Chemical synthesis of marine-derived sulfoglycolipids, a new class of molecular adjuvants

Article abstract of DOI:10.3390/md15090288

Vaccines play a primary role in the protection of human health by preventing infectious and chronic diseases. Recently we have reported 1,2-O-distearoyl-3-O-β-D-sulfoquinovosylglycerol (β-SQDG18), here named Sulfavant A (1), which shows promising properties as a new molecular adjuvant in in vitro and in vivo tests. In the present manuscript, we provide full details about a synthetic strategy for the preparation of 1, including a discussion of chemical determinants of the activity and the major technical hurdles we faced during the study. Synthesis of Sulfavant A (1) is achieved by a versatile procedure based on a trichloroacetimidate methodology and peracetate sugar precursors. The final design opens possibilities for the preparation of a series of interesting analogs for further pharmacological optimization and development, including derivatives containing different saturated and polyunsaturated fatty acids (e.g., 17 and 22).

Full text of DOI:10.3390/md15090288

marine drugs
Article
Chemical Synthesis of Marine-Derived
Sulfoglycolipids, a New Class of
Molecular Adjuvants
1
,
1
1
1
ID
, Carmela Gallo 1
ID
Emiliano Manzo *, Laura Fioretto , Dario Pagano , Genoveffa Nuzzo
,
Raffaele De Palma 2 and Angelo Fontana 1
1
Bio-Organic Chemistry Unit, CNR-Institute of Biomolecular Chemistry, Via Campi Flegrei 34,
IT-80078 Pozzuoli, 80078 Napoli, Italy; laura_fioretto@hotmail.it (L.F.); dariopagano85@gmail.com (D.P.);
nuzzo.genoveffa@icb.cnr.it (G.N.); carmen.gallo@icb.cnr.it (C.G.); afontana@icb.cnr.it (A.F.)
Department of Internal and Experimental Clinic, Clinical Immunology and Allergology,
University of Campania, c/o II Policlinico (Bd. 3), Via S.Pansini 5, 80131 Napoli, Italy;
Raffaele.DEPALMA@unicampania.it
2
*
Correspondence: emanzo@icb.cnr.it; Tel.: +39-0818675310
Received: 27 July 2017; Accepted: 11 September 2017; Published: 20 September 2017
Abstract: Vaccines play a primary role in the protection of human health by preventing infectious
and chronic diseases. Recently we have reported 1,2-O-distearoyl-3-O-β-D-sulfoquinovosylglycerol
(
β
-SQDG18), here named Sulfavant A (
adjuvant in in vitro and in vivo tests. In the present manuscript, we provide full details about
a synthetic strategy for the preparation of , including a discussion of chemical determinants of the
activity and the major technical hurdles we faced during the study. Synthesis of Sulfavant A (
1), which shows promising properties as a new molecular
1
1
)
is achieved by a versatile procedure based on a trichloroacetimidate methodology and peracetate
sugar precursors. The final design opens possibilities for the preparation of a series of interesting
analogs for further pharmacological optimization and development, including derivatives containing
different saturated and polyunsaturated fatty acids (e.g., 17 and 22).
Keywords: glycolipids; sulfolipid; vaccine; adjuvant; immunology; innate immunity; dendritic cells;
immunogenic lipid; Sulfavant A
1
. Introduction
Vaccination consists of the stimulation of immune response by vaccines, historically composed
of attenuated or inactivated biological agents [
peptides or recombinant DNA, which are safer, but often insufficiently immunogenic [
1
]. However, most recent vaccines are constituted by
]. To overcome
1
this problem, modern vaccines contain adjuvants that are necessary to boost the immune response.
To date, except for the monophosphoryl lipid A (MPLA), clinically approved formulations of adjuvants
are restricted to aluminum salts and emulsions of lipids in water [2].
Dendritic cells (DCs) are the most important antigen-presenting cells (APCs), and play a pivotal
role in starting immunogenic reactions and priming a long-lasting and antigen-specific response [
The ability to activate DCs is a key tool for improving the efficacy of vaccination [3].
3].
Recently, in our ongoing research on immunomodulatory substances, we reported the
pharmacological development of the non-natural 1,2-O-distearoyl-3-O- -D-sulfoquinovosylglycerol,
here named Sulfavant A ( ) (Figure 1), as a prototype for a new class of molecular adjuvants
based on a sulfolipid skeleton [ ]. This family of products is structurally inspired by the natural
-D-sulfoquinovosyl-diacylglycerols ( -SQDG), bioactive metabolites bearing a 6-sulfonic function
on D-glucose (sulfoquinovose) linked to a glycerol moiety with two fatty acyl chains [ 20].
β
1
4
α
α
5
–
Mar. Drugs 2017, 15, 288; doi:10.3390/md15090288
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2017, 15, 288
2 of 10
The natural sulfoglycoglycerolipids are marked by the
α
-configuration of the anomeric carbon and the
β-configuration type is not present in nature.
Sulfavant A (
After treatment, DCs show up-regulation of MHC II molecules and other co-stimulatory proteins
CD83, CD86), as well as production of pro-inflammatory cytokines (IL-12 and INF- ). Mice immunized
1) activates human DCs and triggers an efficient immune response in vivo [4].
(
γ
with OVA associated to Sulfavant A produced a release of anti-OVA Ig comparable to traditional
adjuvants, while, in an experimental model of melanoma, vaccination of C57BL/6 mice with Sulfavant
A-adjuvanted hgp10 peptide elicited a protective response leading to a promising reduction in tumor
growth and increase of mice survival [4].
Here we discuss in detail the strategy for the synthesis of Sulfavant A (1), including an overview
of the main technical hurdles we have faced, together with the considerations for the scale-up of the
process and the possibility to expand the class of synthesizable analogs.
O
SO -
3
O
HO
HO
O
O
O
OH
O
1
0
Figure 1. 1,2-O-distearoyl-β-6 -sulfoquinovosylglycerol (Sulfavant A).
2
. Results and Discussion
For the preparation of Sulfavant A, the main challenges lay in the requirement of planning
a chemical strategy comprising the introduction of the β-glycosidic bond at the anomeric carbon and
the insertion of the sulfonic function on C of glucose (Scheme 1). At the same time, the possibility
6
of introducing different and unsaturated fatty acyl residues on the glycerol moiety would make the
strategy expandable to a wide class of analogs. Furthermore, the process had to be suitable to scale-up
for further pharmacological development.
-
+
-
+
SO K
SO K
3
3
OP
O
OAcyl1
OAcyl2
OAcyl1
OAcyl2
OAcyl1
OAcyl2
O
O
HO
HO
O
PO
PO
O
PO
PO
O
OH
OP
OP
-SQDG
OH
OP
O
OH
O
HO
HO
PO
PO
O
OH
OH
OH
OP
D-glucose
Scheme 1. Retrosynthetic scheme for preparation of Sulfavant A (1) and its analogs.
The first crucial point was the choice of orthogonal protecting groups on sugar, whose deprotection
should not affect the diacylglycerol aglycon part and the glycosidic linkage. In this frame,
tert-butyldimethylsilyl and benzyl groups to which the synthesis of
α- and β-SQDGs [14,21,22]
had already been applied, were initially tested for the preparation of the sulfolipid. Unfortunately,
the tert-butyldimethylsilyl group proved to be too labile in the experimental conditions and this,
together with its low deprotection efficiency, led to a considerable decrease in yields; whereas the use
of benzyl excluded the possibility of introducing unsaturated acyl chains to the glycerol, and greatly
reduced the possibility of extending the synthesis to other analogs. For these reasons, we decided to
explore the use of acetate as transient protecting group for the hydroxy functions of the sugar moiety.
The acetoxy derivatives are stable under a wide range of conditions and, most importantly, the presence

Mar. Drugs 2017, 15, 288
3 of 10
0
of the acetate residue at C-2 is capable of ensuring an excellent stereoselectivity in the formation of
the glycosidic bond by anchimeric assistance [23 24]. On the other hand, acetate removal implies
β
,
basic conditions that are not suitable for preserving the ester linkage of glycerol with the acyl chains.
To this end, a careful tuning of hydrolysis by hydrazine was crucial to completing the synthesis of the
sulfolipid [23,24].
As reported in Scheme 2, the total synthesis of Sulfavant A (1) started with acetylation of D-glucose,
followed by selective deacetylation at the anomeric position with benzylamine and derivatization
with trichloroacetimidate. The subsequent coupling with 1,2-O-isopropylidene glycerol by Schmidt
methodology [25,26] gave the derivative 5 in high yields (about 80%). As expected, the acetate groups
determined the high stereoselectivity of the reaction (β/α ratio 95:5) with the β-orientation of the
glycosidic bond by neighboring group participation.
OH
OAc
O
OAc
O
OAc
O
a
b
c
O
HO
HO
AcO
AcO
AcO
AcO
AcO
AcO
OH
OAc
73%
OH
82%
/=98/2
94%
AcO
OH
OAc
OAc

CNHCCl
3
2
3
4
D-glucose
8
0%
d

/=5/95
OAc
O
OAc
OStearoyl
OH
OAc
O
f
e
O
O
AcO
AcO
O
OStearoyl
AcO
AcO
O
OH
AcO
AcO
O
O
9
2%
75%
OAc
OAc
OAc
7
6
5
g
70%
OH
OTr
OTr
OStearoyl
OStearoyl
OStearoyl
OStearoyl
OStearoyl
OStearoyl
h
O
i
O
O
HO
HO
O
HO
HO
O
AcO
AcO
O
70%
90%
OH
OH
OAc
8
9
10
l
82%
OTs
OH
OStearoyl
OStearoyl
OStearoyl
OStearoyl
SAc
OStearoyl
OStearoyl
n
O
m
O
AcO
AcO
O
AcO
AcO
O
O
AcO
AcO
O
80%
93%
OAc
OAc
11
OAc
1
3
12
p
65%
SO
3
-K+
SO3-K+
O
OStearoyl
OStearoyl
1
OStearoyl
OStearoyl
q
4'
O
HO
HO
1' O
AcO
AcO
O
6
8%
OH
3
OAc
1
4
Sulfavant (A) (1)
◦
Scheme 2.
(
a
) Ac O/pyridine; (
b
) BnNH , THF; (
c
) Trichloroacetonitrile, DBU, dichloromethane, 0 C;
2
2
◦
(
d
) BF -Et O, glycerol acetonide, dichloromethane, molecular sieves 4A,
−
10 C; (
e
) Zn(NO ) -6H O,
3
2
3 6
2
◦
acetonitrile, 50 C; (
f) Stearic acid, DCC, DMAP, dichloromethane; (
g
) hydrazine mono-hydrate, EtOH
) Tosyl chloride, pyridine;
) Hydrogen peroxide, acetic acid, potassium acetate,
◦
◦
8
5%, 45 C; (
h
) TrCl, pyridine; (
i
) Ac O/pyridine; (
l
) I -MeOH (1%), 60 C; (
m
2
2
◦
(
4
n
) Potassium thioacetate, 2 butenone, 80 C; (
p
◦
◦
0 C; (q) hydrazine mono-hydrate, EtOH 85%, 44 C.
The next step was the removal of the isopropylidene residue under soft acidic conditions to
preserve the glycosidic linkage. Use of zinc nitrate hexahydrate in acetonitrile allowed hydrolysis
in quantitative yields [27], whereas N,N-dicyclohexylcarbodiimide (DCC)-mediated condensation
with two equivalents of stearic acid yielded the diacyl derivative
introducing the sulfonic function on carbon 6 , a selective removal of sugar acetyl groups without
7
. At this point, with the aim of
0

Mar. Drugs 2017, 15, 288
4 of 10
affecting the acyls on glycerol was necessary to obtain
β-glucosyl-distearoyl-glycerol
8
. This was
achieved by an accurate setting of the basic conditions that drove us to use monohydrate hydrazine in
◦
an amount of 2.4 moles per acetate unit at a temperature below 45 C [23
,24]. After trytilation of the
primary alcohol of glucose (compound
9
, 70 %), acetylation (compound 10, 89%), detrytilation [28
]
0
(compound 11, 82%) and subsequent introduction on C-6 of the tosyl function (compound 12, 80%)
permitted the insertion of a thioacetate group (compound 13, 93%) [29]. Final oxidation by hydrogen
peroxide gave the corresponding sulfonic acid (compound 14, 65%), which was fully deacetylated by
monohydrate hydrazine to give Sulfavant A (1) (68%), whose NMR data are reported in Table 1.
1
13
Table 1. H and C NMR data of Sulfavant A (1) in CDCl :CD OD (1:1), 400 MHz.
3
3
Position
δ
δH, m, J (Hz)
C
0
1
2
3
4
103.4
73.6
76.5
73.1
72.5
53.8
53.8
63.3
63.3
70.5
67.8
67.8
4.34/4.32 (each for 1H), d, 7.8
3.26, m
0
0
3.42, m
3.14, m
3.79–3.75, m
3.32, m
0
0
5
0
0
6
6
a
b
a
b
2
2.98, m
4.47, m
4.19, m
5.29, m
1
1
3
3
a
b
4.13–4.03, m
3.79–3.75, m
Fatty acyls
α-CH2
β-CH2
CH2 (chain)
CH3
34.4
25.1
30.4–29.6
14.1
2.43–2.35, overlapped
1.69–1.54, overlapped
1.32–1.25, overlapped
0.94, overlapped
Synthesis of Sulfavant A (1) was characterized by clean and efficient steps, along with a simple
and easy workup, which makes the procedure suited for subsequent and crucial scaling-up. In fact,
we did not face any problems in the transition from small to larger volumes. Furthermore, the workup
did not require any particular experimental change in order to prepare hundreds of milligrams (400 mg)
of the product. On the basis of this process, we also expect that the synthetic procedure is able to
provide grams of the vaccine adjuvant for in vivo tests and future pharmacological development.
The design of the synthetic strategy also opens possibilities for the preparation of a large
range of analogs, including compounds with mixed composition of fatty acids or unsaturated acyl
chains on glycerol moiety. An example of this is reported in Scheme 3 for the synthesis of 17
,
1
-O-stearoyl-2-O-palmitoyl-3-O- -D-sulfoquinovosylglycerol, which contains two different fatty acids
β
resgiospecifically distributed at the two positions of glycerol.
◦
In this case, use of DCC at 4 C permitted a good control of the step-wise introduction of two
different acyl chains and gave yields of the monoacyl derivative 15 (81%) significantly higher than
those obtained by other methods [30
favored in the first coupling reaction with equimolar amounts of fatty acid, and we did not observe any
byproduct at C-2 of 15, as indicated by the NMR spectra, which showed deshielding of H -1 signals
–32]. Acylation of the primary hydroxy residue was largely
2
at
δ 4.21–4.11 (long range correlation of H -1 to a carboxylic ester at δ 171.1) and δ value of H-2,
H 2 C H
linked to non-acylated oxygen, at 3.91 ppm (Figure 2).

Mar. Drugs 2017, 15, 288
5 of 10
OAc
O
OAc
O
OH
a
OH
AcO
AcO
OH
AcO
AcO
O
OStearoyl
O
81%
OAc
OAc
6
15
b
84%
-
+
SO K
Scheme 2
g-q)
3
OAc
O
OPalmitoyl
OStearoyl
OPalmitoyl
OStearoyl
(
O
HO
HO
O
AcO
AcO
O
OH
OAc
16
17
◦
Scheme 3.
(a
) Stearic acid (1 equiv.), DCC, DMAP, dichloromethane, 4 C. (
b) Palmitic acid (1,2 equiv.),
DCC, DMAP, dichloromethane.
OAc
OH
4'
AcO
AcO
O
1' O
OStearoyl
3
1
2
H ‐6′
OAc
H‐3′
H‐3′ H‐4′
H‐4′
H‐2′
H‐1′
H‐1′
H‐3a H‐5′H‐3b
H‐3a H‐5′H‐3b
1
Figure 2. H-NMR of 15 (CDCl , 400MHz) and enlargement of the area between 5.3 and 3.5 ppm.
3
The introduction of acetate as protecting group also offers access to unsaturated analogs, as long
as it is possible to control the sensitivity of double bonds to both the iodine and the hydrogen
peroxide/acetic acid/acetate systems that are used for removal of the trityl group and sulfur oxidation,
respectively. As shown in Scheme 4, this could be achieved by a slight modification of the synthetic
procedure, and the use of an acidic resin to deprotect the primary hydroxy group and molybdate
+
◦
oxidation for the final step. Soft acidic conditions by Dowex H at 50 C in methanol/water
5/5 allowed a complete removal of the trityl group while preserving the glycosidic bond of 19
9
.
The reaction led to formation of a small amount of other hydrolyzed byproducts, which were removed
by chromatographic purification, but resulted in a slight decrease in the synthetic yield in comparison
with the original step used for Sulfavant A. Molybdate oxidation has been used already for synthesis
of
α
-SQDGs [14], but application in the preparation of the
β
-SQDG required a careful adjustment of
◦
the reaction time. Increase in the temperature from room value to 50 C, together with the use of twice
the amount of ammonium heptamolybdate tetrahydrate and hydrogen peroxide, favoured the course
of the reaction and led to the dioleoyl analog 22 with yield close to 60%.

Mar. Drugs 2017, 15, 288
6 of 10
OAc
OTr
OH
a; Scheme 2 (g-i)
OOleoyl
OOleoyl
O
O
AcO
AcO
OH
AcO
AcO
O
O
OAc
OAc
6
18
b
56%
Scheme 2
m, n)
SAc
O
OH
(
OOleoyl
OOleoyl
OOleoyl
OOleoyl
O
AcO
AcO
AcO
AcO
O
O
OAc
OAc
20
19
c
5
3%
SO -
SO3-
O
3
OOleoyl
OOleoyl
OOleoyl
OOleoyl
d
9%
O
AcO
AcO
HO
HO
O
O
5
OAc
OH
2
1
22
+
Scheme 4. (a) Oleic acid (2.3 equiv.), DCC, DMAP, dichloromethane; (b) Dowex-H , MeOH/H O
(
2
◦ ◦
95/5), 50 C; (c) (NH ) Mo O -4H O, H O , MeOH, 50 C; (d) hydrazine mono-hydrate (2.4 equiv.),
4 6 7 24 2 2 2
EtOH 85%.
3
. Materials and Methods
D- and 2D-NMR spectra were recorded on a Bruker Avance-400 (400.13 MHz) and on a Bruker
DRX-600 equipped with a TXI CryoProbe in CDCl and CD OD ( values are referred to CHCl and
1
δ
3
3
3
13
CH OH at 7.26 and 3.34 ppm respectively) and C NMR spectra were recorded on a Bruker DPX-300
3
(
75.47 MHz) (
δ values are referred to CDCl and CD OD at 77.0 and 49.0 ppm respectively). HRESIMS
3 3
was conducted on a Micromass Q-TOF micro. TLC plates (Kieselgel 60 F254) and silica gel powder
(Kieselgel 60, 0.063–0.200 mm) were from Merck.
All the reagents were purchased from Sigma-Aldrich and used without any further purification.
Preparation and Characterization of Synthetic Molecules
All the experimental procedures and spectral data of Sulfavant A (1) and synthetic intermediates
2
–14 were reported in reference 4.
Compound 15: compound (0.148 g, 0.00035 mol) was dissolved in anhydrous dichloromethane
6
(
5 mL); stearic acid (0.0994 g, 0.00035 mol), dicyclohexylcarbodiimide (0.0721 g, 0.00035 mol) and
◦
DMAP (0.00427 g, 0.000035 mol) were added at 0 C and the reaction mixture was stirred overnight at
4
◦
C; after evaporation under reduced pressure, the mixture was purified by silica gel chromatography
using a gradient of petroleum ether/diethyl ether to give compound 15 (0.165 g, 0.000284 mol, 81%).
1
0
0
0
H-NMR (400 MHz, CDCl3):
δ
5.18 (1H, t, J = 9.6 Hz, H-3 ), 5.04 (1H, t, J = 9.6 Hz, H-4 ), 4.97 (1H,
0
dd, J = 8.1, 9.6 Hz, H-2 ), 4.53 and 4.51 (each for 1H of the two epimers, d, J = 8.1 Hz, H-1 ), 4.20 (1H,
dd, J = 5.2, 12.0 Hz, H-1a), 4.13 (1H, dd, J = 2.4, 12.0 Hz, H-1b), 4.09–4.06 (2H, m, H -6 ), 3.91(1H,
m, H-2), 3.84 (1H, dd, 3.4, 10.3 Hz, H-3a), 3.70, (1H, m, H-5 ), 3.64 (1H, dd, 5.7, 10.3 Hz, H-3b), 2.29
0
2
0
(
2H, t, J = 7.1 Hz,
α-methylene ), 2.07–1.96 (12H, s, 4 OAc), 1.61–1.52 (2H, m, β-methylene), 1.30–1.18
(
acyl chain methylenes), 0.84 (3H, t, 6.8 Hz, CH ); HRESIMS m/z 711.3940 [M + Na]+ (calcd for
3
C H O Na, 711.3932).
35
60 13
Compound 16
:
compound 15 (0.165 g, 0.000284 mol) was dissolved in anhydrous
dichloromethane (6 mL); palmitic acid (0.0738 g, 0.00034 mol), dicyclohexylcarbodiimide (0.07 g,

Mar. Drugs 2017, 15, 288
7 of 10
0.00034 mol) and DMAP (0.0042 g, 0.000034 mol) were added and the reaction mixture was
stirred overnight; after evaporation under reduced pressure, the mixture was purified by silica gel
chromatography using a gradient of petroleum ether/diethyl ether to give compound 16 (0.228 g,
0.00024 mol, 84%); NMR data were identical to those of
7 [4
]; HRESIMS m/z 949.6235 [M + Na]+
(
calcd for C H O Na, 949.6228).
51
90 14
Compound 17: NMR data were identical to those of Sulfavant A (
1) (Table 1); HRESIMS m/z
21.5454 [M − K]− (calcd for C H O S , 821.5454).
−
8
43
81 12
Compound 18: 1H-NMR (400 MHz, CDCl3):
δ 7.47–7.18 (15H, m, trityl portion), 5.36–5.32 (4H,
0
0
0
m, olefinic protons), 5.23 (1H, m, H-2), 5.15 (1H, m, H-4 ), 5.14 (1H, m, H-3 ), 5.05 (1H, m, H-2 ), 4.54
0
(
(
1H, d, J = 7.7 Hz, H-1 ), 4.34 (1H, dd, J = 4.1, 12.0 Hz, H-1a), 4.13 (1H, dd, J = 5.9, 12.0 Hz, H-1b), 4.03
0
1H, dd, J = 4.9, 11.1 Hz, H-3a), 3.74 (1H, dd, J = 5.1, 11.1 Hz, H-3b), 3.55 (1H, m, H-5 ), 3.27 (1H, bd,
0
0
J = 10.2 Hz, H-6 a), 3.09 (1H, dd, J = 2.3, 10.2, H-6 b), 2.31 (2H, t, J = 7.5 Hz,
t, J = 7.8 Hz, -methylene), 2.10–2.01 (9H, s, 3 OAc), 2.03–2.00 (8H, overlapped, allylic protons), 1.54
4H, m, -methylene), 1.28–1.15 (acyl chain), 0.86 (6H, t, J = 7.0 Hz, 2CH ); HRESIMS m/z 1173.7224
α-methylene), 2.16 (2H,
α
(
[
β
+
3
M + Na] (calcd for C H O Na, 1173.7218).
70 102 13
Compound 19: compound 18 (0.1 g, 0.000087 mol) was dissolved in methanol/water (95/5)
+
◦
solution and dowex-H (1.1 g) was added and the reaction was stirred at 50 C overnight; after
filtration and evaporation under reduced pressure, the reaction mixture was purified by silica gel
chromatography using a gradient of petroleum ether/diethyl ether to give compound 19 (0.044 g,
0
0
(
.000048, 56%);
δ
5.37–5.33 (4H, m, olefinic protons), 5.31–5.28 (2H, overlapped, H-2, H-3 ), 5.08–5.00
0
0
0
2H, m, H-2 , H-4 ), 4.61 (1H, d, J = 7.8 Hz, H-1 ), 4.36 (1H, m, H-1a), 4.16 (1H, m, H-1b), 3.92 (1H, m,
H-3a), 3.78 (1H, m, H-3b), 3.74 (1H, m, H-6 a), 3.60 (1H, m, H-6 b), 3.54 (1H, m, H-5 ), 2.41–2.29 (4H, m,
0
0
0
α
-methylene), 2.10–2.01 (9H, s, 3 OAc), 2.04–2.01 (8H, overlapped, allylic protons), 1.62–1.52 (4H, m,
+
β
-methylene), 1.33–1.15 (acyl chain), 0.90 (6H, t, J = 7.0 Hz, 2CH ); HRESIMS m/z 931.6118 [M + Na]
3
(
calcd for C H O Na, 931.6123).
51 88 13
1
Compound 20: H-NMR (400 MHz, CDCl ): δ 5.35–5.31 (4H, m, olefinic protons), 5.18–5.14 (2H,
3
0
0
0
0
m, H-2, H-3 ), 4.96–4.89 (2H, m, H-2 , H-4 ), 4.50 (1H, d, J = 8.0 Hz, H-1 ), 4.28 (1H, dd, J = 4.1, 11.8 Hz,
H-1a), 4.08 (1H, dd, J = 5.7, 11.8 Hz, H-1b), 3.91 (1H, dd, J = 4.5, 11.1 Hz, H-3a), 3.65 (1H, dd, J = 5.4,
1
0
0
1.1 Hz, H-3b), 3.62 (1H, m, H-5 ), 3.24 (1H, bd, J = 11.4 Hz, H-6 a), 3.05 (1H, dd, J = 2.4 Hz, 11.4 Hz),
2
.33–2.29 (4H, m,
α
β
-methylene), 2.13–1.98 (9H, s, 3 OAc), 2.05–2.01 (8H, overlapped, allylic protons),
1
.64–1.57 (4H, m,
-methylene), 1.32–1.23 (acyl chain), 0.91–0.87 (6H, overlapped, 2CH ); HRESIMS
3
+
m/z 989.6007 [M + Na] (calcd for C H O NaS, 989.6000).
53
90 13
Compound 21: to a solution of compound 20 (0.04 g, 0.000041 mol) in methanol (8 mL) ammonium
heptamolybdate tetrahydrate [(NH ) Mo O 4H O] (0.051 g, 1 equiv., 0.000041 mol) in 30% H O
4
6
7
24
2
2
2
◦
(
1.0 mL) was added with stirring. After 24 h at 50 C the mixture was diluted with water and extracted
with ethylacetate; the organic phase was purified by silica gel chromatography using a gradient of
chloroform/methanol to give 21 (0.022 g, 0.000022 mol, 53%); 5.36–5.31 (4 H, m, olefinic protons),
.27 (1H, m, H-2), 5.18 (1H, dd, 8.9, 8.9 Hz, H-3), 5.08–4.99 (2H, m, H-2 , H-4 ), 4.68 (1H, d, 7.3 Hz,
H-1 ), 4.31 (1H, dd, J = 5.1, 11.1 Hz, H-1a), 4.15–4.06 (3H, overlapped, H-1b, H-3a, H-5 ), 3.75 (1H, m,
H-3b), 3.20 (2H, overlapped, H -6 ), 2.33–2.28 (4H, m,
δ
0
0
5
0
0
0
α
-methylene), 2.06–1.98 (9H, s, 3 OAc), 2.05–2.00
2
(
8H, overlapped, allylic protons), 1.64–1.55 (4H, m,
β
-methylene), 1.34–1.22 (acyl chain), 0.91–0.88 (6H,
−
−
51 87 15
overlapped, 2CH ); HRESIMS m/z 971.5764 [M − K] (calcd for C H O S , 971.5771).
3
Compound 22: compound 21 (0.022 g, 0.000022 mol) was dissolved in aq. ethanol (85%) (4 mL),
hydrazine monohydrate (0.00016 mol, 2.4 equiv.) was added, and the reaction mixture was stirred
◦
for 6 h at 44 C. After evaporation under a stream of nitrogen, the mixture was purified by silica gel
chromatography using a gradient of chloroform/methanol to obtain 22 (0.011 g, 0.000013 mol, 59%)
1
H-NMR (400 MHz, CDCl /CD OD 1/1):
δ
5.34–5.27 (5H, overlapped, H-2 and olefinic protons), 4.47
3
3
0
(
1H, m, H-1a), 4.34 and 4.32 (each for 1H, d, 7.8 Hz, H-1 ), 4.19 (1H, m, H-1b), 4.13–4.03 (1H, m, H-1a),
0 0 0 0
3
.79–3.75 (2H, m, H-3b, H-5 ), 3.42 (1H, m, H-3 ), 3.32 (1H, m, H-6 a), 3.26 (1H, m, H-2 ), 3.14 (1H, m,
0
0
H-4 ), 2.98 (1H, m, H-6 b), 2.43–2.35 (4H, m, α-methylene), 2.06–2.01 (8H, m, allylic protons), 1.69–1.58

Mar. Drugs 2017, 15, 288
8 of 10
(
[
4H, m,
β
-methylene), 1.43–1.29 (acyl chain), 0.94 (6H, overlapped, 2CH ); HRESIMS m/z 845.5460
3
M − K]− (calcd for C H O S , 845.5454).
−
45
81 12
4
. Conclusions
Sulfavant A (1) is a synthetic β-sulfoglycolipid with promising potential for immunomodulant
and adjuvant activity. Synthesis of this molecule was achieved by a versatile and simple
chemical procedure involving stereoselective glycosylation of trichloroacetimidate-glucose donor
with 1,2-O-isopropylidene glycerol acceptor. In addition to the simplification of the synthetic steps,
the trichloroacetimidate methodology and the use of acetate as the main protecting group of the
sugar donor permit the synthesis in high yields of saturated and unsaturated
β-anomer SQDG
derivatives with mixed composition of fatty acids (e.g., 17 and 22). The proposed procedure has
versatile applications, and allows access to a wide family of regio-pure compounds. The efficiency and
simplicity of the strategy also allow a scale-up of the synthetic approach, leading to amounts of pure
β-SQDGs for in vivo tests and future pharmacological development.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/15/9/288/s1,
Figure S1: COSY and HMBC spectra of 15.
Acknowledgments: This work was funded by the Italian National Research Council. The authors acknowledge
the contribution of the MIUR (Italian Research Ministry of Research) project “Antigens and adjuvants for vaccines
and immunotherapy” (PON01_00117). The authors thank D. Melck of ICB-NMR service.
Author Contributions: E.M., L.F. and D.P. designed and carried out the chemical synthesis of sulfoquinovosides;
G.N. and C.G. purified the synthetic molecules; E.M. and A.F. provided intellectual input and assisted with
experimental design and writing of the paper; E.M., A.F. and R.D.P. conceived the study and analyzed the
results together.
Conflicts of Interest: The authors declare no competing financial interest.
References
1.
2.
3.
4.
Di Pasquale, A.; Preiss, S.; Da Silva, F.T.; Garcon, N. Vaccine Adjuvants: Form 1920 to 2015 and Beyond.
Vaccines 2015, 3, 320–343. [CrossRef] [PubMed]
Rappuoli, R.; Mandl, C.W.; Black, S.; De Gregorio, E. Vaccines for the twenthy-first century society.
Nat. Rev. Immunol. 2011, 11, 865–872. [PubMed]
Kastenmuller, W.; Kastenmuller, K.; Kurts, C.; Seder, R.A. Dendritic cell-targeted vaccines-hope or hype?
Nat. Rev. Immunol. 2014, 14, 705–711. [CrossRef] [PubMed]
Manzo, E.; Cutignano, A.; Pagano, D.; Gallo, C.; Barra, G.; Nuzzo, G.; Sansone, C.; Ianora, A.; Urbanek, K.;
Fenoglio, D.; et al. A new marine-derived sulfoglycolipid triggers dendritic cell activation and immune
adjuvant response. Sci. Rep. Nat. 2017, in press. [CrossRef] [PubMed]
5.
6.
7.
8.
9.
Sahara, H.; Ishikawa, M.; Takahashi, N.; Ohtani, S.; Sato, N.; Gasa, S.; Akino, T.; Kikuchi, K.
0
0
In vivo anti-tumour effectof 3 -sulphonoquinovosyl 1 -monoacylglyceride isolated from sea urchin
Strongylocentrotus intermedius) intestine. Br. J. Cancer 1997, 75, 324–332. [CrossRef] [PubMed]
(
Golik, J.; Dickey, J.K.; Todderud, G.; Lee, D.; Alford, J.; Huang, S.; Klohr, S.; Eustice, D.; Aruffo, A.; Agler, M.L.
Isolation and Structure Determination of Sulfonoquinovoyl Dipalmitoyl Glyceride, a P-selectin Receptor
Inhibitor from the Alga Dictyochloris fragrans. J. Nat. Prod. 1997, 60, 387–389. [CrossRef] [PubMed]
Loya, S.; Reshef, V.; Mizrachi, E.; Silberstein, C.; Rachamim, Y.; Carmeli, S.; Hizi, A. The Inhibition of
the Reverse Transcriptase of HIV-1 by the Natural Sulfoglycolipids from Cyanobacteria: Contribution of
Different Moieties to Their High Potency. J. Nat. Prod. 1998, 61, 891–895. [CrossRef] [PubMed]
Gustafson, R.; Cardelina, J.H.; Fuller, R.W.; Weislow, O.S.; Kiser, R.F.; Snader, K.M.; Patterson, G.M.L.;
Boyd, M.R. AIDS-Antiviral Sulfolipids From Cyanobacteria (Blue-Green Algae). J. Nat. Cancer Inst. 1989, 81,
1
254–1258. [CrossRef] [PubMed]
Murakami, C.; Yamazaki, T.; Hanashima, S.; Takahashi, S.; Ohta, K.; Yoshida, H.; Sugawara, F.; Sakaguchi, K.;
Mizushina, Y. Structure-function relationship of synthetic sulfoquinovosyl-acylglycerols as mammalian
DNA polymerase inhibitors. Arch. Biochem. Biophys. 2002, 403, 229–236. [CrossRef]

Mar. Drugs 2017, 15, 288
9 of 10
1
1
1
1
1
1
1
1
1
1
2
0. Matsumoto, Y.; Sahara, H.; Fujita, T.; Shimozawa, K.; Takenouchi, M.; Torigoe, T.; Hanashima, S.; Yamazaki, T.;
Takahashi, S.; Sugawara, F.; et al. An immunosuppressive effect by synthetic sulfonolipids deduced from
sulfoquinovosyl diacylglycerols of sea urchin. Transplantation 2002, 74, 261–267. [CrossRef] [PubMed]
1. Matsumoto, Y.; Sahara, H.; Fujita, T.; Hanashima, S.; Yamazaki, T.; Takahashi, S.; Sugawara, F.; Mizushina, Y.;
Ohta, K.; Takahashi, N.; et al. A novel immunosuppressive agent, SQDG, derived from sea urchin.
Transplant. Proc. 2000, 32, 2051–2053. [CrossRef]
2. Ohta, K.; Mizushina, Y.; Yamazaki, T.; Hanashina, S.; Sugawara, F.; Sakaguchi, K. Specific interaction
between an Oligosaccharide on the Tumour Cell Surface and the Novel Antitumour Agents,
Sulfoquinovosylacylglycerols. Biochem. Biophys. Res. Commun. 2001, 288, 893–900. [CrossRef] [PubMed]
3. Reshef, V.; Mizrachi, E.; Maretzki, T.; Silberstein, C.; Loya, S.; Hizi, A.; Carmeli, S. New Acylated
Sulfoglycolipids and Digalactolipids and Related Known Glycolipids from Cyanobacteria with a Potential to
Inhibit the Reverse Transcriptase of HIV-1. J. Nat. Prod. 1997, 60, 1251–1260. [CrossRef] [PubMed]
4. Hanashima, S.; Mizushina, Y.; Yamazaki, T.; Ohta, K.; Takahashi, S.; Sahara, H.; Sagakuchi, K.; Sugawara, F.
Synthesis of sulfoquinovosylacylglycerols, inhibitors of Eukaryotic DNA Polymerase
α and β. Bioorg. Med.
Chem. 2001, 9, 367–376. [CrossRef]
5. Hanashima, S.; Mizushina, Y.; Yamazaki, T.; Ohta, K.; Takahashi, S.; Koshino, H.; Sahara, H.; Sagakuchi, K.;
Sugawara, F. Structural determination of sulfoquinovosyldiacylglycerol by chiral syntheses. Tetrahedron Lett.
2
000, 41, 4403–4407. [CrossRef]
6. Mizushina, Y.; Maeda, N.; Kawasaki, M.; Ichikawa, H.; Murakami, C.; Takemura, M.; Xu, X.; Sugawara, F.;
Fukumori, Y.; Yoshida, H.; Sakaguchi, K. Inhibitory action of emulsified sulfoquinovosyl acylglycerol on
mammalian DNA polymerases. Lipids 2003, 38, 1065–1074. [CrossRef] [PubMed]
7. Hanashima, S.; Mizushina, Y.; Ohta, K.; Yamazaki, T.; Sugawara, F.; Sakaguchi, K. Structure-Activity
Relationship of
a
ovel Group of Mammalian DNA Polymerase Inhibitors, Synthetic
Sulfoquinovosylacylglycerols. Jpn. J. Cancer Res. 2000, 91, 1073–1083. [CrossRef] [PubMed]
8. Mizushina, Y.; Watanabe, I.; Ohta, K.; Takemura, M.; Sahara, H.; Takahashi, N.; Gasa, S.; Sugawara, F.;
Matsukage, A.; Yoshida, S.; et al. Studies on inhibitors of mammalian DNA polymerase
α and β: Sulfolipids
from a pteridophyte, Athyrium niponicum. Biochem. Pharmacol. 1998, 55, 537–541. [CrossRef]
9. Ohta, K.; Hanashima, S.; Mizushina, Y.; Yamazaki, T.; Saneyoshi, M.; Sugawara, F.; Sakaguchi, K. Studies on
a novel DNA polymerase inhibitor group, synthetic sulfoquinovosylacylglycerol: inhibitory action on cell
proliferation. Mutat. Res. 2000, 467, 139–152. [CrossRef]
0. Mizushina, Y.; Xu, X.; Asahara, H.; Takemura, M.; Yamaguchi, T.; Kuroda, K.; Linn, S.; Yoshida, H.; Koiwai, O.;
Saneyoshi, M.; et al. A sulfoquinovosyl diacylglycerol is a DNA polymerase epsilon inhibotor. Biochem. J.
2
003, 370, 299–305. [CrossRef] [PubMed]
2
2
1. Gigg, R.; Penglis, A.A.E.; Conant, R. Synthesis of 3-O-(6-deoxy-6-sulfo-α-D-glucopyranosyl)-1,2-di-O-
hexadecanoyl-L-glycerol, ‘sulphoquinovosyl diglyceride’. J. Chem. Soc. 1980, 2490–2493.
2. Yamazaki, T.; Aoki, S.; Ohta, K.; Hyuma, S.; Sakaguchi, K.; Sugawara, F. Synthesis of an immunosuppressant
SQAG9 and the determination of the binding peptide by T7 phage display. Bioorg. Med. Chem. Lett. 2004
343–4346. [CrossRef] [PubMed]
,
4
2
3. Manzo, E.; Ciavatta, M.L.; Pagano, D.; Fontana, A. An efficient and verstaile chemical synthesis of bioactive
glyco-glycerolipids. Tetrahedron Lett. 2012, 53, 879–881. [CrossRef]
2
4. Pagano, D.; Cutignano, A.; Manzo, E.; Tinto, F.; Fontana, A. Glycolipids synthesis: Improved hydrazinolysis
conditions for preparation of 1,2-polyunsaturated fatty acyl-
016, 424, 21–23. [CrossRef] [PubMed]
5. Schmidt, R.R.; Michel, J. Facile synthesis of α- and β-O-Glycosil Imidates; Preparation of Glycosides and
Disaccharides. Angew. Chem. Int. Ed. Engl. 1980, 19, 731–732. [CrossRef]
6. Schmidt, R.R. New Methods for the Synthesis of Glycosides and Oligosaccharides—Are There Alternatives
to the Koenigs-Knorr Method? [New Synthetic Methods (56)]. Angew. Chem. Int. Ed. 1986, 25, 212–235.
β-monogalactosyl-glycerols. Carbohydr. Res.
2
2
2
[
CrossRef]
7. Vijayasaradhi, S.; Singh, J.; Aidhen, I.S. An Efficient, Selective Hydrolysis of Terminal Isopropylidene Acetal
Protection by Zn(NO ) 6H O in Acetonitrile. Synlett 2000, 110–112.
2
2
3
2
2
8. Wahlstrom, J.L.; Ronald, R.C. Detritylation of Ethers using Iodine-Alcohol Reagents: An Acid Catalytized
Reaction. J. Org. Chem. 1998, 63, 6021–6022. [CrossRef] [PubMed]

Mar. Drugs 2017, 15, 288
10 of 10
2
3
9. Fernandez-Bolanos, J.G.; Morales, J.; Garcia, S.; Dianez, M.J.; Estrada, M.D.; Lopez-Castro, A.; Perez, S.
Synthesis and cristal structure of methyl 2-amino-2,6-dideoxy-α-D-glucopiranoside-6-sulfonic acid.
Carbohydr. Res. 1993, 248, 1–14. [CrossRef]
0. Chen, J.; Profit, A.A.; Prestwich, G.D. Synthesis of Photoactivatable 1,2-O-Diacyl-sn-glycerol Derivatives
of 1-L-Phosphatidyl-D-myo-inositol 4,5-Bisphosphate (PtdInsP2) and 3,4,5-Trisphosphate (PtdInsP3).
J. Org. Chem. 1996, 61, 6305–6312. [CrossRef] [PubMed]
3
1. Dosuna-Rodríguez, I.D.; Gaigneaux, E.M. Glycerol acetylation catalysed by ion exchange resins. Catal. Today
2
012, 195, 14–21. [CrossRef]
3
2. Pruvost, R.; Boulanger, J.; Léger, B.; Ponchel, A.; Monflier, E.; Ibert, M.; Mortreux, A.; Sauthie, M. Biphasic
Palladium-Catalyzed Hydroesterification in aPolyol Phase: Selective Synthesis of Derived Monoesters.
ChemSusChem 2015, 8, 2133–2137. [CrossRef] [PubMed]
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(