 to 5 g/mL methanol solutions and eluted
with methanol (120 L/h). FT-IR spectra of the same compounds were registered on a PerkinElmer
instrument (mod. FT-IR spectrum one, PerkinElmer, Waltham, MA, USA) equipped with universal
attenuated total reflection (ATR) sampling.
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14

1
2
4.1.2. Synthesis of compounds 1a and 1b
3
4
5
6
7
8
4.1.2.1. 1,3-di-O-benzyl-2-O-(6-azido-6-deoxy--D-glucopyranosyl)-sn-glycerol (11)
1,3-Di-O-benzyl-2-O-(6-O-tosyl--D-glucopyranosyl)-sn-glycerol 109 (1.2 g, 2.04 mmol) was
dissolved in DMF (15 mL), NaN3 (0.57 g, 8.77 mmol) was added, the reaction mixture was warmed
to 85 °C and stirred for 6 h under Argon atmosphere (TLC, CH2Cl2:CH3OH, 90:10, v/v). The
suspension was filtered and evaporated under vacuum, yielding a crude compound, which was
purified by flash chromatography (CH2Cl2:CH3OH, 90:10, v/v) to yield azide 11 (0.66 g, 1.44
mmol, 70% yield) as a light yellow oil: []2D0 =  2.6 (c 1, CHCl3); 1H NMR (CDCl3):  3.36-3.52
(m, 6H, H-2’, H-3’, H-4’, H-5’, H-6’a, H-6’b), 3.54-3.66 (m, 4H, H-1a, H-1b, H-3a and H-3b), 4.05
(m, 1H, H-2), 4.45 (d, J1’,2’= 7.8 Hz, 1H, H-1’), 4.45-4.53 (m, 4H, 2 CH2Ph), 7.23-7.34 (m, 10H,
2Ph); 13C-NMR (CDCl3):  = 51.41 (C6’), 70.18 (C1 or C3), 70.45 (C3 or C1), 70.69, 73.25, 73.42
(OCH2Ph), 73.45 (OCH2Ph), 75.30, 76.10, 77.11 (C2), 102.88 (C1’), 127.75 (Ph), 127.78 (Ph),
127.82 (Ph), 127.85 (Ph), 128.38 (Ph), 128.47(Ph), 137.63 (Ph), 137.76 (Ph). ESI-MS (CH3OH,
positive-ion mode): m/z = 482.2 [M+Na]+. Calcd for C23H29N3O7, m/z 459.2 [M].
9
10
11
12
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4.1.2.2. 1,3-di-O-benzyl-2-O-(6-amino-6-deoxy--D-glucopyranosyl)-sn-glycerol (12)
Azide 11 (0.64 g, 1.39 mmol) was dissolved in THF/H2O (90/10, 14 mL) and polymer-bound PPh3,
(3 mmol/g, 0.95 g, 2.85 mmol), used to efficiently remove triphenylphosphine oxide by-product,
was added. The reaction mixture was left under stirring overnight at 40°C (TLC, CH2Cl2:CH3OH,
80:20, v/v), then filtered and the resin repeatedly washed with MeOH. The combined organic layers
were dried over Na2SO4 and evaporated under reduced pressure. The obtained crude compound was
purified by flash chromatography (CH2Cl2:CH3OH, 85:15, v/v, 2% NH3) to afford amine 12 (0.51
g, 1.18 mmol, 85%) as an oil: []2D0 =  10.0 (c 1, CH3OH); 1H NMR (MeOD):  2.68 (dd, J5’,6’a=7.0
Hz, J6’a,6’b=13.3 Hz, 1H, H-6’a), 2.98 (dd, J5’,6’b=2.5 Hz, 1H, H-6’b), 3.12-3.18 (m, 2H, H-4’ and H-
5’), 3.20 (dd, J1’,2’= 7.8 Hz, J2’,3’=9.3 Hz, 1H, H-2’), 3.34 (dd, J3’,4’=9.0 Hz, 1H, H-3’), 3.61-3.72
(m, 4H, H-1a, H-1b, H-3a and H-3b), 4.07 (m, 1H, H-2), 4.47 (d, 1H, H-1’), 4.50-4.56 (m, 4H, 2
CH2Ph), 7.24-7.35 (m, 10H, 2Ph); 13C-NMR (MeOD):  = 41.75 (C6’), 69.04 (C1 or C3), 69.38
(C3 or C1), 71.21 (C4’), 72.32 (OCH2Ph), 72.46 (OCH2Ph), 73.20 (C2’), 75.55 (C5’), 75.69 (C3’),
76.19 (C2), 101.98 (C1’), 126.76 (Ph), 126.92 (Ph), 126.99 (Ph), 127.40(Ph), 137.44 (Ph), 137.49
(Ph). ESI-MS (CH3OH, positive-ion mode): m/z = 434.1 [M+1]+. Calcd for C23H31NO7, m/z 433.21
[M].
15

1
2
4.1.2.3. General procedure for the synthesis of amides 13a and 13b
3
4
5
6
7
8
9
To a solution of compound 12 (0.23 g, 0.53 mmol) in dry pyridine (3.5 mL) at 0 °C the proper acyl
chloride (0.79 mmol) was added and the reaction mixture was stirred at rt for 4 h (hexanoyl
chloride) or 7 h (octadecanoyl chloride) (TLC, CH2Cl2:CH3OH, 80:20, v/v). After quenching with
CH3OH, the solvent was evaporated under vacuum by repeated adding of toluene and the obtained
crude was submitted to flash column chromatography (CH2Cl2:CH3OH, from 90:10, to 80:20 v/v).
10
11
12
13
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4.1.2.3.1. 1,3-di-O-benzyl-2-O-(N-hexanoyl-6-amino-6-deoxy--D-glucopyranosyl)-sn-glycerol
13a: 0.20 g (0.38 mmol, 72% yield); oil: []2D0 =  19.8 (c 1, CH3OH); 1H NMR (CDCl3):  = 0.87
(t, J=7.0 Hz, 3H, CH3), 1.21-1.34 (m, 4H, 2CH2), 1.57 (m, 2H, CH2), 2.13 (m, 2H, CH2), 3.04 (ddd,
J5’,6’a=2.7 Hz, J6’a,6’b=14.8 Hz, J6’a,NH= 4.6, 1H, H-6’a), 3.12 (dd, J3’,4’=9.6 Hz, J4’,5’=9.6 Hz, 1H, H-
4’), 3.21 (ddd, J5’,6’b=2.7 Hz, 1H, H-5’), 3.36 (dd, J1’,2’= 7.8 Hz, J2’,3’=9.1 Hz, 1H, H-2’), 3.52-3.62
(m, 5H, H-1a, H-1b, H-3a, H-3b and H-3’), 3.92 (ddd, J6’b, NH= 8.4 Hz, 1H, H-6’b), 4.04 (m, 1H, H-
2), 4.45 (d, 1H, H-1’), 4.52 (br s, 4H, 2 CH2Ph), 5.83 (dd, 1H, NH), 7.25-7.35 (m, 10H, 2Ph); 13C-
NMR (CDCl3):  = 13.88 (CH3), 22.30 (CH2), 25.25 (CH2), 31.38 (CH2), 36.31 (CH2), 39.81
(C6’), 69.74 (C4’), 70.12 (C1 or C3), 70.23 (C3 or C1), 73.26 (OCH2Ph), 73.52 (OCH2Ph), 73.95
(C2’), 74.81 (C3’), 75.13 (C5’), 78.46 (C2), 103.74 (C1’), 127.52 (Ph), 127.74 (Ph), 127.82 (Ph),
127.90 (Ph), 128.36 (Ph), 128.48 (Ph), 137.46 (Ph), 137.96 (Ph), 175.56 (CO). ESI-MS (CH3OH,
negative-ion mode): m/z = 530.5 [M1]. Calcd for C29H41NO8, m/z 531.28 [M].
4.1.2.3.2. 1,3-di-O-benzyl-2-O-(N-octadecanoyl-6-amino-deoxy-6--D-glucopyranosyl)-sn-
glycerol 13b: 0.25 g (0.36 mmol, 68% yield): oil; []2D0 =62.0 (c 1, CHCl3); 1H NMR (CDCl3): 
= 0.86 (t, J=7.0 Hz, 3H, CH3), 1.13-1.37 (m, 28H, 14CH2), 1.58 (m, 2H, CH2), 2.14 (m, 2H, CH2),
3.02 (ddd, J5’,6’a=2.6 Hz, J6’a,6’b=14.9 Hz, J6’a,NH= 4.7, 1H, H-6’a), 3.12 (dd, J3’,4’=9.3 Hz, J4’,5’=9.3
Hz, 1H, H-4’), 3.22 (ddd, J5’,6’b=2.6 Hz, 1H, H-5’), 3.37 (dd, J1’,2’= 7.8 Hz, J2’,3’=9.1 Hz, 1H, H-2’),
3.51-3.62 (m, 5H, H-1a, H-1b, H-3a, H-3b and H-3’), 3.95 (ddd, J6’b, NH= 8.5 Hz, 1H, H-6’b), 4.05
(m, 1H, H-2), 4.45 (d, 1H, H-1’), 4.53 (br s, 4H, 2 CH2Ph), 5.85 (dd, 1H, NH), 7.25-7.34 (m, 10H,
2Ph); 13C-NMR (CDCl3):  = 14.12 (CH3), 22.69 (CH2), 25.61 (CH2), 29.85-29.20 (12 CH2),
31.92 (CH2), 36.39 (CH2), 39.75 (C6’), 69.51 (C4’), 70.10 (C1 or C3), 70.28 (C3 or C1), 73.29
16

(OCH2Ph), 73.57 (OCH2Ph), 74.03 (C2’), 74.65 (C3’), 75.25 (C5’), 78.70 (C2), 103.97 (C1’),
127.55 (Ph), 127.79 (Ph), 127.87 (Ph), 127.98 (Ph), 128.40 (Ph), 128.54 (Ph), 137.38 (Ph), 137.95
(Ph), 175.80 (CO). ESI-MS (CH3OH, negative-ion mode): m/z = 698.5 [M1]. Calcd for
C41H65NO8, m/z 699.47 [M].
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4.1.2.4. General procedure for benzylic groups removal (preparation of 1a and 1b)
To a solution of compound 13a or 13b (0.31 mmol) in CH3OH (10 mL) 10% Pd/C (0.15 g) was
added under Argon atmosphere and the reaction mixture was stirred under H2 (1 atm) for 8 h.
Dilution with CH3OH, filtering through a celite pad, washing with pyridine followed by CH3OH
and evaporation of the solvent to dryness afforded, after purification by flash chromatography
(CH2Cl2:CH3OH, 90:10, v/v), pure compounds 1a and 1b.
4.1.2.4.1. 2-O-(N-hexanoyl-6-amino-6-deoxy--D-glucopyranosyl)-sn-glycerol (1a): 0.098 g
(0.28 mmol, 90% yield); white solid, m.p.: 144-145 °C; []2D0 =  24.4 (c 1, CH3OH); 1H NMR
(MeOD):  = 0.91 (t, J=7.0 Hz, 3H, CH3), 1.26-1.39 (m, 4H, 2CH2), 1.61 (m, 2H, CH2), 2.21 (m,
2H, CH2), 3.12 (dd, J3’,4’=9.1 Hz, J4’,5’=9.5 Hz, 1H, H-4’), 3.21 (dd, J1’,2’=7.8 Hz, J2’,3’=9.1 Hz, 1H,
H-2’), 3.3 (ddd, 1H, H-5’), 3.36 (dd, 1H, H-3’), 3.39 (dd, J5’,6’a=6.6 Hz, J6’a,6’b=14.0 Hz, 1H, H-6’a),
3.57 (dd, J5’,6’b=2.8 Hz, 1H, H-6’b), 3.61-3.70 (m, 4H, H-1a, H-1b, H-3a and H-3b), 3.74 (m, 1H,
H-2), 4.39 (d, 1H, H-1’); 13C-NMR (MeOD):  = 12.27 (CH3), 21.43 (CH2), 24.74 (CH2), 30.58
(CH2), 35.00 (CH2), 39.50 (C6’), 60.75 (C1 or C3), 61.17 (C3 or C1), 70.84 (C4’), 73.31 (C2’),
74.02 (C5’), 75.31 (C3’), 81.06 (C2), 102.36 (C1’), 175.01 (CO). ESI-MS (CH3OH, negative-ion
mode): m/z = 350.3 [M1]. Calcd for C15H29NO8, m/z 351.19 [M]. ESI-HRMS (CH3OH, positive-
ion mode): m/z = 374.17843 [M+Na]+ (error, 0.3 ppm). Calcd for C15H29NO8Na, m/z = 374.17854
(monoisotopic mass). FT-IR (ATR): max 3312, 2918, 1650, 1534 cm-1.
4.1.2.4.2. 2-O-(N-octadecanoyl-6-amino-6-deoxy--D-glucopyranosyl)-sn-glycerol (1b): 0.083 g
(0.16 mmol, 51% yield): white solid, m.p.: 138 -141°C; []2D0 =  16.6 (c 0.6, Py); 1H NMR (Pyd5):
 = 0.85 (t, J=7.0 Hz, 3H, CH3), 1.15-1.36 (m, 28H, 14CH2), 1.78 (m, 2H, CH2), 2.40 (m, 2H,
17

CH2), 3.85 (ddd, 1H, H-5’), 3.91 (dd, J3’,4’=9.4 Hz, J4’,5’=9.4 Hz, 1H, H-4’), 4.00-4.11 (m, 3H, H-
6’a, H-6’b and H-2’), 4.15-4.26 (m, 5H, H-1a, H-1b, H-3a, H-3b and H-3’), 4.37 (m, 1H, H-2), 5.11
(d, J1’,2’= 7.8 Hz, 1H, H-1’), 8.85 (dd, J6’a,NH= 5.7 Hz, J6’b, NH= 5.7 Hz, 1H, NH); 13C-NMR (Pyd5): 
= 13.77 (CH3), 22.43 (CH2), 25.79 (CH2), 29.05-29.58 (12 CH2), 31.62 (CH2), 36.04 (CH2), 40.85
(C6’), 62.20 (C1 or C3), 62.55 (C3 or C1), 72.10 (C4’), 75.02 (C2’), 75.91 (C5’), 77.04 (C3’),
83.02 (C2), 104.48 (C1’), 174.09 (CO). ESI-MS (CH3OH, negative-ion mode): m/z = 518.5
[M1]. Calcd for C27H53NO8, m/z 519.38 [M]. ESI-HRMS (CH3OH, positive-ion mode): m/z =
542.36595 [M+Na]+ (error, 0.7 ppm). Calcd for C27H53NO8Na, m/z = 542.36634 (monoisotopic
mass). FT-IR (ATR): max 3290, 2916, 2848, 1641, 1554 cm-1.
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4.1.3. Enzymatic synthesis of 1-O-esters 2a and 2b
4.1.3.1. 1-O-hexanoyl-2-O-(N-hexanoyl-6-amino-6-deoxy--D-glucopyranosyl)-sn-glycerol (2a)
Compound 1a (0.05 g, 0.14 mmol) was dissolved in dry pyridine (2.5 mL) and trifluoroethyl
hexanoate (0.35 g, 1.77 mmol) and LPS (0.5 g) were added in the order. The suspension was stirred
at 45°C overnight (TLC, CH2Cl2:CH3OH, 90:10 v/v). After 24 h (about 90% conversion by TLC)
the reaction was stopped by filtering off the enzyme which was washed with pyridine. After
removing the solvent under vacuum, the crude was submitted to flash column chromatography
(CH2Cl2:CH3OH, from 95:5 to 80:20, v/v) yielding pure 2a: 0.051 g (0.114 mmol, 80% yield) as an
oil; []2D0 =  26.0 (c 1, CH3OH); 1H NMR (MeOD):  = 0.91 (m, 6H, 2CH3), 1.27-1.38 (m, 8H,
4CH2), 1.57-1.65 (m, 4H, 2CH2), 2.21 (m, 2H, CH2), 2.34 (m, 2H, CH2), 3.12 (dd, J3’,4’=9.2 Hz,
J4’,5’=9.2 Hz, 1H, H-4’), 3.17 (dd, J1’,2’= 7.8 Hz, J2’,3’=9.2 Hz, 1H, H-2’), 3.32 (ddd, 1H, H-5’), 3.35
(dd, 1H, H-3’), 3.38 (dd, J6’a,5’= 6.7 Hz, J6’a,6’b=14.0 Hz, 2H, H-6’a), 3.58 (dd, J6’b,5’= 2.7 Hz, 1H,
H-6’b), 3.65 (dd, J3a,2= 5.3 Hz, J3a,3b=11.7 Hz, 1H, H-3a), 3.67 (dd, J3b,2= 4.7 Hz, 1H, H-3b), 3.95
(m, 1H, H-2), 4.19 (dd, J1a,2= 5.0 Hz, J1a,1b= 11.6 Hz, 1H, H-1a), (dd, J1b,2= 5.8 Hz, 1H, H-1b), 4.40
(d, 1H, H-1’); 13C-NMR (MeOD):  12.26 (CH3), 12.30 (CH3), 21.39 (CH2), 21.44 (CH2), 23.67
(CH2), 24.77 (CH2), 30.43 (CH2), 30.59 (CH2), 32.93 (CH2), 35.00 (CH2), 39.50 (C6’), 61.14 (C3),
62.54 (C1), 70.83 (C4’), 73.00 (C2’), 74.04 (C5’), 75.34 (C3’), 76.95 (C2), 102.10 (C1’), 173.40
(CO), 174.97 (CO). ESI-MS (CH3OH, negative-ion mode): m/z = 448.3 [M1]. Calcd for
18

C21H39NO9, m/z 449.26 [M]. ESI-HRMS (CH3OH, positive-ion mode): m/z = 472.25112 [M+Na]+
(error, 1.2 ppm). Calcd for C21H39NO9Na, m/z = 472.25170 (monoisotopic mass). FT-IR (ATR):
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3
4
5
6

max 3301, 2931, 1741, 1644, 1547 cm-1.
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8
9
4.1.3.2. 1-O-octadecanoyl-2-O-(N-octadecanoyl-6-deoxy-6-amino--D-glucopyranosyl)-sn-
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11
12
13
14
15
16
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18
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20
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65
glycerol (2b)
With the same procedure, apart from repeated flash chromatographies to remove stearic acid,
starting from compound 1b (0.05 g, 0.096 mmol), 0.029 g (0.037 mmol, 39% yield) of pure 2b
were obtained as a white solid: m.p.: 151-152°C; []2D0 =  20.0 (c 1, Py). 1H NMR (Pyd5):  = 0.87
(m, 6H, 2CH3), 1.18-1.33 (m, 56H, 28CH2), 1.65 (m, 2H, CH2), 1.79 (m, 2H, CH2), 2.35-3.43 (m,
4H, 2CH2), 3.86 (ddd, 1H, H-5’), 3.90 (dd, J3’,4’=8.5 Hz, J4’,5’=9.4 Hz, 1H, H-4’), 3.96 (dd, J1’,2’
=
7.8 Hz, J2’,3’=8.5 Hz, 1H, H-2’), 4.04-4.09 (m, 2H, H-6’a, H-6’b), 4.09-4.14 (m, 2H, H-3a and H-
3b), 4.18 (dd, 1H, H-3’), 4.44 (m, 1H, H-2), 4.67 (dd, J1a,2= 4.8 Hz, J1a,1b= 11.6 Hz, 1H, H-1a), 4.70
(dd, J1b,2= 5.6 Hz, 1H, H-1b), 5.02 (d, 1H, H-1’), 8.60 (dd, J6’a,NH= 6.0 Hz, J6’b, NH= 6.0 Hz, 1H,
NH); 13C-NMR (Pyd5):  = 13.77 (2CH3), 22.44 (2CH2), 24.75 (CH2), 25.81 (CH2), 28.8-29.8
(24CH2), 31.63 (2CH2), 33.88 (CH2), 36.07 (CH2), 40.93 (C6’), 62.20 (C3), 63.82 (C1), 72.15
(C4’), 74.53 (C2’), 75.84 (C5’), 77.07 (C3’), 78.55 (C2), 104.23 (C1’), 173.15 (CO), 173.96 (CO).
ESI-MS (CH3OH, negative-ion mode): m/z = 784.5 [M1]Calcd for C45H87NO9, m/z 785.64 [M].
ESI-HRMS (CH3OH, positive-ion mode): m/z = 808.62704 [M+Na]+ (error, 0.3 ppm). Calcd for
C45H87NO9Na, m/z = 808.62730 (monoisotopic mass). FT-IR (ATR): max 3320, 2919, 2850, 1738,
1651, 1550 cm-1.
4.1.4. Configuration assignment of 1-O-hexanoylamide 2a
Converging synthesis of 1-O-hexanoyl-3-O-trityl-2-O-(N-hexanoyl-6-amino-6-deoxy--D-
glucopyranosyl)-sn-glycerol 18 was obtained starting from known tosylate 149 or from 2a as here
reported. In this section the synthesis of octadecanoyl azide 20 starting from tosylate 199 is also
reported.
4.1.4.1. From 1-O-hexanoyl-2-O-(6-O-tosyl--D-glucopyranosyl)-sn-glycerol (14)
4.1.4.1.1. 1-O-hexanoyl-2-O-(6-azido-6-deoxy--D-glucopyranosyl)-sn-glycerol (15)
19

Tosylate 14 (1.00 g, 1.97 mmol) was dissolved in DMF (10 mL), NaN3 (0.55 g, 8.46 mmol) was
added under stirring, the reaction mixture was warmed to 85 °C and stirred for 5 h under Argon
atmosphere (TLC, CH2Cl2:CH3OH, 90:10 v/v). The mixture was filtered, the DMF was co-
evaporated with cyclohexane at reduced pressure and the crude was submitted to flash column
chromatography (CH2Cl2:CH3OH, 95:5, v/v), yielding 15 (0.52 g, 1.38 mmol, 70% yield) as an oil;
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7
8
9
[] 2D0 = 25.6 (c 1, CHCl3). 1H NMR (CDCl3):  = 0.87 (t, J=7.0 Hz, 3H, CH3), 1.21-1.35 (m, 4H,
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2CH2), 1.59 (m, 2H, CH2), 2.31 (m, 2H, CH2), 3.37-3.58 (m, 6H, H-2’, H-3’, H-4’, H-5’, H-6’a and
H-6’b), 3.66 (dd, J3a,2=5.3 Hz, J3a,3b=12.0 Hz, 1H, H-3a), 3.75 (dd, J3b,2=2.0 Hz, 1H, H-3b), 3.96
(m, 1H, H-2), 4.19 (dd, J1a,2=6.2 Hz, J1a,1b=11.6 Hz, 1H, H-1a), 4.24 (dd, J1b,2=5.5 Hz, 1H, H-1b),
4.46 (d, J1’,2’=7.7 Hz, 1H, H-1’); 13C-NMR (CDCl3):  = 13.86 (CH3), 22.26 (CH2), 24.49 (CH2),
31.23 (CH2), 34.11 (CH2), 51.33 (C6’), 62.51 (C1 or C3), 62.54 (C3 or C1), 70.59 (C4’), 73.03
(C2’), 75.16 (C5’), 75.98 (C3’), 77.77 (C2), 102.16 (C1’), 174.30 (CO). ESI-MS (CH3OH,
negative-ion mode): m/z =376.0 [M1]. Calcd for C15H27N3O8, m/z 377.18 [M].
4.1.4.1.2. 1-O-octadecanoyl-2-O-(6-azido-6-deoxy--D-glucopyranosyl)-sn-glycerol (20)
With the same procedure, starting from tosylate 19 (0.10 g, 0.148 mmol), 0.06 g of 20 (0.11 mmol,
74% yield) were obtained as white solid (m.p.: 70-71 °C); [] 2D0 = 18.7 (c 1, CH3OH). 1H NMR
(CD3OD):  = 0.89 (t, J=7.0 Hz, 3H, CH3), 1.20-1.38 (m, 28H, 14CH2), 1.61 (m, 2H, CH2), 2.35
(m, 2H, CH2), 3.20 (dd, J1’,2’=7.8 Hz, J2’,3’=9.0 Hz, 1H, H-2’), 3.25 (dd, J4’,3’=9.0 Hz, J4’,5’=9.0 Hz,
1H, H-4’), 3.34 (dd, 1H, H-3’), 3.41 (dd, J6’a,6’b=12.6 Hz, J6’a,5’=6.6 Hz, 1H, H-6’a), 3.43 (ddd,
J5’,6’b=1.8 Hz, 1H, H-5’), 3.51 (dd, 1H, H-6’b), 3.65 (dd, J3a,2=5.3 Hz, J3a,3b=11.6 Hz, 1H, H-3a),
3.70 (dd, J3b,2=4.7 Hz, 1H, H-3b), 3.97 (m, 1H, H-2), 4.23 (dd, J1a,2=5.9 Hz, J1a,1b=11.7 Hz, 1H, H-
1a), 4.27 (dd, J1b,2=4.7 Hz, 1H, H-1b), 4.47 (d, J1’,2’=7.8 Hz, 1H, H-1’); 13C-NMR (CD3OD):  =
12.45 (CH3), 21.72 (CH2), 23.97 (CH2), 28.15-28.90 (12CH2) 31.06 (CH2), 32.97 (CH2), 50.72
(C6’), 61.09 (C3), 62.65 (C1), 70.29 (C4’), 72.87 (C2’), 74.86 (C5’), 75.53 (C3’), 76.48 (C2),
101.95 (C1’), 173.46 (CO). ESI-MS (CH3OH, negative-ion mode): m/z = 544.3 [M1]. Calcd for
C27H51N3O8, m/z 545.37 [M].
4.1.4.1.3. 1-O-hexanoyl-3-O-trityl-2-O-(6-azido-6-deoxy--D-glucopyranosyl)-sn-glycerol (16)
Azide 15 (0.29 g, 0.77 mmol) was dissolved in dry pyridine (5.0 mL), trityl chloride (0.43 g, 1.54
mmol) was added under stirring, the reaction mixture was warmed to 100 °C and stirred for 5 h
(TLC, CH2Cl2:CH3OH, 90:10 v/v). The solvent was evaporated under reduced pressure and the
20

crude was submitted to flash column chromatography (CH2Cl2:CH3OH, from 95:5 to 90:10, v/v) to
1
2
3
afford 16 (0.322 g, 0.52 mmol, 68% yield) as an oil: [] 2D0 =  10.8 (c 1, CHCl3). 1H NMR
4
5
6
7
8
9
(CDCl3):  = 0.91 (t, J=7.0 Hz, 3H, CH3), 1.25-1.37 (m, 4H, 2CH2), 1.59 (m, 2H, CH2), 2.28 (m,
2H, CH2), 3.27 (dd, J3a,2= 7.5 Hz, J3a,3b=9.5 Hz, 1H, H-3a), 3.32 (dd, J1’,2’= 7.7 Hz, J2’,3’=8.9 Hz,
1H, H-2’), 3.35-3.50 (m, 6H, H-3b, H-4’, H-5’, H-6’a and H-6’b), 3.53 (dd, J3’,4’= 8.9 Hz, 1H, H-
3’), 3.97 (m, 1H, H-2), 4.26 (dd, J1a,2= 5.7 Hz, J1a,1b= 11.8 Hz, 1H, H-1a), 4.36 (d, 1H, H-1’), 4.55
(dd, J1b,2= 3.0 Hz, 1H, H-1b), 7.23-7.48 (m, 15H, 3Ph); 13C-NMR (CDCl3):  = 13.88 (CH3), 22.25
(CH2), 24.52 (CH2), 31.23 (CH2), 34.24 (CH2), 51.27 (C6’), 62.84 (C3), 63.78 (C1), 70.53 (C4’),
73.56 (C2’), 74.99 (C5’), 76.21 (C3’), 77.83 (C2), 86.97 (OCPh3), 103.18 (C1’), 127.07 (3C, Ph),
127.80 (6C, Ph), 128.71 (6C, Ph), 143.70 (3C, Ph), 174.39 (CO). ESI-MS (CH3OH, positive-ion
mode): m/z = 642.3 [M+Na]+. Calcd for C34H41N3O9, m/z 619.29 [M].
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4.1.4.1.4. 1-O-hexanoyl-3-O-trityl-2-O-(6-amino-6-deoxy--D-glucopyranosyl)-sn-glycerol (17)
Azide 16 (0.10 g, 0.16 mmol) was dissolved in THF/H2O (90/10, 1.5 mL) and polymer-bound PPh3
(3 mmol/g, 0.12 g, 0.36 mmol) was added. The reaction mixture was left under stirring overnight at
40 °C (TLC, CH2Cl2:CH3OH, 90:10 v/v), then filtered washing with CH3OH. The organic layer was
dried over Na2SO4 and evaporated under reduced pressure. The obtained crude compound was
purified by flash chromatography (CH2Cl2:CH3OH, 90:10, v/v) to afford 17 (0.074 g, 0.125 mmol,
1
72%) as a solid; m.p.: 53-54 °C; []2D0 = +4.3 (c 1, CHCl3) H NMR (CDCl3): = 0.85 (t, J=7.0 Hz,
.
3H, CH3), 1.18-1.31 (m, 4H, 2CH2), 1.53 (m, 2H, CH2), 2.23 (m, 2H, CH2), 2.84-2.91 (m, 2H, H-
6’a and H-6’b), 3.16-3.22 (m, 2H, H-5’ and H-3a), 3.25 (dd, J1’,2’= 7.8 Hz, J2’,3’=9.0 Hz, 1H, H-2’),
3.31 (dd, J3b,2= 5.2 Hz, J3b,3a= 9.6 Hz, 1H, H-3b), 3.42 (dd, J3’,4’= 9.0 Hz, J4’,5’= 9.0 Hz, 1H, H-4’),
3.49 (dd, 1H, H-3’), 3.89 (m, 1H, H-2), 4.18 (dd, J1a,2= 5.9 Hz, J1a,1b= 11.8 Hz, 1H, H-1a), 4.30 (d,
1H, H-1’), 4.43 (dd, J1b,2= 3.3 Hz, 1H, H-1b), 7.17-7.43 (m, 15H, 3Ph); 13C-NMR (CDCl3):  =
13.90 (CH3), 22.25 (CH2), 24.50 (CH2), 31.23 (CH2), 34.19 (CH2), 43.25 (C6’), 63.18 (C3), 63.88
(C1), 72.13 (C4’), 73.64 (C2’), 75.05 (C5’), 76.12 (C3’), 77.95 (C2), 86.88 (OCPh3), 103.61 (C1’),
127.11 (3C, Ph), 127.81 (6C, Ph), 128.66 (6C, Ph), 143.71 (3C, Ph), 174.17 (CO). ESI-MS
(CH3OH, positive-ion mode): m/z = 616.3 [M+Na]+. Calcd for C34H43NO8, m/z 593.3 [M].
4.1.4.1.5. 1-O-hexanoyl-3-O-trityl-2-O-(N-hexanoyl-6-amino-6-deoxy--D-glucopyranosyl)-sn-
glycerol (18)
21

To a solution of compound 17 (0.06 g, 0.10 mmol) in dry pyridine (1.0 mL) at 0 °C hexanoyl
chloride (0.02 mL, 0.14 mmol) was added and the reaction mixture was stirred at rt for 5 h (TLC,
CH2Cl2:CH3OH, 85:15 v/v). After quenching with MeOH, the solvent was evaporated under
vacuum by repeated adding of toluene and the obtained crude was submitted to flash column
chromatography (CH2Cl2:CH3OH, from 95:5 to 90:10, v/v) yielding 18 (0.04 g, 0.058 mmol, 58%
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yield) as an oil. []2D0
59.5 (c 1, CHCl3). 1H NMR (CDCl3):  = 0.82-0.88 (m, 6H, 2 CH3), 1.14-
= 
1.33 (m, 8H, 4CH2), 1.50 (m, 2H, CH2), 1.60 (m, 2H, CH2), 2.00 (m, 2H, CH2), 2.25 (m, 2H, CH2),
2.93 (ddd, J5’,6’a= 2.8 Hz, J6a’,6’b= 14.8 Hz, J6a’,NH= 4.5 Hz, 1H, H-6’a), 3.09 (dd, J3’,4’= 9.4 Hz,
J4’,5’= 9.6 Hz, 1H, H-4’), 3.18 (dd, J2,3a= 5.5 Hz, J3a,3b= 9.7 Hz, 1H, H-3a), 3.21 (ddd, 1H, H-5’),
3.28-3.34 (m, 2H, H-2’ and H-3b), 3.57 (dd, J2’,3’= 9.2 Hz, 1H, H-3’), 3.91-4.00 (m, 2H, H-2 and H-
6’b), 4.17 (dd, J1a,2= 6.4 Hz, J1a,1b= 11.8 Hz, 1H, H-1a), 4.33 (dd, J1b,2= 3.8 Hz, 1H, H-1b), 4.37 (d,
1H, H-1’), 5.58 (dd, J6b,NH= 8.4 Hz, 1H, NH), 7.20-7.47 (m, 15H, 3Ph); 13C-NMR (CDCl3):  =
13.87 (2CH3), 22.24 (2CH2), 24.47 (CH2), 25.16 (CH2), 31.20 (CH2), 31.35 (CH2), 34.13 (CH2),
36.20 (CH2), 39.53 (C6’), 63.76 (C3), 63.99 (C1), 69.56 (C4’), 73.80 (C2’), 74.53 (C3’), 74.87
(C5’), 77.82 (C2), 86.85 (OCPh3), 103.73 (C1’), 127.20 (3C, Ph), 127.80 (6C, Ph), 128.70 (6C, Ph),
143.75 (3C, Ph), 174.00 (CO), 175.65 (CO). ESI-MS (CH3OH, negative-ion mode): m/z = 690.4
[M1]. Calcd for C40H53NO9, m/z 691.37 [M]. ESI-HRMS (CH3OH, positive-ion mode): m/z =
714.36023 [M+Na]+ (error, 1.4 ppm). Calcd for C40H53NO9Na, m/z = 714.36125 (monoisotopic
mass). FT-IR (ATR): max 3426, 3058, 2927, 1737, 1657, 1524 cm-1.
4.1.4.2. From 1-O-hexanoyl-2-O-( N-hexanoyl-6-amino-6-deoxy--D-glucopyranosyl)-sn-
glycerol (2a)
Compound 2a (0.02 g, 0.044 mmol) was dissolved in dry pyridine (0.6 mL), trityl chloride (0.024 g,
0.086 mmol) was added under stirring, the reaction mixture was warmed to 100 °C and stirred for 6
h (TLC, CH2Cl2:CH3OH, 90:10, v/v). The solvent was evaporated under reduced pressure and the
crude was submitted to flash column chromatography (CH2Cl2:CH3OH, 95:5, v/v) yielding pure 1-
O-hexanoyl-3-O-trityl-2-O-(N-hexanoyl-6-amino-6-deoxy--D-glucopyranosyl)-sn-glycerol 18
(0.024 g, 0.035 mmol, 79% yield), identical in its physico-chemical characteristics, NMR and MS
spectra to compound 18 previously obtained from the known tosylate 14.
22

4.1.5. Synthesis of compounds 3 and 4
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3
4.1.5.1. Octyl 2,3,4-tri-O-acetyl-6-O-tosyl--D-glucopyranoside (26)
Tosyl chloride (0.76 g, 4.0 mmol) was added to a solution of octyl -D-glucopyranoside (1g, 3.42
mmol) in pyridine (22 mL) at -5°C and left overnight at this temperature. After about 90%
conversion of the starting material by TLC (ethyl acetate 100%), acetic anhydride (4.85 mL, 51.3
mmol) was added at 0°C under stirring. After 1 h the reaction mixture was left to gradually reach
the room temperature (23°C). After 4 h the reaction was completed (TLC, petroleum ether:ethyl
acetate 60:40 v/v), poured into ice/water 100 mL and extracted with CH2Cl2 (30 mL x 5). The
collected organic layers were then washed with 5M hydrochloric acid (35 mL x 2), sodium
bicarbonate (50 mL) and water (50 mL x 2), dried with sodium sulphate and concentrated under
reduced pressure. Crystallization of the crude material (1.87 g) with a mixture of ethanol (18 mL)
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and CH2Cl2 (1 mL) yielded 1.332 g (2.33 mmol, 68% yield) of pure 26. Mp: 109-110°C; [] 20 = -
D
4.6 (CHCl3, c=1.0); 1H NMR (CDCl3):  = 0.86 (t, J= 6.7, 3H, CH3), 1.18-1.33 (m, 10H, 5 CH2),
1.50 (m, 2H, CH2), 1.96, 1.97 and 1.99 (3s, 9H, 3 CH3CO), 2.43 (s, 3H, CH3), 3.39 (m, 1H, OCHa),
3.71 (ddd, 1H, H-5), 3.75 (m, 1H, OCHb), 4.04 (dd, J5,6a= 5.9 Hz, J6a,6b= 10.9 Hz, 1H, H-6a), 4.09
(dd, J5,6b= 3.0 Hz, 1H, H-6b), 4.41 (d, J1,2= 7.9 Hz, 1H, H-1), 4.86 (dd, J2,3= 9.5 Hz, 1H, H-2), 4.87
(dd, J3,4= 9.5 Hz, J4,5= 9.7 Hz, 1H, H-4), 5.14 (dd, 1H, H-3). 13C NMR (CDCl3):  = 14.03 (CH3),
20.50, 20.54 and 20.55 (3 CH3CO), 21.62 (CH3Ph), 22.61 (CH2), 25.80 (CH2), 29.22 (CH2), 29.24
(CH2), 29.35 (CH2), 31.77 (CH2), 67.91 (C6), 68.87 (C4), 70.15 (OCH2), 71.23 (C2), 71.58 (C5),
72.62 (C3), 100.60 (C1), 128.05 (2C Ph), 129.86 (2C Ph), 132.59 (1C Ph), 145.08 (1C Ph), 169.14,
169.46 and 170.16 (3 CO). ESI-MS (CH3OH, positive-ion mode): m/z = 595.0 [M+Na]+. Calcd for
C27H40O11S, m/z 572.23 [M].
4.1.5.2. Octyl 2,3,4-tri-O-acetyl-6-azido-6-deoxy--D-glucopyranoside (27)
Tosylate 26 (0.50 g, 0.87 mmol) was dissolved in DMF (5.0 mL), NaN3 (0.25 g, 3.80 mmol) was
added under stirring, the reaction mixture was warmed to 85°C and stirred for 3 h under Argon
atmosphere (TLC, petroleum ether:AcOEt 70:30 v/v). The solution was diluted with water,
extracted with CH2Cl2 (3 x 30 mL), the organic layer washed with water, dried over Na2SO4 and
DMF was then co-evaporated with cyclohexane at reduced pressure, yielding a crude compound,
which was purified by flash chromatography (petroleum ether:AcOEt, 80:20, v/v) to yield azide 27
(0.37 g, 0.84 mmol, 97% yield) as a wax: m.p.: 55-56°C; []2D0 =  31.4 (c 1, CHCl3). 1H NMR
(CDCl3):  = 0.85 (t, J=6.8, 3H, CH3), 1.17-1.34 (m, 10H, 5CH2), 1.54 (m, 2H, CH2), 1.98, 2.00
and 2.01 (3s, 9H, 3 CH3CO), 3.15 (dd, J5,6a= 2.5 Hz, J6a,6b= 13.3 Hz, 1H, H-6a), 3.39 (dd, J5,6b= 7.6
23

Hz, 1H, H-6b), 3.46 (m, 1H, OCHa), 3.65 (ddd, 1H, H-5), 3.86 (m, 1H, OCHb), 4.50 (d, J1,2= 8.0
Hz, 1H, H-1), 4.93 (dd, J3,4= 9.6 Hz, J4,5= 9.6 Hz, 1H, H-4), 4.96 (dd, J2,3= 9.6 Hz, 1H, H-2), 5.18
(dd, 1H, H-3). 13C NMR (CDCl3):  = 14.04 (CH3), 20.57 (3 CH3CO), 22.61 (CH2), 25.80 (CH2),
29.21 (2 CH2), 29.34 (CH2), 31.77 (CH2), 51.20 (C6), 69.83 (C4), 70.11 (OCH2), 71.37 (C2), 72.64
(C3), 73.62 (C5), 100.60 (C1), 169.21, 169.50 and 170.24 (3 CO). ESI-MS (CH3OH, positive-ion
mode): m/z = 466.0 [M+Na]+. Calcd for C20H33N3O8, m/z 443.23 [M].
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4.1.5.3. Octyl 6-azido-6-deoxy--D-glucopyranoside (28)
CH3ONa (3 mL, 1 M solution in CH3OH) was added to a solution of compound 27 (0.35 g, 0.79
mmol) in dry CH3OH (1.5 mL). The reaction was stirred overnight at room temperature (TLC
CH2Cl2:CH3OH 90:10 v/v), then it was neutralized with an ion-exchange resin (Dowex–50, H+
form). The resin was filtered-off and the solution was concentrated under reduced pressure. The
crude was purified by flash-chromatography (CH2Cl2:CH3OH 90:10 v/v) to give the de-acetylated
product 28 (0.215 g, 0.68 mmol, 86% yield) as a solid: m.p.: 71-72°C; [] 2D0 =  48.7 (c 1, CHCl3);
1H NMR (CDCl3): = 0.86 (t, J=6.8, 3H, CH3), 1.18-1.35 (m, 10H, 5 CH2), 1.61 (m, 2H, CH2),
3.33-3,56 (m, 7H, H-2, H-4, H-5, H6a, H-6b, H-3 and OCHa), 3.85 (m, 1H, OCHb), 4.27 (d, J1,2=
8.0 Hz, 1H, H-1). 13C NMR (CDCl3):  = 14.06 (CH3), 22.63, 25.89, 29.26, 29.38, 29.61 and 31.82
(6 CH2), 51.42 (C6), 70.36 (OCH2), 70.89 (C4), 73.47 (C2), 75.22 (C5), 76.20 (C3), 102.37 (C1).
ESI-MS (CH3OH, negative-ion mode): m/z = 315.9 [M1]. Calcd for C14H27N3O5, m/z 317.2 [M].
4.1.5.4. Octyl 6-amino-6-deoxy--D-glucopyranoside (3)
Azide 28 (0.20 g, 0.63 mmol) was dissolved in THF/H2O (90/10, 10 mL) and polymer-bound PPh3
(3 mmol/g, 0.42 g, 1.26 mmol) was added. The reaction mixture was left under stirring overnight at
40°C, then filtered (TLC, CH2Cl2:CH3OH 90:10, NH3 1% v/v). The organic layer was dried over
Na2SO4 and evaporated under reduced pressure. The obtained crude compound was purified by
flash chromatography (CH2Cl2:CH3OH 90:10, NH3 1% v/v) to afford amine 3 (0.17 g, 0.58 mmol,
92%) as an oil; [] 2D0 =  36.7 (c 1, CHCl3). 1H NMR (CDCl3): = 0.86 (t, J=6.9, 3H, CH3), 1.18-
1.36 (m, 10H, 5 CH2), 1.60 (m, 2H, CH2), 3.01 (dd, J5,6a= 6.4 Hz, J6a,6b= 12.8 Hz, 1H, H-6a), 3.09
(dd, J5,6b= 5.8 Hz, 1H, H-6b), 3.25 (ddd, 1H, J4,5= 8.9 Hz, H-5), 3.35 (dd, 1H, J1,2= 7.8 Hz, J2,3= 8.9
Hz, H-2), 3.49 (m, 1H, OCHa), 3.50 (dd, J3,4= 8.9 Hz, 1H, H-4), 3.55 (dd, 1H, H-3), 3.85 (m, 1H,
24

OCHb), 4.26 (dd, 1H, H-1). 13C NMR (CDCl3):  = 14.07 (CH3), 22.64 (CH2), 25.97 (CH2), 29.30
(CH2), 29.48 (CH2), 29.78 (CH2), 31.84 (CH2), 42.48 (C6), 70.17 (OCH2), 71.01 (C4), 73.51 (C2),
76.39 (C5), 77.00 (C3), 102.93 (C1). ESI-MS (CH3OH, negative-ion mode): m/z =290.0 [M1].
Calcd for C14H29NO5, m/z 291.2 [M]. ESI-HRMS (CH3OH, positive-ion mode): m/z = 314.19361
[M+Na]+ (error, 0.6 ppm). Calcd for C14H29NO5Na, m/z = 314.19379 (monoisotopic mass). FT-IR
(ATR): max 3368, 2922, 2854, 1573 cm-1.
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4.1.5.5. Octyl N-hexanoyl-6-amino-6-deoxy--D-glucopyranoside (4)
To a solution of compound 3 (0.14 g, 0.48 mmol) in dry pyridine (3 mL) at 0 °C hexanoyl chloride
(0.1 mL, 0.72 mmol) was added and the reaction mixture was stirred at rt for 3 h (TLC,
CH2Cl2:CH3OH 90:10, v/v). After quenching with CH3OH, the solvent was evaporated under
vacuum by repeated adding of toluene and the obtained crude was submitted to flash column
chromatography (CH2Cl2:CH3OH, 95:5, v/v) to afford pure compound 4 (0.12 g, 0.30 mmol, 62%
yield), white crystals; m.p.: 142-144°C; [] 2D0 =  13.7 (c 1, CHCl3). 1H NMR (CDCl3):  = 0.83-
0.91 (m, 6H, 2CH3), 1.19-1.37 (m, 14H, 7CH2), 1.56-1.67 (m, 4H, 2CH2), 2.17-2.23 (m, 2H, CH2),
3.12 (ddd, J5,6a= 2.6 Hz, J6a,6b= 15.0 Hz, J6a,NH= 4.6 Hz, 1H, H-6a), 3.14 (dd, J3,4= 9.5 Hz, J4,5= 9.2
Hz, H-4), 3.26 (ddd, 1H, H-5), 3.34 (dd, J1,2= 7.8 Hz, J2,3= 9.2 Hz, 1H, H-2), 3.51 (m, 1H, OCHa),
3.59 (dd, 1H, H-3), 3.85 (m, 1H, OCHb), 4.05 (ddd, J5,6b= 2.6 Hz, J6b,NH= 8.3 Hz, 1H, H-6b), 4.28
(d, 1H, H-1), 5.93 (dd, 1H, NH); 13C NMR (CDCl3):  = 13.89 (CH3), 14.06 (CH3), 22.33 (CH2),
22.63 (CH2), 25.30 (CH2), 25.92 (CH2), 29.20 (CH2), 29.36 (CH2), 29.60 (CH2), 31.41 (CH2),
31.79 (CH2), 36.40 (CH2), 39.78 (C6), 69.73 (C4), 70.66 (OCH2), 73.84 (C2), 74.88 (C3), 74.99
(C5), 103.14 (C1), 175.86 (CO). ESI-MS (CH3OH, negative-ion mode): m/z = 388.4 [M1]. Calcd
for C20H39NO6, m/z 389.28 [M]. ESI-HRMS (CH3OH, positive-ion mode): m/z = 412.26656
[M+Na]+ (error, 1.0 ppm). Calcd for C20H39NO6Na, m/z = 412.26696 (monoisotopic mass). FT-IR
(ATR): max 3313, 2922, 2872, 1642, 1553 cm-1.
4.2. Biological methods
4.2.1. Short term in vitro bioassay for anti-tumor promoters
25

Inhibition was tested using a short term in vitro assay for EBV activation in Raji cells (obtained first
from Prof. G. Klein, Karolinska Institute, Stockholm - Sweden) cultivated in RPMI 1640 medium
containing 10% fetal calf serum, and induced by TPA as described previously.3,21 Raji cells
(1×106/mL) were incubated at 37°C for 48 h in 1mL of a medium containing n-butyric acid (4mM),
32 pmol of TPA in DMSO, and a known amount of the test compound in DMSO. The cells were
stained by high titer EBV-positive sera from nasopharyngeal carcinoma patients and fluorescein-
isothiocyanate-labelled anti-human IgG. After staining, they were detected by a conventional
indirect immunofluorescence technique. The assays were performed in triplicate for each compound
in which at least 500 cells were counted. The average EBV-EA inhibitory activity of the test
compounds was compared to that of control experiments (100%) with butyric acid (4 mM) and TPA
(32 pmol) in which EBV-EA induction was typically around 30%. The viability of the cells was
assayed against treated cells using the Trypan Blue staining method. For an accurate determination
of cytotoxicity, the cell viability was required to be more than 60% 3 days after treatment with the
compounds.
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4.2.2. In vivo two-stage mouse skin carcinogenesis test
Female SENECAR mice were obtained at 5-6 weeks of age from SLC Co. Ltd. (Shizuoka, Japan).
Groups of animals (15 animals per group) were housed in bunches of five in polycarbonate cages.
Mice were permitted free access to MP solid diet (Oriental yeast Co., Ltd. Chiba, Japan) and
drinking water at all times during the study. The back of each mouse was shaved with surgical
clippers before the first day of initiation. Tumors on the back of the mice were initiated with
dimethylbenz[a]anthracene (DMBA; 390 nmol) in acetone (0.1 mL). One week after initiation, they
were promoted twice a week by application of TPA (1.7 nmol) in acetone (0.1 mL). For the animals
in the test compound treated groups the mice were treated with the test compounds (85 nmol) in
acetone (0.1 mL) 1 h before each TPA treatment. The incidence of papillomas was observed weekly
for 20 weeks. The differences in mouse skin papillomas between control and experiments were
analyzed by means of the Student’s t-test at 20 weeks of promotion.
Acknowledgements
This study was supported in part by the University of Milan (Italy) and in part by Grants-in-Aid
26

from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare
(Japan). We also gratefully acknowledge Fondazione CARIPLO under project 2011-0490 for
financial support. The authors thank Mr Andrea Lorenzi for MS technical support and Shahrzad
Reza Elahi (Euticals, Quinto de’ Stampi – Rozzano (MI), Italy) for FT-IR experiments.
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28

CAPTIONS TO SCHEMES AND FIGURES
1
2
Scheme 1
3
4
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6
a) NaN3, DMF; b) PPh3 polymer-bound, THF/H2O (90/10); c) RCOCl, Py; d) H2, Pd/C, MeOH; e)
LPS, Py, CF3CH2OCOR.
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Scheme 2
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a) NaN3, DMF; b) CClPh3, Py 100°C; c) PPh3 polymer-bound, THF/H2O (90/10); d) C5H11COCl,
Py ; e) CClPh3 ,Py 100°C
Scheme 3
a) NaN3, DMF; b) PPh3 polymer-bound, THF/H2O (90/10)
Scheme 4
a) NaN3, DMF; b) MeONa, MeOH; c) PPh3 polymer-bound, THF/H2O (90/10); d) C5H11COCl, Py.
Figure 1
Structures of natural monogalactosyldiacylglycerols (MGDG), sulfoquinovosylacylglycerols
(SQAG) and 6-amino-6-deoxyglycoglycerolipids (AGGL).
Figure 2
Structures of the studied compounds 1-4 and of the reference compounds 5-10.
Figure 3
1H-NMR resonances of glycerol H-2 of compounds 21, 23 and 25 in deuterated methanol at room
temperature. The spectra were collected at 0, 44 and 186 hours.
Figure 4.
Inhibitory effects of compounds 1a and 2a (85 nmol) on DMBA-TPA mouse skin carcinogenesis.
All mice were initiated with DMBA (390 nmol) and promoted with TPA (1.7 nmol) twice a week
starting one week after initiation. A: percentage of mice with papillomas (papilloma incidence); B:
averaged number of papillomas per mouse (papilloma multiplicity). (, TPA alone; ∆, TPA + 1a;
, TPA + 2a,  TPA + 7 ). At 20 weeks of promotion the averaged number of papillomas per
mouse was reduced, with respect to the control group (7.9 ± 1.8), to 5.8 ± 1.1 for 1a (P < 0.1), 6.2 ±
1.3 for 2a (P <0.1 ) and 4.0 ± 0.9 (P <0.001 ) for 7 (control group 9.1 ± 1.6) 23.
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